Fucoidin as well as preparation method and application thereof

Abstract

The invention provides fucoidin and a preparation method and application thereof.The fucoidin comes from sargassum horneri and can be used for constructing a drug carrier, and the fucoidin mainly contains fucose, galactose and mannose and further contains a small amount of glucuronic acid, rhamnose, glucose, xylose and glucosamine. The drug carrier is stable in blood microenvironment pH and is split in tumor microenvironment pH, and efficient delivery of antitumor drugs in vivo can be realized; the polymer has high affinity with P-selectin, and can deliver loaded antitumor drugs in a targeted manner; the inhibition activity on human breast cancer cells MDA-MB-231 is far higher than that of doxorubicin and doxorubicin and no-load nanoparticles with the same equivalent, that is, after doxorubicin is carried by the fucoidin drug carrier, the anti-tumor effect can be better achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of marine biopolysaccharide nanocarriers, and in particular relates to fucoidan and a preparation method and application thereof. Background Art

[0002] Cancer is one of the major diseases that endanger human health and ranks second among the causes of death in the global population. According to WHO statistics, there will be approximately 19.3 million new cancer cases and 10 million cancer deaths in 2020. Therefore, cancer is still a global problem, and cancer prevention and treatment are urgent. Cancer treatment methods mainly include surgery, radiotherapy, chemotherapy, etc. These methods often produce serious side effects during use, which not only reduce the patient's own immunity, but also cause a series of complications. It is urgent to find new anti-cancer therapies with strong targeting, high selectivity and few side effects. The targeted drug delivery system proposed in recent years mainly uses carriers with certain tumor targeting to locate drugs in target organs, selectively kill tumor cells and exert therapeutic effects. It has become a hot spot in pharmaceutical research at home and abroad. Among them, nanoparticle carrier systems, as the research focus of targeted drug delivery systems, have received widespread attention.

[0003] P-selectin is an adhesion molecule that mediates the adhesion of tumor cells to platelets and endothelial cells, thereby promoting the metastasis of tumor cells. It is highly expressed in a variety of human tumor cells and tumor vascular systems. Targeted delivery of tumor therapeutic drugs to tumors expressing P-selectin through nanoparticle carriers can effectively exert a therapeutic effect.

[0004] Fucoidan can target and bind to P-selectin and be used to construct nanoparticle tumor-targeted drug carriers. However, at present, commercialized Fucoidan from Fucus vesiculosus is mostly used in targeted research and carrier construction. The use of fucoidan from other sources is relatively rare or has not been reported. Moreover, what kind of fucoidan structure is more suitable for constructing a tumor-targeted delivery system, that is, the common structural characteristics of fucoidan that can be used to construct a good drug carrier have not received attention. The patent "A Tumor-Targeted Fucoidan Sulfate and Its Preparation Method" with application number ZL202011436361.2 previously applied for and authorized by the inventor discloses a kind of Sargassum fusiformis fucoidan and its fine structure that can be used for the construction of tumor-targeted drug carriers, which can be used for the preparation of targeted delivery systems for anti-tumor drugs or anti-tumor active substances. However, it is of great significance for in-depth research on fucoidan-based drug carriers to develop better drug carriers based on fucoidan, and to explore the common structural characteristics of fucoidan used to construct drug carriers through structural analysis of a variety of fucoidans used to construct good drug carriers. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-tumor drug carrier with better effect.

[0006] In order to achieve the above-mentioned object, the present invention provides a fucoidan and a preparation method and application thereof. The technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a fucoidan, which mainly contains fucose, galactose, mannose, and also contains a small amount of glucuronic acid, rhamnose, glucose, xylose, and glucosamine; its main chain is composed of 1,3-, 1,4-α-L-Fucp and 1,2-, 1,2,3-β-D-Galp, 1,6-, 1,4,6-β-D-Manp, and the sulfate groups are located at the C-2 position of the fucosyl 1,3-α-L-Fucp and the C-2 and C-3 positions of the 1,4-α-L-Fucp, among which the double sulfated substituted fucosyl accounts for a relatively high proportion, and the C-3 position of the galactosyl 1,2-β-D-Galp and the C-4 position of the mannosyl 1,6-β-D-Manp also contain sulfate groups.

