Method for large-scale preparation of high-purity floridoside and isomer monomer thereof

Abstract

The invention relates to the technical field of floridoside and an isomer monomer thereof, and discloses a method for preparing high-purity floridoside and an isomer monomer thereof on a large scale, which is used for carrying out large-scale preparation and purification research on the high-purity floridoside and the isomer monomer thereof and preparing the floridoside and the isomer monomer thereof from a porphyra haitanensis crude extract. And the structure and purity of the compound are identified. The method comprises the following steps: firstly, by utilizing a DART-MS technology, through a single factor experiment, optimizing experiment parameters such as extraction times and elution, and obtaining optimal extraction efficiency; and secondly, based on a two-step purification method of a prep-HPLC (Prep-High Performance Liquid Chromatography) technology, enriching the target compound by using a reversed-phase (C18) mode stationary phase in the first step, and separating the pair of isomers by using a normal-phase (NH3) mode stationary phase in the second step. The obtained floridoside and isomer monomers thereof are subjected to structure and purity identification by mass spectrometry (MS) and nuclear magnetism (NMR). Results are as follows: the two compounds are identified as floridoside and isomers thereof through high-resolution mass spectrometry, and the purities of the floridoside and the isomers thereof are up to 99.5% or above.

Description

Technical Field

[0001] The present invention relates to the technical field of furoside and its isomer monomers, in particular to a method for scalably preparing high-purity furoside and its isomer monomers. Background Art

[0002] Glycosides, as secondary metabolites widely found in plant tissues, possess a variety of pharmacological activities, including anti-inflammatory, anti-infective, anti-cancer, and antioxidant activities, and have garnered significant attention in the field of drug synthesis. Given the abundance of marine algae, the development of new glycoside drugs derived from marine organisms has become an important research direction for the treatment of complex metabolic diseases.

[0003] Rhodosides are monoglycosides with potential for development in marine algae. They primarily exist in two configurations: rhodoside (α-D-galactopyranosyl-D-glycerol) and isorhodoside (α-D-galactopyranosyl-L-glycerol). They are abundant in Porphyra haitanensis. Studies have reported that they exhibit antioxidant activities, inhibit tumor cell migration, improve intestinal immune function, and promote the growth of human fetal liver cells. Their polyhydroxylated structure makes them promising candidates as lead compounds for novel clinical glycoside drugs.

[0004] However, the preparation process of red algae glycoside and its isomer monomer compounds is currently missing. Existing research mainly focuses on stress enrichment in algae and in vitro efficacy verification, and there are deficiencies in preparation and purification. For example, Gao Yuli et al. used response surface analysis to optimize the extraction process, but the effect was limited in the separation and purification of the target compound; Yue Juan et al. used subcritical fluid extraction technology to achieve large-scale preparation, but did not effectively solve the problem of monomer separation; Shi Luqiu et al. produced red algae glycoside through genetic engineering technology, but faced difficulties such as high culture conditions and ineffective solutions to the post-fermentation extraction and purification process. Therefore, it is urgent to develop a method for the efficient preparation of red algae glycoside and its isomer monomers.

[0005] This case is proposed to solve or improve the shortcomings or deficiencies of the existing technology. Summary of the Invention

[0006] The present invention is achieved by taking the following technical solutions:

[0007] Preparation of crude liquid: Porphyra taurensis was collected from Nanji Island, Wenzhou, freeze-dried, ground and stored at low temperature. +The extract was activated using an anion exchange resin (Dowex 1×8, 100-200 mesh, Cl- form). Porphyra haitanensis powder was weighed and added with 72.3% ethanol at a liquid-to-solid ratio of 1:14 (g:mL). Extraction was performed three times in a 60°C water bath, each for 4 hours. After filtration and concentration, the extract was extracted three times with ethyl acetate. The upper aqueous phase was collected and concentrated to volume. Purification was performed sequentially through activated cation exchange resin columns and anion exchange resin columns. The eluate was collected and concentrated to dryness to obtain a crude extract of red algae glycoside compounds with a purity of 49.5%. The ion exchange resin can be regenerated with acid and alkali and reused repeatedly.

