Application of golden squid ink polysaccharide SIP4 in preparing products for treating breast cancer

Abstract

The present invention discloses the use of golden squid ink polysaccharide SIP4 in the preparation of products for treating breast cancer. The golden squid ink polysaccharide SIP4 provided by the present invention can inhibit the proliferation and migration of breast cancer cells both in vivo and in vitro, and mediate Ca 2+ The influx promotes apoptosis of breast cancer cells, and is particularly effective in inducing early apoptosis. The present invention provides a new technical option for therapeutic drugs and chemotherapy-assisted treatment of breast cancer, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to application of golden cuttlefish ink polysaccharide SIP4 in preparing products for treating breast cancer. Background Art

[0002] Breast cancer is a highly heterogeneous disease. Targeted therapies have shown promising efficacy in breast cancers positive for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). However, for triple-negative breast cancer (TNBC), which is highly aggressive and lacks these receptors, there are still no effective targeted drugs, and chemotherapy remains the mainstay of treatment. TNBC accounts for approximately 15% of breast cancer cases, 12% of which eventually develop distant metastases, such as to the lungs, liver, and central nervous system. Therefore, new treatments are urgently needed for TNBC, which has a poor prognosis, is prone to recurrence, metastasis, and a high mortality rate (5-year survival is only 26%), especially for patients with metastatic TNBC.

[0003] Ink polysaccharides are polysaccharide compounds in some marine organisms (such as golden cuttlefish). For example, sepia ink polysaccharide (SIP) is a multifunctional marine active substance that not only has chemotherapy protection and anticoagulant functions, but also has anti-tumor and chemotherapy sensitization activities. SIP can inhibit tumor cell growth, proliferation and metastasis, and also block new angiogenesis. The prior art (application number 200410032014.8) discloses that squid ink extract has a significant inhibitory effect on cancer cells, but its main function is to prevent precancerous lesions of malignant tumors. The prior art (application number: 202310189174.6) discloses the efficacy of golden cuttlefish ink polysaccharide in improving high-glucose damage to endothelial cells and treating vascular inflammation. Although the prior art has reported that ink polysaccharides from some marine organisms have potential value in the development of anti-tumor drugs, further exploration of their efficacy in treating breast cancer is still needed. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides the use of golden cuttlefish ink polysaccharide SIP4 in preparing a product for treating breast cancer.

[0005] The first object of the present invention is to provide the use of golden cuttlefish ink polysaccharide SIP4 in preparing a product for treating breast cancer.

[0006] The second object of the present invention is to provide the use of golden cuttlefish ink polysaccharide SIP4 in preparing products for preventing breast cancer.

[0007] The third object of the present invention is to provide the use of golden cuttlefish ink polysaccharide SIP4 in the preparation of a product for inhibiting the progression of breast cancer.

[0008] The fourth object of the present invention is to provide the use of golden cuttlefish ink polysaccharide SIP4 in the preparation of products for inhibiting breast cancer metastasis.

[0009] The fifth object of the present invention is to provide the use of golden cuttlefish ink polysaccharide SIP4 in the preparation of products for inhibiting breast cancer cell proliferation, migration and / or inducing breast cancer cell apoptosis.

[0010] The sixth object of the present invention is to provide the use of golden cuttlefish ink polysaccharide in the preparation of products for treating breast cancer.

[0011] The seventh object of the present invention is to provide the use of golden cuttlefish ink polysaccharide in the preparation of products for preventing breast cancer.

[0012] The eighth object of the present invention is to provide the use of golden cuttlefish ink polysaccharide in the preparation of a product for inhibiting the progression of breast cancer.

[0013] The ninth objective of the present invention is to provide the use of golden cuttlefish ink polysaccharide in the preparation of a product for inhibiting breast cancer metastasis.

[0014] The tenth object of the present invention is to provide the use of golden cuttlefish ink polysaccharide in the preparation of a product for inhibiting breast cancer cell proliferation, migration and / or inducing breast cancer cell apoptosis.