[0008] In the embodiment of the present invention, the total molar ratio of fucose, galactose and mannose is 81.69%, and the remainder is glucuronic acid, rhamnose, glucose, xylose and aminoglucose; the molar ratio of the double sulfated substituted fucosyl in the sulfate group is 26.45%.

[0009] In an embodiment of the present invention, the fucoidan is derived from copper algae.

[0010] In a second aspect, the present invention provides a method for preparing the fucoidan, comprising the following steps:

[0011] After defatting the copper algae powder, collect the precipitate and dry it;

[0012] Add CaCl2 solution to the defatted powder and take the supernatant;

[0013] The supernatant was concentrated, precipitated with alcohol, and the precipitate was collected;

[0014] The precipitate is redissolved and separated by ion exchange chromatography, and then the target solution is concentrated, desalted, concentrated and dried to obtain copper algae fucoidan.

[0015] In the third aspect, the present invention provides an anti-tumor drug nanocarrier based on copper algae fucoidan, the release rate of the anti-tumor drug of the nanocarrier in the tumor microenvironment (pH5.5) is much higher than the release rate in the blood environment at pH7.4, and the nanocarrier has a high affinity with P-selectin, which can achieve efficient and targeted delivery of anti-tumor drugs in vivo; the inhibitory activity of the nanoparticles loaded with anti-tumor drugs on human breast cancer cells MDA-MB-231 is much higher than that of the same equivalent amount of doxorubicin and doxorubicin + empty nanoparticles, that is, doxorubicin can exert a better anti-tumor effect after being transported by the copper algae fucoidan nanocarrier.

[0016] In a fourth aspect, the present invention provides a method for preparing an anti-tumor drug nanocarrier based on copper algae fucoidan, comprising the following steps:

[0017] Preparation of nanoparticles: The copper algae fucoidan and chitosan are prepared into a certain concentration, chitosan is added dropwise into the fucoidan, and a cell ultrasonic disruptor is used to disperse the fucoidan to obtain nanoparticles.

[0018] The mass ratio of fucoidan to chitosan is 3-1:1-1.5, preferably 1.5:1.

[0019] In a fifth aspect, the present invention provides an application of an anti-tumor drug nanocarrier based on copper algae fucoidan in the preparation of tumor-targeted drugs. The prepared nanoparticles are encapsulated with anti-tumor drugs to prepare tumor-targeted drugs.

[0020] Furthermore, the anti-tumor drug includes doxorubicin.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention provides a copper algae fucoidan for constructing a drug carrier, which has a novel structure and mainly contains fucose, galactose, and mannose, the sum of the molar ratios of the three being 81.69%, and also contains a small amount of glucuronic acid, rhamnose, glucose, xylose, and aminoglucose. It is different from the fucoidan structure provided by the previous patent.

[0023] 2. The present invention provides an anti-tumor drug carrier based on copper algae fucoidan, which is stable in the pH of the blood microenvironment and cleaved in the pH of the tumor microenvironment, and can achieve efficient delivery of anti-tumor drugs in vivo; it has high affinity with P-selectin and can deliver the loaded anti-tumor drugs in a targeted manner; the inhibitory activity on human breast cancer cells MDA-MB-231 is much higher than that of the same equivalent of doxorubicin and doxorubicin + empty nanoparticles, that is, doxorubicin can better play an anti-tumor effect after being carried by copper algae fucoidan drug carrier. In addition, under the condition of the same doxorubicin equivalent, the inhibitory activity of copper algae fucoidan nanosystem loaded with doxorubicin on human breast cancer cells MDA-MB-231 is higher than that of Sargassum fusiformis fucoidan nanosystem loaded with doxorubicin, indicating that under the preparation evaluation system of this application, copper algae fucoidan nanocarriers are superior to Sargassum fusiformis fucoidan nanocarriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings constituting a part of the present disclosure are used to provide a further understanding of the present disclosure. The illustrative embodiments of the present disclosure and their descriptions are used to explain the present disclosure and do not constitute an improper limitation on the present disclosure.

