Preparation method of fucoidan from Sargassum hemifolia and its application in the preparation of anti-hepatocellular carcinoma drugs

Abstract

This invention discloses a method for preparing fucoidan from *Sargassum hemifolia* and its application in the preparation of anti-hepatocellular carcinoma drugs. Experimental results show that *Sargassum hemifolia* fucoidan Fb has a strong inhibitory effect on HepG2 hepatocellular carcinoma cells, significantly stronger than the inhibitory effects of *Sargassum henryi* polysaccharide Fh and *Sargassum fusiforme* polysaccharide SFPS on HepG2 hepatocellular carcinoma cells. After treatment of HepG2 hepatocellular carcinoma cells with *Sargassum hemifolia* fucoidan DF1 and DF2, apoptosis is ultimately induced through the interaction and cooperation of exogenous (death receptor pathway) and endogenous (mitochondrial pathway). *Sargassum hemifolia* fucoidan possesses strong antitumor activity and therefore can be used to prepare anti-hepatocellular carcinoma drugs.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drugs, specifically relating to the preparation method of fucoidan from Sargassum hemifolia and its application in the preparation of anti-liver cancer drugs. Background Technology

[0002] Cancer is one of the most serious diseases in the world today. It is caused by various carcinogenic factors affecting the body, leading to abnormal growth of a local cell at the gene level, resulting in abnormal proliferation and the formation of a tumor. Cancer treatment typically involves chemotherapy, which has severe side effects. Therefore, developing a natural drug with fewer toxic side effects is crucial.

[0003] Numerous experimental studies have demonstrated that fucoidan sulfate, an anionic polysaccharide containing sulfate groups, is mainly found in brown algae and some marine invertebrates. It shows promising applications in antitumor, immunomodulatory, and anti-aging applications, with minimal toxicity. Therefore, the search for highly effective and low-toxicity antitumor drugs from Sargassum polysaccharides is a current hot topic in cancer research, possessing both theoretical and practical significance.

[0004] In recent years, Sargassum polysaccharides have been proven to have anti-tumor effects. Sargassum algae are abundant, distributed throughout my country from the Liaodong Peninsula in the north to the Leizhou Peninsula in the south. The physicochemical properties and biological activities of polysaccharides vary among different Sargassum species. Current literature mainly focuses on Sargassum extraction and its applications in terms of activity. There are few reports on the intracellular targets of Sargassum fucoidan, and even fewer reports on Sargassum fucoidan with high activity for treating different tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing fucoidan from *Sargassum fusiforme* with antitumor activity and its application in the preparation of anti-hepatocellular carcinoma drugs, thereby addressing the problems existing in the prior art. Experimental results of this invention show that fucoidan (Fb) from *Sargassum fusiforme* has a strong inhibitory effect on HepG2 hepatocellular carcinoma cells, significantly stronger than the inhibitory effects of *Sargassum henleyi* polysaccharide (Fh) and *Sargassum fusiforme* polysaccharide (SFPS) on HepG2 hepatocellular carcinoma cells. Further analysis of its mechanism of inhibiting the proliferation of HepG2 hepatocellular carcinoma cells revealed that after treatment of HepG2 hepatocellular carcinoma cells with fucoidan (DF1, DF2), apoptosis is ultimately induced through the interaction and joint participation of exogenous pathways (death receptor pathway) and endogenous pathways (mitochondrial pathway). In summary, fucoidan from *Sargassum fusiforme* has a strong antitumor effect and therefore can be used to prepare anti-hepatocellular carcinoma drugs.

[0006] The first objective of this invention is to provide a method for preparing fucoidan from Sargassum hemifolia, comprising the following steps:

[0007] S1. Sargassum hemifolia was pulverized and sieved, then extracted with water, and after sonication and concentration, it was precipitated with alcohol to remove fat, removed with Sevage to remove protein, dialyzed and freeze-dried under vacuum to obtain Sargassum hemifolia fucoidan (Fb).