[0015] In order to achieve the above object, the present invention is implemented through the following scheme:

[0016] The golden cuttlefish ink polysaccharide SIP4 described in the present invention is the "SIP-IV" in the prior art "Effects of a sulfatedglycosaminoglycan from Sepia esculenta ink on transcriptional and metabolic profiles of Saccharomyces cerevisiae" (DOI: 10.1016 / j.carbpol.2021.118715).

[0017] The present invention seeks protection for the following:

[0018] Application of golden squid ink polysaccharide SIP4 in the preparation of products for treating breast cancer.

[0019] Application of golden squid ink polysaccharide SIP4 in the preparation of products for preventing breast cancer.

[0020] Application of golden squid ink polysaccharide SIP4 in the preparation of products for inhibiting the progression of breast cancer.

[0021] Application of golden squid ink polysaccharide SIP4 in the preparation of products for inhibiting breast cancer metastasis.

[0022] Preferably, the breast cancer is triple-negative breast cancer. Use of golden squid ink polysaccharide SIP4 in the preparation of a product for inhibiting breast cancer cell proliferation, migration and / or inducing breast cancer cell apoptosis.

[0023] Preferably, the breast cancer cells are triple-negative breast cancer cells.

[0024] More preferably, the triple-negative breast cancer cells are MDA-MB-231 cells.

[0025] The invention discloses an application of golden cuttlefish ink polysaccharide in preparing a product for preventing breast cancer. The golden cuttlefish ink polysaccharide is composed of fucose, galactosamine, glucosamine, mannose and glucuronic acid in a molar ratio of 5.1:7.3:3.8:1:4.4.

[0026] The invention discloses an application of golden cuttlefish ink polysaccharide in preparing a product for inhibiting the progression of breast cancer. The golden cuttlefish ink polysaccharide is composed of fucose, galactosamine, glucosamine, mannose and glucuronic acid in a molar ratio of 5.1:7.3:3.8:1:4.4.

[0027] The invention discloses an application of golden cuttlefish ink polysaccharide in preparing a product for inhibiting breast cancer metastasis. The golden cuttlefish ink polysaccharide is composed of fucose, galactosamine, glucosamine, mannose and glucuronic acid in a molar ratio of 5.1:7.3:3.8:1:4.4.

[0028] Preferably, the breast cancer is triple-negative breast cancer.

[0029] The invention discloses an application of golden cuttlefish ink polysaccharide in preparing a product for inhibiting breast cancer cell proliferation, migration and / or inducing breast cancer cell apoptosis. The golden cuttlefish ink polysaccharide is composed of fucose, galactosamine, glucosamine, mannose and glucuronic acid in a molar ratio of 5.1:7.3:3.8:1:4.4.

[0030] Preferably, the breast cancer cells are triple-negative breast cancer cells.

[0031] More preferably, the triple-negative breast cancer cells are MDA-MB-231 cells.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The squid ink polysaccharide SIP4 provided by the present invention can inhibit the proliferation and migration of breast cancer cells both in vivo and in vitro, and mediate Ca 2+ The influx promotes apoptosis of breast cancer cells, and is particularly effective in inducing early apoptosis. The present invention provides a new technical option for therapeutic drugs and chemotherapy-assisted treatment of breast cancer, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The in vitro proliferation of MDA-MB-231 cells treated with ink polysaccharide; A is SIP4 in Example 1; B is SIP1 in Comparative Example 1.

[0035] Figure 2 The in vitro migration of MDA-MB-231 cells treated with ink polysaccharide; A is the in vitro migration imaging of MDA-MB-231 cells; B is the statistical results of the in vitro migration distance of MDA-MB-231 cells; **p<0.01, ***p<0.001.