[0025] Figure 1is the infrared spectrum of the fucoidan nanoparticles of the present invention;

[0026] Figure 2 The nanocarriers loaded with the anti-tumor drug doxorubicin of the present invention release the doxorubicin at pH 7.4 and pH 5.5; wherein (a) is the release rate at pH 7.4, and (b) is the release rate at pH 5.5;

[0027] Figure 3 is the result of the affinity test between the fucoidan nanoparticles and P-selectin of the present invention; wherein 1, 2, 3, and 4 represent the mass ratios of fucoidan to chitosan of 1.0 / 1.5, 1.0 / 1.0, 1.5 / 1.0, and 3.0 / 1.0, respectively;

[0028] Figure 4 The results of the inhibitory activity test of the nanocarrier of the present invention on human breast cancer cells MDA-MB-231 are shown in FIG. 1 ; wherein (a) is a cell survival rate graph at doxorubicin equivalent, and (b) is a cell survival rate graph at different concentrations of the marked substances in the graph;

[0029] Figure 5 The copper algae fucoidan of the present invention 1 H and 13 C NMR spectrum;

[0030] Figure 6 The present invention is the copper algae fucoidan 1 H- 1 H COSY spectrum;

[0031] Figure 7 The copper algae fucoidan of the present invention 1 H- 13 C HSQC spectrum;

[0032] Figure 8 The copper algae fucoidan of the present invention 1 H- 1 H NOESY spectrum. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present disclosure. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present disclosure belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present disclosure. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] In the first aspect, the present invention provides a copper algae fucoidan, which mainly contains fucose, galactose, mannose, and also contains a small amount of glucuronic acid, rhamnose, glucose, xylose, and glucosamine; its main chain is composed of 1,3-, 1,4-α-L-Fucp and 1,2-, 1,2,3-β-D-Galp, 1,6-, 1,4,6-β-D-Manp, and the sulfate groups are located at the C-2 position of the fucosyl 1,3-α-L-Fucp and the C-2 and C-3 positions of the 1,4-α-L-Fucp, among which the double sulfated substituted fucosyl accounts for a relatively high proportion, with a molar ratio of about 26.45%, and the C-3 position of the galactosyl 1,2-β-D-Galp and the C-4 position of the mannosyl 1,6-β-D-Manp also contain sulfate groups.

[0036] In some embodiments, the fucoidan has a molecular weight of about 300 kDa.

[0037] In some embodiments, the sum of the molar ratios of fucose, galactose, and mannose is 81.69%.

[0038] In some embodiments, the molar ratios of 1,3-α-L-Fucp, 1,4-α-L-Fucp, 1,2,3,4-α-L-Fucp, 1,2,3-α-L-Fucp, 1,2,3-β-D-Galp and 1,4,6-β-D-Manp in the glycosyl fragments of the fucoidan are 5.90%, 8.00%, 26.45%, 6.47%, 30.08% and 23.10%.

[0039] In a second aspect, the present invention provides a method for preparing the fucoidan, comprising the following steps:

[0040] After defatting the copper algae powder, collect the precipitate and dry it;

[0041] Add CaCl2 solution to the defatted powder and take the supernatant;

[0042] The supernatant was filtered, concentrated, and precipitated with alcohol, and the precipitate was collected;

[0043] The precipitate is redissolved and separated by ion exchange chromatography, and then the target solution is concentrated, desalted, concentrated and dried to obtain the product.

[0044] In some embodiments, the solution used for defatting is 95% ethanol.

[0045] Furthermore, the volume ratio of the defatted solution to the copper algae powder is 20-30:1.

[0046] Furthermore, the degreasing time is 20-30 hours, and the degreasing temperature is 20-35°C.

[0047] Furthermore, the number of times of degreasing is 2-3 times.

[0048] Furthermore, after defatting, centrifugal separation is performed.