[0008] Purification Step 1: Dissolve the crude extract in ultrapure water to a 100 mg / mL solution, then pass it through a 0.45 μm organic membrane. Use a C18 (50 mm × 250 mm, 10 μm) preparative column with a gradient elution of water and methanol as the mobile phase (0-5 min, methanol increased from 2% to 5%; 5-8 min, methanol increased from 5% to 100% and held for 4 min; 12-12.1 min, methanol rapidly returned to 2% and held for 6 min). The flow rate was 15 mL / min, the injection volume was 350 μL, and the detection wavelength was 200 nm. Fractions with a retention time of 4.25-5.0 min were collected and concentrated to dryness by rotary evaporation. The mixture was then 93.8% pure.

[0009] Purification Step 2: The sample obtained in Step 1 was dissolved in pure water at a concentration of 100 mg / mL. A Shim-pack GIST-NH2 column (250 mm × 20 mm, 5 μm) was used as the preparative column, with ACN / H2O = 90 / 10 (v / v) as the eluent, a flow rate of 15 mL / min, an injection volume of 300 μL, and detection at a wavelength of 200 nm. Fractions with retention times of 30.0-31.3 min and 33.2-34.7 min were collected, rotary evaporated, and freeze-dried to yield a white flocculent powder with a monomer purity exceeding 99.5%.

[0010] Structure and purity identification: Mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques were used to identify the structure and purity of the obtained compounds. The mass spectrometry used ESI source and DART ion source, measured in negative ion conversion mode, and set relevant parameters; the NMR was determined by dissolving the sample in D2O. 1 H NMR (600 MHz).

[0011] The advantages and positive effects of the present invention are:

[0012] 1. The two-step purification method based on preparative high performance liquid chromatography (prep-HPLC) established in the present invention successfully prepared high-purity fucoidan and isofucoidan monomers with a purity of more than 99.5%.

[0013] This method combines the reverse phase (C18) and normal phase (NH3) modes, and the two-step purification cooperates with each other to effectively achieve the enrichment and preparation of the target monomer compound, providing a key material basis for subsequent in-depth research on the pharmacological activity of red algae glycoside and its isomers and the development of new glycoside drugs, which will help promote the development of innovative marine drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described below with reference to the accompanying drawings and examples.

[0015] Figure 1 The effects of different extraction times (1, 2, 3 and 4) on the extraction efficiency of red algae glycoside compounds of the present invention;

[0016] Figure 2 The distribution of the red algae glycoside compounds in each tube after purification by the cation exchange resin of the present invention, and the peak area content value of the target compound in the tube (n=3);

[0017] Figure 3 This is the EIC diagram of the target compound in the corresponding tube of the present invention;

[0018] Figure 4 The distribution of the red algae glycoside compounds in each tube after purification by the anion exchange resin of the present invention (measured in peak area, n=3);

[0019] Figure 5 This is the separation spectrum of the red algae glycoside compounds of the present invention on a preparative liquid reverse phase C18 column.

[0020] Figure 6 It is the separation effect of impurities and target compounds under different injection volumes of the present invention.

[0021] Figure 7 The chromatographic separation effects of fucoidan and its isomers on three different chromatographic columns of the present invention include: Hypersile Gold C8 (150 mm × 2.1 mm, 3 μm); Syncronis C18 (2.1 mm × 150 mm, 1.7 μm) and ACQUITY HSS T3 (2.1 mm x 100 mm, 1.8 μm).

[0022] Figure 8 The chromatographic separation effect of the red algae glycoside and its isomers of the present invention is shown on an Accucore HILIC (2.1 mm x 100 mm, 2.6 μm) and a ShimNex HE NH2 column (150 mm x 4.6 mm, 5.0 μm).