[0036] Figure 3 The following table shows the toxic effects of SIP4 in wild-type zebrafish treated with different concentrations of Example 1; A is a photo of a 96 hpf zebrafish; B is the heart rate statistics of a 96 hpf zebrafish; C is the survival rate statistics of a 96 hpf zebrafish; D is the deformity rate statistics of a 96 hpf zebrafish; E is the number statistics of 96 hpf zebrafish with uninflated air sacs; F is the number statistics of 96 hpf zebrafish with bent body axes; ns is p>0.05, **p<0.01.

[0037] Figure 4 The proliferation of MDA-MB-231 cells in a zebrafish breast cancer model under treatment with different concentrations of SIP4 from Example 1; A is fluorescence imaging of MDA-MB-231 cells in 96 hpf zebrafish; B is the fluorescence analysis result of the relative number of MDA-MB-231 cells in A; **p<0.01, ***p<0.001.

[0038] Figure 5 The migration of MDA-MB-231 cells in a zebrafish breast cancer model under treatment with different concentrations of SIP4 from Example 1; A is fluorescence imaging of MDA-MB-231 cells in 96 hpf zebrafish; B is the statistical result of the number of migration foci of MDA-MB-231 cells in 96 hpf zebrafish; C is the statistical result of the maximum migration distance of MDA-MB-231 cells in 96 hpf zebrafish; *p<0.05, **p<0.01, ***p<0.001.

[0039] Figure 6Flow cytometry was used to detect the in vitro apoptosis of MDA-MB-231 cells treated with ink polysaccharide; A is the flow cytometric detection results of the control group, SIP4 group and SIP1 group; B is the statistical results of the apoptosis rate obtained based on the flow cytometric detection results in A; **p<0.01, ***p<0.001.

[0040] Figure 7 The Hoechst 33342 / PI cell apoptosis staining kit was used to detect the in vitro apoptosis of MDA-MB-231 cells treated with SIP4 in Example 1; ***p<0.001.

[0041] Figure 8 This is the intracellular calcium ion concentration of MDA-MB-231 cells under SIP4 treatment in Example 1. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0043] Example 1 A kind of golden squid ink polysaccharide

[0044] This example provides a golden cuttlefish ink polysaccharide SIP4, which is "SIP-IV" in the prior art "Effects of a sulfated glycosaminoglycan from Sepia esculenta ink on transcriptional and metabolic profiles of Saccharomyces cerevisiae" (DOI: 10.1016 / j.carbpol.2021.118715), and can be prepared according to the following method:

[0045] (1) Fresh golden squid ink was added with 1 volume of ice-cold phosphate buffered saline (PBS, pH 7.2, concentration 0.01 M), fully ground, and then ultrasonicated at 500 W for 5 s, 10 s intervals, and 20 times.

[0046] (2) The suspension obtained after ultrasonic treatment was stirred and soaked at 4°C for 12 hours, and then centrifuged at 4°C for 60 minutes at a centrifuge speed of 8000 rpm;

[0047] (3) Remove the precipitate from the centrifuged solution, add papain to a final concentration of 0.15 v / v% to the supernatant, and incubate in a 55°C water bath for 60 min;

[0048] (4) Heat in a water bath at 100°C for 20 min, cool, and centrifuge at 4°C for 60 min at 8000 rpm.

[0049] (5) Remove the precipitate from the centrifuged liquid and add 1 / 4 volume of a mixture of chloroform and n-butanol (i.e., the volume ratio of chloroform to n-butanol is 1:1) to the supernatant, mix thoroughly, and let stand at 4°C for 20 min.

[0050] (6) Centrifuge at 4°C for 60 min at 8000 rpm, and remove the upper yellow supernatant for later use.

[0051] (7) Repeat steps (5) to (6) three times, combine all the yellow clear liquids, and concentrate to 1 / 4 volume on a rotary evaporator;

[0052] (8) Add 4 times the volume of anhydrous ethanol to the concentrate, mix thoroughly, and let stand at 4°C for 60 min;

[0053] (9) Centrifuge at 4°C for 10 min at 5000 rpm, and dissolve the precipitate in distilled water (final concentration: 20 mg / mL).