[0049] In some embodiments, the mass percentage of the CaCl2 solution is 1.8%-2.2%.

[0050] Furthermore, the volume of CaCl2 solution added to each gram of copper algae powder is 20-30 ml.

[0051] Further, add CaCl2 solution to the copper algae powder, stir at 60-70°C for 2-4 hours, centrifuge at the end of stirring, repeat at least 2 times, and combine the supernatant.

[0052] In some embodiments, after the supernatant is filtered, the liquid volume is concentrated to 1 / 10 to 1 / 20 of the original volume, and then alcohol precipitation is performed.

[0053] Furthermore, the solution used for alcohol precipitation is 95% ethanol.

[0054] In some embodiments, the solvent used to reconstitute the precipitated portion is distilled water.

[0055] In some embodiments, the ion exchange chromatography is DEAE-Sepharose Fast Flow ion exchange chromatography.

[0056] Furthermore, when performing ion exchange chromatography separation, sodium chloride aqueous solution is used for elution, and the eluate is collected, desalted, and dried to obtain the fucoidan.

[0057] Furthermore, the desalination is dialysis desalination with a molecular weight cut-off of 10000Da.

[0058] In the third aspect, the present invention provides an anti-tumor drug nanocarrier based on copper algae fucoidan, the release rate of the anti-tumor drug of the nanocarrier in the tumor microenvironment (pH5.5) is much higher than the release rate in the blood environment at pH7.4, and the nanocarrier has a high affinity with P-selectin, which can achieve efficient and targeted delivery of anti-tumor drugs in vivo; the inhibitory activity of the nanoparticles loaded with anti-tumor drugs on human breast cancer cells MDA-MB-231 is much higher than that of the same equivalent amount of doxorubicin and doxorubicin + empty nanoparticles, that is, doxorubicin can exert a better anti-tumor effect after being transported by the copper algae fucoidan nanocarrier.

[0059] In some embodiments, the doxorubicin loading rate of the anti-tumor drug nanocarrier is 30-35%;

[0060] In some embodiments, the doxorubicin release rate of the anti-tumor drug nanocarrier in the tumor microenvironment (pH 5.5) for 12 hours is 47-60%, and the release rate in the blood environment (pH 7.4) for 12 hours is 33-36%. Preferably, the nanocarrier has a release rate of 60% in the tumor microenvironment and a release rate of 36% in the blood environment.

[0061] In some embodiments, the inhibition rate of doxorubicin-loaded nanoparticles on human breast cancer cells MDA-MB-231 was 41%, while the inhibition rates of the same amount of doxorubicin and doxorubicin + empty nanoparticles were 31% and 28% respectively (doxorubicin equivalent 5 μg / mL).

[0062] In a fourth aspect, the present invention provides a method for preparing an anti-tumor drug nanocarrier based on copper algae fucoidan, comprising the following steps:

[0063] 1) Preparation of nanoparticles: The fucoidan and chitosan are prepared into a certain concentration, chitosan is added dropwise to the fucoidan, and a cell ultrasonic disruptor is used to disperse the fucoidan to obtain nanoparticles.

[0064] 2) Determination of affinity between nanoparticles and P-selectin: Nanoparticles were prepared and simultaneously encapsulated with the fluorescent dye IR-783, and the interaction between nanoparticles and P-selectin was determined by ELISA;

[0065] 3) Determination of the inhibitory activity of doxorubicin-loaded nanoparticles on human breast cancer cells MDA-MB-231: Nanoparticles were prepared and encapsulated with the anti-tumor drug doxorubicin, and the inhibitory activity of loaded nanoparticles, doxorubicin, and empty nanoparticles + doxorubicin on human breast cancer cells MDA-MB-231 was determined.

[0066] In some embodiments, the nanoparticles in step (1) are formed by a polyelectrolyte self-assembly method, and the mass ratio of fucoidan to chitosan in the nanoparticles is 3.0:1, 1.5:1, 1.0:1, 1.0:1.5.