[0008] S2. Dissolve Sargassum fusiforme fucoidan (Fb) in distilled water to prepare a 0.1% (w / w) aqueous solution, pack it onto a DEAE-52 cellulose column, elute with distilled water first, then elute with a linear gradient of 0.2–1.8 M sodium chloride aqueous solution at a flow rate of 1 mL / min. Collect the eluent in test tubes using an automatic collector, and collect the 0.6 M and 1.5 M sodium chloride aqueous solutions as F1 and F2, respectively; then perform dialysis, concentration, and lyophilization, respectively.

[0009] S3. F1 and F2 were degraded by hydrogen peroxide and ascorbic acid, respectively, and the mixtures were obtained by centrifugation. The supernatant was dialyzed, concentrated, and lyophilized. Finally, it was purified by Separose CL-6B gel column to obtain Sargassum fucosum DF1 and Sargassum fucosum DF2, respectively.

[0010] Preferably, step S1 is as follows: Sargassum fusiforme is pulverized and passed through a 0.18mm sieve. Water is added at a ratio of 1g:30mL, and the mixture is then extracted in an 80℃ constant temperature water bath for 3.5h. The mixture is then sonicated at 60℃ and 350W for 50min, and centrifuged at 4000r / min for 20min to concentrate. Anhydrous ethanol is added to the concentrated solution, with a volume ratio of 7:3. The mixture is thoroughly mixed, and after 24h, the supernatant is obtained by centrifugation. Anhydrous ethanol is then added until the volume fraction of the supernatant reaches 80%, and the mixture is thoroughly mixed. After homogenization, centrifugation was performed after 24 hours to obtain a precipitate. The precipitate was washed twice each with acetone and anhydrous ethanol, alternating between the two. The precipitate was dissolved in a small amount of water to form a polysaccharide solution. Sevage was added to the polysaccharide solution at a volume ratio of 5:1. The mixture was shaken vigorously for 1 hour. After separation in a separatory funnel, the precipitate was removed. The above steps were repeated 6 times until no denatured proteins appeared. The Sevage was obtained by mixing n-butanol and chloroform at a volume ratio of 1:4. The mixture was dialyzed for 48 hours and then freeze-dried under vacuum to obtain fucoidan (Fb) from Sargassum fusiforme.

[0011] Preferably, in step S2, the DEAE-52 cellulose column has a specification of 2.6×50cm and is in Cl- form.

[0012] Preferably, in step S3, the degradation of F1 and F2 using hydrogen peroxide and ascorbic acid respectively includes the following steps: preparing an aqueous solution of F1 and F2 with distilled water at a concentration of 15 g / L, placing it in a constant temperature water bath at 30°C, adding 0.2590 g of ascorbic acid for every 0.3 g of F1 or F2 raw material based on the mass of the raw material used, adding 1 mL of 5% hydrogen peroxide aqueous solution while stirring, and stirring the reaction for 2 hours.

[0013] Preferably, the Separose CL-6B gel column has a size of 1.6 × 80 cm.

[0014] This invention also provides the application of fucoidan from Sargassum hemifolia in the preparation of anti-liver cancer drugs.

[0015] Preferably, the fucoidan from Sargassum fusiforme is Sargassum fusiforme DF1 and Sargassum fusiforme DF2, which are prepared according to the preparation method described above.

[0016] Preferably, the fucoidan from Sargassum hemifolia is Sargassum hemifolia fucoidan DF2.

[0017] Preferably, the application is the use of fucoidan from Sargassum fusiforme in the preparation of an anti-liver cancer drug that induces tumor cell apoptosis through the death receptor pathway and the mitochondrial pathway.

[0018] The present invention also provides an anti-liver cancer drug containing the aforementioned Sargassum fucoidan DF1 and / or Sargassum fucoidan DF2 and a drug-acceptable carrier.