[0054] (10) The solution obtained in the previous step was purified using a DEAE52 cellulose ion exchange chromatography column (2.5 cm × 20 cm). Impurities were eluted with 100 ml of a 0.2 mol / L NaCl solution, followed by elution with a 0.5 mol / L NaCl solution. The polysaccharide elution peak measured by the sulfuric acid phenol method was collected and freeze-dried to obtain SIP4. The resulting SIP4 consisted of fucose, galactosamine, glucosamine, mannose, and glucuronic acid in a molar ratio of 5.1:7.3:3.8:1:4.4.

[0055] Comparative Example 1: A golden squid ink polysaccharide

[0056] This comparative example provides a golden cuttlefish ink polysaccharide SIP1, which is "SIP-1" in the prior art "Effects of a sulfated glycosaminoglycan from Sepia esculenta ink on transcriptional and metabolic profiles of Saccharomyces cerevisiae" (DOI: 10.1016 / j.carbpol.2021.118715), and can be prepared according to the following method:

[0057] The preparation method of golden cuttlefish ink polysaccharide SIP4 is basically the same as that of Example 1, except that:

[0058] (10) The solution obtained in the previous step was purified using a DEAE52 cellulose ion exchange chromatography column (2.5 cm × 20 cm), eluted with double distilled water, and the components of the elution peak at OD190 nm were collected and freeze-dried to obtain SIP1. The obtained SIP1 was composed of monosaccharides such as galactose, arabinose, fucose, and xylose in a molar ratio of 4.6:4.48:1.0:0.48.

[0059] Application Example 1 Effects of Ink Polysaccharide on the Proliferation and Migration of Triple-Negative Breast Cancer in Vitro

[0060] 1. Experimental methods

[0061] (1) Cell culture

[0062] MDA-MB-231 cells were seeded at 4,000 cells / well into a 96-well cell plate and cultured in DMEM containing 10% FBS in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0063] (2) Drug treatment and cell proliferation detection

[0064] When the MDA-MB-231 cells reached a density of 80%, SIP4 from Example 1 and SIP1 from Comparative Example 1 were added to the culture supernatant of the MDA-MB-231 cells at different final concentrations (0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, or 2 mg / mL). MDA-MB-231 cells treated with cell culture medium served as a control group (denoted as control).

[0065] The proliferation of MDA-MB-231 cells was detected by CCK8 assay at 24, 48 and 72 hours after drug treatment. The proliferation inhibition rate and drug concentration were fitted and analyzed, and the IC 50 value.

[0066] (3) Drug treatment and cell migration detection

[0067] When the MDA-MB-231 cell density reached 80%, SIP4 from Example 1 and SIP1 from Comparative Example 1 were added to the culture supernatant of the MDA-MB-231 cells at a final concentration of 400 mg / mL. MDA-MB-231 cells incubated with cell culture medium served as a control group (denoted as control).

[0068] The migration of MDA-MB-231 cells was detected by scratch test 6 hours and 12 hours after drug treatment.

[0069] 2. Experimental results

[0070] CCK8 test results Figure 1 As shown in A, after 24, 48 and 72 hours of treatment, the inhibition rate of SIP4 in Example 1 on MDA-MB-231 cell proliferation was positively correlated with the concentration, IC 50 0.38mg / mL, 0.33mg / mL and 0.23mg / mL respectively; Figure 1 As shown in B, the inhibition rate of SIP1 in comparative example 1 on MDA-MB-231 cell proliferation is positively correlated with its concentration, IC 50 The results show that SIP4 in Example 1 significantly inhibits the growth and proliferation of MDA-MB-231 cells, and its inhibitory ability is significantly stronger than that of SIP1 in Comparative Example 1.