[0067] In some embodiments, the fluorescent dye or doxorubicin in step (1) is first mixed and dissolved with fucoidan.

[0068] In some embodiments, the particle size of the doxorubicin-loaded nanoparticles in step (1) is 360-400 nm.

[0069] In some embodiments, in step (2), the fluorescence is finally determined by ELISA.

[0070] In some embodiments, the loading rate of doxorubicin in the loaded nanoparticles is 30%-35%.

[0071] In some embodiments, CCK8 cell viability assay was used to determine the inhibitory activity of loaded nanoparticles, doxorubicin, and empty nanoparticles + doxorubicin on human breast cancer cells MDA-MB-231, and the inhibition rates were 41%, 31%, and 28%, respectively (doxorubicin equivalent 5 μg / mL).

[0072] Example 1, a method for preparing an anti-tumor drug carrier based on fucoidan, comprising the following steps:

[0073] (1) Take 200g of copper algae powder, add 4L of 95% ethanol to defatted, stir at room temperature for 24h, repeat 3 times. After defatted, centrifuge, collect the precipitate, and dry naturally.

[0074] (2) Add 6L of 2% CaCl2 solution to the defatted algal powder and stir at 60-70°C for 3h. Centrifuge at the end of stirring. Repeat twice and combine the supernatant.

[0075] (3) The supernatant was ultrafiltered with a molecular weight cutoff of 100 kDa. When the liquid volume was concentrated to 1 / 10 of the original volume, 4 volumes of 95% ethanol were added for alcohol precipitation. The mixture was allowed to stand at 4°C overnight and centrifuged. The precipitate was collected and dried at 40°C.

[0076] (4) The polysaccharide described in (3) is separated by DEAE-Sepharose Fast Flow ion exchange chromatography, eluted with sodium chloride aqueous solution, the eluate is collected, dialyzed for desalination and then dried to obtain the copper algae fucoidan.

[0077] (5) Preparation of nanoparticles: The fucoidan and chitosan are prepared into an aqueous solution, wherein the fucoidan contains 1 mg / mL IR-783 or 2 mg / mL doxorubicin, and the mass ratio of fucoidan to chitosan is 3.0:1, 1.5:1, 1.0:1, 1.0:1.5. The chitosan solution is added dropwise to the fucoidan and dispersed using a cell ultrasonic disruptor to obtain nanoparticles.

[0078] When the ratio was 1.5:1, the doxorubicin loading rate of the anti-tumor drug nanocarrier was 34.7, and when the ratio was 3.0:1, the doxorubicin loading rate of the anti-tumor drug nanocarrier was 30.2.

[0079] Example 2, product performance measurement

[0080] (1) Determination of affinity between nanoparticles and P-selectin: The interaction between nanoparticles and P-selectin was determined by ELISA, with fucoidan nanoparticles (Sigma, USA) as the positive control and dextran sulfate nanoparticles as the negative control (literature reports that fucoidan has a good affinity for P-selectin, while dextran sulfate has no affinity for P-selectin).

[0081] Determination of the inhibitory activity of doxorubicin-loaded nanoparticles on human breast cancer cells MDA-MB-231: Doxorubicin-loaded nanoparticles were prepared, and the CCK8 cell viability assay was used to determine the inhibitory activity of loaded nanoparticles, doxorubicin, and empty nanoparticles + doxorubicin on human breast cancer cells MDA-MB-231. At the same time, the cytotoxicity of fucoidan and fucoidan empty nanoparticles on human breast cancer cells was investigated to exclude the inhibitory activity of fucoidan and fucoidan empty nanoparticles themselves on human breast cancer cells.

[0082] The characteristics of the nanocarriers based on fucoidan anti-tumor drugs were tested and analyzed, including infrared spectroscopy analysis of the nanocarriers, affinity analysis with P-selectin, physicochemical property analysis, inhibitory activity analysis on human breast cancer cells MDA-MB-231, and molecular weight analysis, monosaccharide composition analysis, methylation analysis, and nuclear magnetic resonance spectroscopy analysis of copper algae fucoidan.