[0023] Figure 9 This is the chromatographic separation effect of the red algae glycoside and its isomers of the present invention.

[0024] Figure 10 It is the secondary mass spectrum of the red algae glycoside and its isomers of the present invention and the fragmentation pathway thereof. DETAILED DESCRIPTION

[0025] The present invention will now be described in further detail with reference to the accompanying drawings, which are simplified schematic diagrams that illustrate the basic structure of the present invention in a schematic manner.

[0026] The embodiments of the present invention are further described below in conjunction with the accompanying drawings:

[0027] If there are terms related to directional indications or positional relationships in the embodiments of this application (such as up, down, left, right, front, back, inside, outside, top, bottom, center, vertical, horizontal, longitudinal, transverse, length, width, counterclockwise, clockwise, axial, radial, circumferential, etc.), such terms are only used to explain the relative positional relationship, movement, etc. between the components in a specific posture (as shown in the accompanying drawings); if the specific posture changes, the directional indication or positional relationship will also change accordingly. In addition, the terms "first" and "second" in the embodiments of this application are only used for the purpose of convenience of description and should not be understood as indicating or implying relative importance.

[0028] like Figure 1-10 As shown, the method of the present invention for preparing high-purity fucoidan glycoside and its isomer monomers on a large scale,

[0029] Instruments and consumables: A Shimadzu LC-16P liquid chromatograph and its associated equipment, various chromatographic columns, a Q-Exactive quadrupole orbitrap high-resolution mass spectrometer, and a Bruker Avance NEO 600 MHz nuclear magnetic resonance instrument were also prepared. Consumables included chromatographically grade ammonium acetate, analytically grade NaOH and HCl, chromatographically grade methanol and acetonitrile, Milli-Q ultrapure water, a Dowex 50W x 8 cation exchange resin column, a Dowex 1 x 8 anion exchange resin column, and a standard of fucoside.

[0030] Crude fluid preparation

[0031] Pretreatment of Porphyra haitanensis: Porphyra haitanensis was collected from a specific area of ​​Nanji Island, Wenzhou, freeze-dried to constant weight, ground into powder, and stored at -20°C.

[0032] Resin activation: Activate the cation exchange resin and anion exchange resin according to the method described above.

[0033] Extraction and Purification: Accurately weigh 100-500g of Porphyra haitanensis powder and add 72.3% ethanol at the specified liquid-to-solid ratio (1:14) (g:mL). Extract three times in a 60°C waterbath for 4 hours each time. Filter through multiple layers of gauze, rotary concentrate to near dryness, and transfer purified water to a separatory funnel. Extract three times with an equal volume of ethyl acetate. Collect the upper aqueous phase and rotary concentrate to volume at 60°C. Purify the extract sequentially through a cation exchange resin column and then an anion exchange resin column. Collect the eluate and concentrate to dryness to obtain 100ml of crude extract of red algal glycoside compounds.

[0034] First purification step: The crude extract is prepared into a solution as required, passed through a membrane, and then purified on a C18 column. Strictly follow the set gradient elution program, flow rate, injection volume, and detection wavelength. Fractions at a specific retention time are collected and concentrated to obtain a preliminarily purified mixture.

[0035] Second purification step: Dissolve the sample from the first purification step and purify it using a specific preparative column, eluent, flow rate, injection volume, and detection wavelength. Collect fractions with corresponding retention times, rotary evaporate, and freeze-dry to obtain high-purity fucoidan and its isomer monomers.

[0036] Structure and purity identification: The structure and purity of the obtained monomer were identified according to the set mass spectrometry and nuclear magnetic resonance conditions to confirm that the obtained compound was red algae glycoside and its isomers and the purity met the requirements.