[0071] The scratch test results are as follows Figure 2 As shown in Figures A and B, after 6 hours of scratch treatment, the scratch width of SIP4-treated MDA-MB-231 cells was not significantly different from that of the control group, but the cell number was significantly reduced. After 12 hours, the scratch width of SIP4-treated MDA-MB-231 cells was significantly greater than that of the control group, and the cell number was further reduced. Although the scratch width of SIP1-treated MDA-MB-231 cells in Comparative Example 1 was significantly greater than that of the control group at 12 hours, the scratch width at 6 and 12 hours was significantly lower than that of SIP4-treated cells. This shows that SIP4 in Example 1 significantly inhibits the migration and proliferation of MDA-MB-231 cells, and its inhibitory ability is significantly stronger than that of SIP1.

[0072] Application Example 2 Effects of Ink Polysaccharide on the Proliferation and Migration of Triple-Negative Breast Cancer in Vivo

[0073] 1. Determination of the SIP4 dosing concentration in Example 1

[0074] (1) Drug treatment of experimental fish

[0075] Wild-type zebrafish (AB strain) were used as experimental animals. Healthy wild-type zebrafish at 6 hpf (6 hours post-fertilization) were cultured in E3 culture medium containing different concentrations of SIP4, with final concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, or 800 μg / mL. An equal volume of E3 culture medium was added as a control (denoted as control). The solution was changed every 24 hours. At 96 hours, the growth of all zebrafish breast cancer models was monitored. At the end of the experiment, the heart rate and mortality of each fish were counted. The deformities of each fish were observed and the proportion of deformities, uninflated swim bladders, yolk cysts, and body axis curvature was calculated.

[0076] (2) Experimental results

[0077] like Figure 3 As shown in Figures A to F, 96 hpf wild-type zebrafish treated with 50 μg / mL, 400 μg / mL, and 800 μg / mL of SIP4 from Example 1 showed no significant difference in heart rate compared to the control group. At the highest concentration (800 μg / mL) of SIP4 from Example 1, 96 hpf wild-type zebrafish showed no obvious mortality, but exhibited a number of deformities, primarily inflated swim bladders, curvature of the body axis, and yolk cysts. Therefore, the final concentrations of SIP4 from Example 1 were determined to be 50 μg / mL, 100 μg / mL, and 200 μg / mL for subsequent dosing and testing.

[0078] 2. Effect of SIP4 in Example 1 on the proliferation and migration of triple-negative breast cancer in vivo

[0079] (1) Construction of zebrafish breast cancer model

[0080] Transgenic zebrafish (Casper gene knockout) were used as experimental animals. MDA-MB-231 cells were first treated with CM-DiL fluorescent dye to uniformly carry the dye. The successfully stained MDA-MB-231 cells were then injected at a dose of 200 cells / cell into the yolk sac of 2-day-post-fertilization (dpf) transgenic zebrafish to create a zebrafish breast cancer model.

[0081] (2) Dosing treatment

[0082] After the zebrafish breast cancer model has grown for 1 day (1 dpi, 1 day after injection of MDA-MB-231 cells), SIP4 from Example 1 was added to the E3 culture medium of the reared fish at a final concentration of 50 μg / mL, 100 μg / mL, or 200 μg / mL, respectively. An equal volume of E3 culture medium was added as a control (denoted as control).

[0083] (3) Cell proliferation and migration detection

[0084] Three days after treatment, fluorescence intensity in the yolk sac of a zebrafish breast cancer model was measured, and whole-body cell migration fluorescence was photographed. Quantitative analysis of fluorescence intensity in the yolk sac, maximum cell migration distance, and number of cell migration foci was performed to investigate the effects of SIP4 on the proliferation and migration of breast cancer MDA-MB-231 cells.

[0085] (4) Experimental results

[0086] like Figure 4 As shown in Figures A and B, compared to the control group, the fluorescence intensity of MDA-MB-231 cells in the zebrafish breast cancer model was significantly reduced when treated with 50 μg / mL, 100 μg / mL, and 200 μg / mL of SIP4 from Example 1. Furthermore, the fluorescence intensity gradient decreased as the SIP4 concentration gradient increased. This indicates that SIP4 from Example 1 can effectively inhibit the growth and proliferation of MDA-MB-231 cells in zebrafish in a dose-dependent manner.