[0083] (2) The following are the research results based on fucoidan anti-tumor drug carriers, including the analysis of the properties of nanocarriers and the analysis of the structural characteristics of fucoidan.

[0084] Infrared spectra of nanoparticles ( Figure 1 ) shows the characteristic absorption peaks of chitosan and fucoidan, indicating that there is a non-covalent interaction between chitosan and fucoidan, 1640 cm -1 The stretching vibration absorption peak of chitosan amide C=O is 1565cm -1 The bending vibration absorption peak of chitosan NH is 1245 cm -1 It is the S=O stretching vibration absorption peak of the sulfate group of fucoidan.

[0085] The pH of blood is about 7.35-7.45, and the pH of tumor microenvironment is about 4.5-5.5. The release rate of doxorubicin from loaded nanoparticles under the pH of blood and tumor microenvironment was measured. Figure 2As shown, the nanocarriers with low doxorubicin release rate at blood pH and high doxorubicin release rate at tumor microenvironment pH were screened, which can achieve efficient delivery of doxorubicin in vivo. The results of this experimental study showed that the loaded nanoparticles with a ratio of 1.5:1 and 3.0:1 can achieve efficient delivery of doxorubicin in vivo, and the effect is best when the ratio is 1.5:1.

[0086] The results of the affinity test between nanoparticles and P-selectin are as follows Figure 3 As shown, compared with the blank group (bovine serum albumin BSA group), the positive control Fucus vesiculosus fucoidan nanoparticles showed good affinity with P-selectin, while the negative control dextran sulfate nanoparticles had no affinity with P-selectin, indicating that the experimental results are credible. All proportions of copper algae fucoidan nanoparticles have affinity with P-selectin, and when the ratio is 1.5:1, the binding effect between the two is the strongest. The results of this experimental study show that the fucoidan has a high affinity with P-selectin.

[0087] The inhibitory activity of loaded nanoparticles, doxorubicin, and empty nanoparticles + doxorubicin on human breast cancer cells MDA-MB-231 was determined. Figure 4 As shown, the inhibition rates were 41%, 31%, and 28% (doxorubicin equivalent 5 μg / mL, Figure 4 In (a), the inhibition rate of loaded nanoparticles is higher than that of doxorubicin and empty nanoparticles + doxorubicin, indicating that doxorubicin can achieve better antitumor activity after being transported by fucoidan nanocarriers. At the same time, it can be seen that these substances themselves have no antitumor effect, excluding the antitumor effect brought by themselves ( Figure 4 In addition, the inhibitory activity of Cu-algae-fucoidan nanocarriers loaded with doxorubicin on human breast cancer cells MDA-MB-231 was higher than that of Sargassum-fucoidan nanocarriers.

[0088] In order to explore the structural characteristics of fucoidan constituting the anti-tumor drug carrier, the present embodiment conducted a structural analysis of the fucoidan. The glycosyl connection mode of the fucoidan was first determined by methylation analysis. Fucose in fucoidan before desulfurization is mainly 1,3-, 1,4-, 1,2,3,4-, 1,2,3-linked fragments, galactose is mainly 1,2,3-linked fragments, and mannose is mainly 1,4,6-linked fragments, with a molar ratio of 5.90:8.00:26.45:6.47:30.08:23.10. After desulfurization, 1,3- and 1,4-linked fucose increased, 1,2,3,4-linked fucose decreased, 1,2,3-linked fucose disappeared, 1,2,3-linked galactose and 1,4,6-linked mannose decreased, and 1,2-linked galactose and 1,6-linked galactose appeared, indicating that the sulfate group was located at the C-2 and C-3 positions of 1,4-linked fucose, the C-2 position of 1,3-linked fucose, the C-3 position of 1,2-linked galactose, and the C-4 position of 1,6-linked mannose. According to the nuclear magnetic resonance spectrum analysis described later, the 1,2,3,4-linked fucose in fucoidan is all →4)-α-L-Fucp(2,3SO4)-(1→, Therefore, the proportion of double sulfated fucosyl in fucoidan is 26.45%, which is relatively high.