[0037] Instrument conditions of the examples

[0038] ESI source mass spectrometry conditions: The mass spectrometer was measured in full scan mode in negative ion conversion mode, with a mass range of m / z 100-1000, a resolution of 70,000, and an automatic gain control (AGC) target value of 5×105; the voltage was 2700 V, the ion transfer tube temperature was 300°C, the sheath gas pressure (N2) was 35 arb, the auxiliary gas pressure (N2) was 10 arb, and the vaporizer temperature was 350°C; the instrument was positively and calibrated before sample operation; the secondary mode was automatic trigger mode, with a resolution of 35,000, an AGC target value of 2×105, a collision energy range of 25%-40%, and a retention time acquisition range of ±1.00 min based on the retention time (RT) of the target substance in the primary chromatogram.

[0039] DART ion source parameters: helium volumetric flow rate 3.0 L / min; grid voltage (GV): 100 V; ion source gas temperature set to 300°C. The distance between the DART ion source orifice and the mass spectrometer inlet was 1 cm, and the traversal speed was 0.6 mm / s. Mass spectrometry parameters: negative ion mode, ion spray voltage: -2700 V, scan mode: Target-MS, m / z 253.0925.

[0040] NMR conditions: Dissolve an appropriate amount of sample in D2O and measure 1H NMR (600 MHz).

[0041] Analytical column chromatography parameters: ShimNex HE NH2 column (150 mm × 4.6 mm, 5.0 μm), column temperature 40 ° C, flow rate 0.8 mL / min, injection volume 1 μL; mobile phase ACN / H2O (90:10, v / v) isocratic elution 40 min.

[0042] Qualitative analysis: The precise mass error is required to be less than 5*10-6. Compound Discoverer software simultaneously compares retention time, isotope distribution, main secondary fragments and secondary mass spectrum similarity, and makes a comprehensive judgment to obtain accurate qualitative results and avoid false positive results.

[0043] Quantitative Analysis: Accurately weigh 10.0 mg of the standard and dilute it with pure water to a 1000 mg / L standard stock solution. Prepare a standard curve for fucosides with a concentration range of 0, 10.0, 20.0, 50.0, 100, and 200 μg / L, using concentration as the abscissa and standard peak area as the ordinate, as the basis for target quantification. For other isofrucosides detected (not commercially available or not purchased due to insufficient purity), relative quantification was performed using this standard curve due to their structural similarities.

[0044] The purity of the analyte in the sample is calculated according to the following formula (1):

[0045] X=C*V / m*1000*100, where:

[0046] X - purity of the substance to be tested in the sample, in %;

[0047] C - concentration of the analyte in the sample treatment solution, calculated based on the matrix standard curve, in μg / LV - constant volume, in L;

[0048] m - sample mass, in mg.

[0049] In this embodiment, ethanol-water system is selected as the extraction solvent. Ethanol has good solubility and strong permeability, and can effectively destroy the plant cell wall, release target compounds such as red algae glycosides in the cell, and enable the highly polar target compounds to be quickly extracted into the aqueous solution. The ethanol-water system has become a commonly used solvent for the extraction of active ingredients in plants. Referring to the extraction process of red algae glycoside compounds in Porphyra haitanensis optimized by Gao Yuli et al., that is, ethanol concentration of 72.3%, extraction temperature of 60°C, extraction time of 4h, liquid-to-material ratio of 1:14 (ɡ:mL). Taking the extraction amount as an indicator, the effect of the number of extractions (1, 2, 3, 4 times) on the extraction rate of red algae glycoside compounds was further investigated. The extracts obtained at different times were combined, and the aqueous solution was diluted to the same volume (4000mL). The DART-MS without matrix effect interference was used to perform mass spectrometry analysis on the target content (m / z 253.0925) in the complex matrix, and provide immediate detection results (3 parallel, measured in peak area). The results show that ( Figure 1 The extraction yield increased significantly with the number of extractions, reaching a maximum after three extractions and then tending to stabilize (P < 0.05). In summary, the extraction conditions determined were: 72.3% ethanol concentration, 60°C extraction temperature, 1:14 solid-liquid ratio (g:mL), 4h extraction time, and 3 extractions.