[0087] like Figure 5 As shown in Figures A to C, compared to the control group, treatment with 50 μg / mL, 100 μg / mL, and 200 μg / mL of SIP4 from Example 1 significantly reduced the number of MDA-MB-231 cell foci and maximum migration distance in the zebrafish breast cancer model. This demonstrates that SIP4 from Example 1 effectively inhibits the migration of MDA-MB-231 cells in zebrafish in a dose-dependent manner, indicating that breast cancer MDA-MB-231 cells are sensitive to SIP4.

[0088] Application Example 3 Effect of Ink Polysaccharide on Apoptosis of Triple-Negative Breast Cancer in Vitro

[0089] 1. Experimental methods

[0090] (1) Cell culture

[0091] MDA-MB-231 cells were seeded at 4,000 cells / well into a 96-well plate in a DMEM / 10% FBS culture medium at 37°C in a 5% CO2, saturated humidity cell culture incubator.

[0092] (2) Drug treatment and cell apoptosis detection

[0093] When the MDA-MB-231 cell density reached 80%, SIP4 from Example 1 (denoted as the SIP4 group) and SIP1 from Comparative Example 1 (denoted as the SIP1 group) were added to the culture supernatant of the MDA-MB-231 cells at a final concentration of 400 mg / mL. MDA-MB-231 cells incubated with culture medium served as the control group (denoted as the control).

[0094] 24 hours after drug treatment, the cell apoptosis in the SIP4 group, SIP1 group and control group was detected by flow cytometry using the Annexin V-FITC apoptosis detection kit.

[0095] After 24 hours of drug treatment, the apoptosis of MDA-MB-231 cells in the SIP4 group and the control group was detected by Hoechst33342 / PI cell apoptosis staining kit.

[0096] (4) Intracellular calcium ion concentration detection

[0097] After 24 hours of drug treatment, the intracellular calcium concentration of MDA-MB-231 cells in the SIP4 group and the control group was detected by calcium ion fluorescence probe method.

[0098] 2. Experimental results

[0099] Flow cytometry results Figure 6 As shown in Figures A and B, the apoptosis rate of MDA-MB-231 cells treated with SIP4 in Example 1 was significantly higher than that in the control group; the apoptosis rate of MDA-MB-231 cells treated with SIP1 in Comparative Example 1 was also significantly higher than that in the control group, but significantly lower than that in the SIP4-treated group.

[0100] The results of Hoechst33342 / PI cell apoptosis staining are as follows Figure 7 As shown, the fluorescence intensity of MDA-MB-231 cells treated with SIP4 in Example 1 was significantly higher than that of the control group, indicating that SIP4 in Example 1 effectively induced apoptosis in MDA-MB-231 cells.

[0101] The results of intracellular calcium ion concentration detection are as follows Figure 8 As shown, the intracellular Ca 2+ The concentration was significantly higher than that of the control group, indicating that the SIP4 in Example 1 promoted the apoptosis of MDA-MB-231 cells via the mitochondrial pathway by mediating calcium ion influx.