[0089] Figure 5 The nuclear magnetic resonance hydrogen spectrum and carbon spectrum of the fucoidan are shown in Figure 5.0-5.5ppm, the anomeric hydrogen signal of α-pyrano-fucose residue, the anomeric hydrogen signal of β-pyrano-galactose residue, and the anomeric hydrogen signal of β-pyrano-mannose residue at 4.41ppm, which are marked as 5.41 (A), 5.30 (B), 5.18 (C), 5.12 (D), 4.50 (E and F) and 4.41ppm (G and H), respectively. The high field region of 1.31ppm is the fucosyl methyl hydrogen H-6 signal, and the rest are mainly sugar H-2 to H-6 signals. In the carbon spectrum, 100.93 and 101.98 ppm are the terminal carbon signals of fucose, 104.50 ppm is the terminal carbon signal of galactose and mannose, 17.23 ppm in the high field area is the C-6 signal of fucosyl methyl carbon, and the rest are mainly sugar C-2 to C-6 signals. Figure 6 is the homonuclear chemical shift correlation spectrum of the fucoidan ( 1 H- 1 H COSY), Figure 7 is the heteronuclear single quantum chemical shift correlation spectrum of the fucoidan ( 1 H- 13 CHSQC), Figure 8 is the nuclear Overhauser effect correlation spectrum of the fucoidan ( 1 H- 1 H NOESY). Figure 6-8, and considering the results of methylation analysis, it can be seen that the anomeric hydrogen signal of sugar group A at 5.41ppm is correlated with the anomeric carbon signal at 100.93ppm, and the chemical shift signals at C-2, C-3, and C-4 move to the downfield, indicating that there are substitutions at C-2, C-3, and C-4. Among them, the hydrogen signals at C-2 and C-3 move to the downfield, indicating that there are double sulfate substitutions at C-2 and C-3, and it is inferred that it is →4)-α-L-Fucp(2,3SO4)-(1→. Similarly, B is →3)-α-L-Fucp(2SO4)-(1→, C is →3)-α-L-Fucp-(1→, and D is →4)-α-L-Fucp-(1→. The anomeric hydrogen signals of galactose residues are concentrated at 4.50ppm, including sulfated galactose. Its anomeric hydrogen signal is correlated with the anomeric carbon signal at 104.50ppm, and its C-2, C- The chemical shift signals at three positions move downfield, indicating that there are substitutions at C-2 and C-3. Among them, part of the hydrogen signal at C-3 moves downfield, indicating that there is sulfate substitution at C-3. Therefore, the galactose residues are →2,3)-β-D-Galp-(1→, →2)-β-D-Galp(3SO4)-(1→. The anomeric hydrogen signals of mannose residues are concentrated at 4.41 ppm, including sulfated mannose. Its anomeric hydrogen signal is related to the anomeric carbon signal of 104.50 ppm, and its chemical shift signals at C-4 and C-6 move downfield, indicating that there are substitutions at C-4 and C-6. Among them, part of the hydrogen signal at C-4 moves downfield, indicating that there is sulfate substitution at C-4. Therefore, the galactose residues are →4,6)-β-D-Manp-(1→, →6)-β-D-Manp(4SO4)-(1→. According to 1 H- 1 H NOESY spectra, cross signals A(H1) / E(H2)-F(H2), A(H1) / B(H3)-C(H3)-E(H3), and A(H1) / G(H6)-H(H6) indicated the presence of the following glycosyl fragments: →4)-α-L-Fucp(2,3SO4)-(1→2,3)-β-D-Galp-(1→,→4)-α-L-Fucp(2,3SO4)-(1→2)-β-D-Galp(3SO4)-(1→,

[0090] →4)-α-L-Fucp(2,3SO4)-(1→3)-α-L-Fucp(2SO4)-(1→,

[0091] →4)-α-L-Fucp(2,3SO4)-(1→3)-α-L-Fucp-(1→,

[0092] →4)-α-L-Fucp(2,3SO4)-(1→2,3)-β-D-Galp-(1→,→4)-α-L-Fucp(2,3SO4)-(1→4,6)-β-D-Manp-(1→and