[0050] The combined extracts were then rotary evaporated to remove ethanol and replaced with an aqueous solution. Liquid-liquid extraction with ethyl acetate was performed before application to an ion exchange resin column to minimize interference from fat-soluble compounds (primarily fat-soluble chlorophyll). This study compared the color of the upper aqueous phase after 1, 2, 3, 4, and 5 extractions with the same volume of ethyl acetate. For extractions ≤ 3, the color of the upper aqueous phase gradually faded (from dark green to light green). With increasing extractions (4 and 5), the color remained constant. To save time and cost, this study ultimately settled on 3 ethyl acetate extractions.

[0051] After the extracted aqueous phase is concentrated to 100 mL, it is loaded onto a cation exchange resin column, and the flow rate is controlled at ≤1 drop / second to ensure maximum adsorption of positive ion interfering compounds. Then, 500 mL of pure water is used for elution, and the mixture is collected tube by tube (15 mL / tube). Since target compounds such as red algae glycoside are highly water-soluble, as water gradually infiltrates, the target compounds will be preferentially eluted from the column. DART-MS is used to perform rapid mass spectrometry analysis (3 times in parallel, based on peak area) on the target compound (m / z 253.0925) in the collection tube to ensure that the target compound is completely collected and the interference of impurities is eliminated to the greatest extent possible. The results show ( Figure 2), the target compound was primarily concentrated in tubes 2 to 15, with a decreasing distribution trend. The color of the tubes progressed from light to dark (tubes 1 to 18), gradually fading to colorless (tubes 19 to 27). Therefore, the eluates of the target compound (tubes 2 to 15) were combined and concentrated to 100 mL via rotary evaporation for further purification.

[0052] The eluate was further purified by anion exchange resin column using the same conditions as for cation elution. The target compound (m / z 253.0925) in the collection tube was analyzed by rapid mass spectrometry using DART-MS (3 replicates, calculated by peak area). The results showed ( Figure 3 The target compound was concentrated in tubes 2 to 14, with a decreasing concentration and a pale yellow color. The eluates containing the target compound were combined and rotary concentrated to dryness. High-performance liquid chromatography-mass spectrometry analysis revealed a purity of 49.5% for the mixture of fucoside and its isomers.

[0053] The target compound's purity in the crude product was low, and the sample's composition remained relatively complex, necessitating further purification. We first performed preliminary purification using reversed-phase chromatography, selecting the commonly used C18 stationary phase. Given that the target compound is a small, water-soluble monosaccharide, its retention on the C18 column is weak, and effective separation of fucoside and its isomers was not achieved. Therefore, the primary objective of this step was to enrich the target component.

[0054] First, the flow rate was calculated based on the size of the C18 preparative column (250mm*20mm, 10μm), and the optimal flow rate was determined to be 15mL / min. Methanol (A) / water (B) was used as the mobile phase, and the elution gradient was further optimized. By extending the elution time of the high water phase, the target compound was effectively separated from the highly polar compounds. The results showed that ( Figure 4 ): 0-5min, the proportion of phase A increases from 2% to 5%, which can achieve complete elution of the target compound. By collecting the preparation solution at different time points and combining high-resolution mass spectrometry and ultraviolet absorption chromatography (200nm), it was further determined that the elution time of the target compound was 4.25-5.0min. This process effectively removed the interference of highly polar compounds (elution time was 3.86min); 5-8min, the proportion of phase A increased from 5% to 100% and maintained for 4min, which can ensure that most compounds are completely eluted and effectively remove the residues of non-polar compounds on the column. Finally, from 12 to 12.1min, phase A quickly returned to its initial state (2%) and was further maintained for 6min, allowing the column pressure to return to a steady state, enabling continuous sample preparation.