[0102] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. The application of golden squid ink polysaccharide SIP4 in the preparation of a product for treating triple-negative breast cancer, characterized in that: The preparation method of the golden squid ink polysaccharide SIP4 comprises the following steps: (1) Take fresh golden squid ink, add 1 volume of ice-cold phosphate buffer, grind thoroughly, and then perform ultrasonic treatment at 500W, 5s treatment time, 10s interval, and 20 times; the phosphate buffer is PBS with a pH value of 7.2 and a concentration of 0.01M; (2) The suspension obtained after ultrasonic treatment was stirred and soaked at 4°C for 12 hours, and then centrifuged at 4°C for 60 minutes at a centrifuge speed of 8000 rpm; (3) Remove the precipitate from the centrifuge obtained in the previous step, add papain to a final concentration of 0.15 v / v% to the supernatant, and incubate in a 55°C water bath for 60 min; (4) Heat in a water bath at 100°C for 20 min, cool, and centrifuge at 4°C for 60 min at 8000 rpm. (5) removing the precipitate from the centrifuge obtained in the previous step, adding 1 / 4 volume of a mixture of chloroform and n-butanol to the supernatant, mixing thoroughly, and standing at 4°C for 20 minutes; the chloroform and n-butanol mixture is obtained by mixing chloroform and n-butanol in a volume ratio of 1:1; (6) Then centrifuge at 4°C for 60 min at 8000 rpm, and remove the upper yellow clear liquid for later use; (7) Repeat steps (5) to (6) three times, combine all the yellow clear liquids, and concentrate to 1 / 4 volume on a rotary evaporator; (8) Add 4 times the volume of anhydrous ethanol to the concentrated solution obtained in the previous step, mix thoroughly, and let stand at 4°C for 60 min; (9) Centrifuge at 4°C for 10 min at 5000 rpm. Dissolve the precipitate in distilled water to a final concentration of 20 mg / mL. (10) The solution obtained in the previous step was purified using a 2.5 cm × 20 cm DEAE52 cellulose ion exchange chromatography column. Impurities were eluted with 100 ml of 0.2 mol / L NaCl solution, and then eluted with 0.5 mol / L NaCl solution. The polysaccharide elution peak measured by the sulfuric acid phenol method was collected and freeze-dried to obtain the product.

2. The application of golden squid ink polysaccharide SIP4 in the preparation of a product for preventing triple-negative breast cancer, characterized in that: The preparation method of the golden squid ink polysaccharide SIP4 comprises the following steps: (1) Take fresh golden squid ink, add 1 volume of ice-cold phosphate buffer, grind thoroughly, and then perform ultrasonic treatment at 500W, 5s treatment time, 10s interval, and 20 times; the phosphate buffer is PBS with a pH value of 7.2 and a concentration of 0.01M; (2) The suspension obtained after ultrasonic treatment was stirred and soaked at 4°C for 12 hours, and then centrifuged at 4°C for 60 minutes at a centrifuge speed of 8000 rpm; (3) Remove the precipitate from the centrifuge obtained in the previous step, add papain to a final concentration of 0.15 v / v% to the supernatant, and incubate in a 55°C water bath for 60 min; (4) Heat in a water bath at 100°C for 20 min, cool, and centrifuge at 4°C for 60 min at 8000 rpm. (5) removing the precipitate from the centrifuge obtained in the previous step, adding 1 / 4 volume of a mixture of chloroform and n-butanol to the supernatant, mixing thoroughly, and standing at 4°C for 20 minutes; the chloroform and n-butanol mixture is obtained by mixing chloroform and n-butanol in a volume ratio of 1:1; (6) Then centrifuge at 4°C for 60 min at 8000 rpm, and remove the upper yellow clear liquid for later use; (7) Repeat steps (5) to (6) three times, combine all the yellow clear liquids, and concentrate to 1 / 4 volume on a rotary evaporator; (8) Add 4 times the volume of anhydrous ethanol to the concentrated solution obtained in the previous step, mix thoroughly, and let stand at 4°C for 60 min; (9) Centrifuge at 4°C for 10 min at 5000 rpm. Dissolve the precipitate in distilled water to a final concentration of 20 mg / mL. (10) The solution obtained in the previous step was purified using a 2.5 cm × 20 cm DEAE52 cellulose ion exchange chromatography column. Impurities were eluted with 100 ml of 0.2 mol / L NaCl solution, and then eluted with 0.5 mol / L NaCl solution. The polysaccharide elution peak measured by the sulfuric acid phenol method was collected and freeze-dried to obtain the product.

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