[0093] →4)-α-L-Fucp(2,3SO4)-(1→6)-β-D-Manp(4SO4)-(1→. In addition, according to the cross signal E,F(H1) / G,H(H6), the glycosyl fragment →2,3)-β-D-Galp-(1→4,6)-β-D-Manp-(1→,

[0094] →2,3)-β-D-Galp-(1→6)-β-D-Manp(4SO4)-(1→,→2)-β-D-Galp(3SO4)-(1→4,6)-β-D-Manp-(1→ and →2)-β-D-Galp(3SO4)-(1→6)-β-D-Manp(4SO4)-(1→) may also exist.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fucoidan, characterized in that: It mainly contains fucose, galactose, mannose, and also contains a small amount of glucuronic acid, rhamnose, glucose, xylose, and glucosamine; its main chain is composed of 1,3-, 1,4-α-L-Fucp and 1,2-, 1,2,3-β-D-Galp, 1,6-, 1,4,6-β-D-Manp, and the sulfate groups are located at the C-2 position of the fucosyl 1,3-α-L-Fucp and the C-2 and C-3 positions of the 1,4-α-L-Fucp, among which the double sulfated substituted fucosyl accounts for a relatively high proportion. The C-3 position of the galactosyl 1,2-β-D-Galp and the C-4 position of the mannosyl 1,6-β-D-Manp also contain sulfate groups.

2. The fucoidan according to claim 1, characterized in that The fucoidan is derived from copper algae; Preferably, the molecular weight of the fucoidan is about 300 kDa; Preferably, the sum of the molar ratios of fucose, galactose and mannose is 81.69%; Preferably, the molar ratio of the disulfated substituted fucosyl groups in the sulfate groups is 26.45%; Preferably, the molar ratios of 1,3-α-L-Fucp, 1,4-α-L-Fucp, 1,2,3,4-α-L-Fucp, 1,2,3-α-L-Fucp, 1,2,3-β-D-Galp and 1,4,6-β-D-Manp in the glycosyl fragments of the fucoidan are 5.90%, 8.00%, 26.45%, 6.47%, 30.08% and 23.10%.

3. The method for preparing fucoidan according to claim 1, characterized in that: The steps include: After defatting the copper algae powder, collect the precipitate and dry it; Add CaCl2 solution to the defatted powder and take the supernatant; The supernatant was concentrated, precipitated with alcohol, and the precipitate was collected; The precipitate is redissolved and separated by ion exchange chromatography, and then the target solution is concentrated, desalted, concentrated and dried to obtain copper algae fucoidan.

4. An anti-tumor drug nanocarrier based on copper algae fucoidan, characterized in that: Contains the fucoidan according to any one of claims 1 to 3 and chitosan.

5. The method for preparing the anti-tumor drug nanocarrier based on copper algae fucoidan according to claim 4, characterized in that: The steps include: Preparation of nanoparticles: The copper algae fucoidan and chitosan are prepared into a certain concentration, chitosan is added dropwise into the fucoidan, and a cell ultrasonic disruptor is used to disperse the fucoidan to obtain nanoparticles.

6. The method for preparing the anti-tumor drug nanocarrier based on copper algae fucoidan according to claim 5, characterized in that: The mass ratio of fucoidan to chitosan is 3-1:1-1.

5.

7. Use of the copper algae fucoidan-based anti-tumor drug nanocarrier according to claim 4 in the preparation of tumor-targeted drugs.

8. The use according to claim 7, characterized in that: The prepared nanoparticles are wrapped with anti-tumor drugs to prepare tumor-targeted drugs; Furthermore, the anti-tumor drug includes doxorubicin.

9. The use according to claim 8, characterized in that: The particle size of the doxorubicin-loaded nanoparticles is 360-400 nm.

10. The use according to claim 8, characterized in that: The loading rate of doxorubicin in the loaded nanoparticles was 30%-35%.

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