[0055] Then optimize the sample loading volume. The sample loading volume is an important indicator for preparation. The sample is dissolved in water with a concentration of 100 mg / mL. The injection volume is gradually increased (100-500 μL). When the injection volume is 350 μL, the separation degree between impurities and target compounds is still good ( Figure 5 The first step of purification involves repeated injections to enrich the target compound, so good reproducibility is crucial for ensuring effective enrichment. Three consecutive injections yielded reproducible spectra, demonstrating the method's excellent reproducibility. Based on the preparative spectra and high-resolution mass spectrometry results, at a sample concentration of 100 mg / mL, an injection volume of 350 μL, and a fraction collection time of 4.25 to 5.0 minutes, the purity of the mixture of fucoside and its isomers was determined to be 93.8%. This results in a calculated impurity removal rate of 93.92%.

[0056] Due to the significant differences in their separation mechanisms, hydrophilic chromatography and reversed-phase chromatography exhibit excellent complementarity. This method effectively addresses the inadequate separation of certain peaks in conventional chromatography, significantly improving the purity of target compounds. This experiment employed the experimental conditions of a ShimNex HE NH2 analytical column (150 mm × 4.6 mm, 5.0 μm), employing a mobile phase gradient of ACN / H2O (v:v, 90:10). Subsequently, flow rate optimization revealed that this analytical column could achieve complete separation of fucoside and its isomers at a flow rate of 0.8 mL / min. Based on the dimensions of a Shim-pack GIST-NH2 preparative column (250 mm × 20 mm, 5 μm) with the same particle size, the optimal preparative flow rate for this column was determined to be 15 mL / min. To ensure efficiency and purity during the preparative process, fractions were collected at various time points (at 0.5-min intervals). After mass spectrometry detection, the fraction collection time of the target monomer compound was further refined, and the elution time of erythroside was finally determined to be 30.0-31.3 min, and the elution time of isorhizoside was 33.2-34.7 min. The chromatographic separation of the two pairs of isomers was 2.14, which was obtained according to the following calculation formula (2):

[0057] X = 2*(RT2-RT1) / (W1+W2), where:

[0058] X-chromatographic separation of rhodoside and isorhodoside;

[0059] RT1-chromatographic peak retention time of red algae glycoside, in min;

[0060] RT2—chromatographic peak retention time of isorhodoside, in min;

[0061] W1-peak width of the chromatographic peak of red algae glycoside, in min;

[0062] W2—chromatographic peak width of isorhodoside, in min.

[0063] Next, the sample volume was optimized. The mixture of red algal glycoside and its isomers obtained above was dissolved in water at a concentration of 100 mg / mL, and the injection volume was gradually increased (100-500 μL). The results showed that when the injection volume was in the range of 100-300 μL, the separation of red algal glycoside and its isomers remained stable. In addition, the second step of the purification process also involved repeated injections to achieve the enrichment of the target compound.

[0064] Therefore, good reproducibility is crucial for ensuring effective separations. Three consecutive injections at a sample concentration of 100 mg / mL and an injection volume of 300 μL demonstrated excellent spectral reproducibility, with no significant change in the elution time of the target compound during subsequent processing. The collected fractions were combined, rotary evaporated, and lyophilized to yield the fucoside and its isomer monomers, with purity levels of 99.5% and 99.7%, respectively.

[0065] Six columns of varying specifications, covering five packing ranges, were compared: 1) a silica-bonded short-chain C8 functional group Hypersile Gold C8 column (2.1 mm x 150 mm, 3.0 μm); 2) a silica-bonded medium-chain C18 functional group Syncronis C18 column (2.1 mm x 150 mm, 1.7 μm); 3) an ACQUITY HSS T3 column (2.1 mm x 100 mm, 1.8 μm); 4) a hydrophilic Accucore HILIC column (2.1 mm x 100 mm, 2.6 μm); and 5) a normal-phase amino column Ultimate XB-NH2 (3.0 mm x 100 mm, 3.5 μm) and ShimNex HE NH2 (150 mm x 4.6 mm, 5.0 μm). The chromatographic separation of fucosides and their isomers was particularly investigated. The results showed that the silica-bonded short-chain C8 column, C18 functional group column and T3 column had low retention for polar compounds. Despite trying a variety of mobile phases and gradient combinations, the target compounds could not be effectively separated ( Figure 6 However, the 3D ion mobility spectrum shows that two isomers with different mobility values ​​exist under this chromatographic peak, so the chromatographic column needs to be replaced to achieve the desired separation effect.

[0066] It should be emphasized that the embodiments described in the present invention are illustrative rather than restrictive. Therefore, the present invention is not limited to the embodiments described in the specific embodiments. Any other embodiments derived by those skilled in the art based on the technical solutions of the present invention also fall within the scope of protection of the present invention.

Claims

1. A method for scalably preparing high-purity fucoidan and its isomer monomers, characterized in that: The following steps are involved: Step 1: Crude fluid preparation: Porphyra haitanensis was collected, freeze-dried, ground and stored at low temperature; Activate cation exchange resin and anion exchange resin; weigh Porphyra haitanensis powder, add 72.3% ethanol at a liquid-to-solid ratio of 1:14 (g:mL), extract in a 60°C water bath three times for 4 hours each time, filter and concentrate, then extract three times with ethyl acetate, collect the upper aqueous phase and concentrate to 100 ml, purify sequentially through a cation exchange resin column and an anion exchange resin column, collect the eluate and concentrate to dryness to obtain a crude extract of red algal glycoside compounds; Step 2: The crude extract was dissolved in ultrapure water to prepare a 100 mg / mL solution, and the solution was passed through a 0.45 μm organic membrane; a C18 (50 mm × 250 mm, 10 μm) preparative column was used, and water and methanol were used as the mobile phase for gradient elution at a flow rate of 15 mL / min, an injection volume of 350 μL, and a detection wavelength of 200 nm. The fractions with a retention time of 4.25-5.0 min were collected and concentrated to dryness by rotary evaporation; Step 3: The sample obtained from the first step of purification was dissolved in pure water at a concentration of 100 mg / mL; a Shim-pack GIST-NH2 column (250 mm × 20 mm, 5 μm) was used as a preparative column, ACN / H2O = 90 / 10 (V / V) was used as the eluent, the flow rate was 15 mL / min, the injection volume was 300 μL, the detection wavelength was 200 nm, and the fractions with retention times of 30.0-31.3 min and 33.2-34.7 min were collected, and the erythroside and its isomer monomers were obtained by rotary evaporation and freeze-drying; Structure and purity identification: Mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques were used to identify the structure and purity of the obtained compound.

2. The method for scalably preparing high-purity fucoidan and its isomer monomers according to claim 1, characterized in that: The cation exchange resin is Dowex 50W×8 (100-200 mesh, H + The anion exchange resin is Dowex 1×8 (100-200 mesh, Cl- form), and the ion exchange resin can be repeatedly used after acid and alkali regeneration treatment.

3. The method for scalably preparing high-purity fucoidan and its isomer monomers according to claim 2, characterized in that: In the first step of purification, the mobile phase gradient elution program is: 0-5 min, methanol increased from 2% to 5%; 5-8 min, methanol increased from 5% to 100% and maintained for 4 min; 12-12.1 min, methanol quickly returned to 2% and maintained for 6 min.

4. The method for scalably preparing high-purity fucoidan and its isomer monomers according to claim 2, characterized in that: The mass spectrometer uses ESI source and DART ion source, and is measured in negative ion conversion mode. The nuclear magnetic resonance is measured by dissolving the sample in D2O. 1 H NMR (600 MHz).

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