[0019] This invention employs ultrasonic-assisted hot water extraction, ethanol fractionation precipitation, and Sevage deproteinization to extract three Sargassum polysaccharide components: Fh (Sargassum henschneidera polysaccharide), Fb (Sargassum hemifolia fucoidan), and SFPS (Sargassum fusiforme polysaccharide). Further chemical content analysis revealed that Fh, Fb, and SFPS are all heteropolysaccharides containing sulfate groups and some uronic acid. Fb had the highest total sugar content (75.33±3.99)% among the three Sargassum polysaccharides, and its L-fucose content (15.63±3.37)% and sulfate group content (29.16±0.86)% were both higher than Fh. Further infrared spectroscopy analysis showed that components Fh, Fb, and SFPS all contained characteristic absorption peaks of polysaccharides and contained both carboxyl and sulfate groups, indicating that they are all sulfated polysaccharides containing uronic acid. Data showed that the sulfate groups in Fh, Fb, and SFPS were all attached to C2 or C3 positions in equatorial bonds. Furthermore, all three Sargassum polysaccharides exhibited inhibitory effects on HepG2 liver cancer cells, with Fb showing the strongest effect. Further analysis of their mechanism of inhibiting HepG2 liver cancer cell proliferation revealed that DF1 and DF2 (Sargassum hemifolia fucoidan) treatment of HepG2 liver cancer cells induced apoptosis through the interaction of exogenous (death receptor pathway) and endogenous (mitochondrial pathway). Therefore, Sargassum hemifolia fucoidan possesses strong antitumor activity and can be used to prepare anti-liver cancer drugs. Attached Figure Description

[0020] Figure 1 The FT-IR spectra of Fh, Fb, and SFPS are shown.

[0021] Figure 2 The cell viability of HepG2 and LO2 cells after 72 h of treatment with Fh, Fb and SFPS is shown in Figure A: Fh; Figure B: Fb; Figure C: SFPS. Within-group comparisons of different concentrations of the same substance, any identical letter indicates no significant difference (P>0.05).

[0022] Figure 3 The elution curve of Fb by DEAE-52 ion exchange chromatography;

[0023] Figure 4 Figure A shows the high performance liquid chromatograms of F1 and F2; Figure B shows F1 and F2 respectively.

[0024] Figure 5 Figure A shows the elution curves of DF1 and DF2 using CL-6B gel column chromatography. Figure B shows the elution curves of DF1 and DF2.

[0025] Figure 6 Figure A shows the high performance liquid chromatograms of DF1 and DF2; Figure B shows DF1 and DF2 respectively.

[0026] Figure 7 The effect of different concentrations of fucoidan from Sargassum hemifolia on the number of apoptotic cells in HepG2 cells was shown in Figure A: flow cytometry and Figure B: statistical graph. Within-group comparisons of different concentrations of the same substance, any group containing the same letter indicates no significant difference (P > 0.05).

[0027] Figure 8 The effect of different concentrations of fucoidan from Sargassum hemifolia on the activity of caspase family proteins is shown in Figure A: protein activity statistics and Figure B: Western blot. For comparisons within the same group at different concentrations of the same substance, any group containing the same letter indicates no significant difference (P > 0.05).

[0028] Figure 9 The effect of different concentrations of fucoidan from Sargassum hemifolia on ROS content in HepG2 cells was shown in Figure A: fluorescence image under an inverted microscope, and Figure B: statistical graph. Within-group comparisons of different concentrations of the same substance, any group containing the same letter indicates no significant difference (P > 0.05). Detailed Implementation

[0029] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0030] Example 1

[0031] 1. Materials and Methods

[0032] 1.1 Materials and Reagents

[0033] Sargassum fusiforme and Sargassum heinzmannii were harvested in April 2021 from Naozhou Island, Zhanjiang City, Guangdong Province. Rotten and yellow leaves were removed from the harvested Sargassum fusiforme, which was repeatedly rinsed with tap water, dried at 60℃, and stored in sealed bags for later drying and storage. Dried Sargassum fusiforme products were produced in Xiapu, Fujian Province. Human HepG2 liver cancer cells were provided by the American College of Cell Bank (ATCC); normal human liver LO2 cells were provided by the Shanghai Cell Bank of the Chinese Academy of Sciences; fetal bovine serum (catalog number 10270-106), PBS (catalog number C10010500BT), DMEM high-glucose medium (catalog number C11995500BT), 0.25% trypsin solution (catalog number 25200072), and penicillin-streptomycin solution (catalog number 15140122) were all purchased from Gibco, a brand of Thermo Fisher Scientific (China) Co., Ltd.; fucose (catalog number CCFD200617), fucoidan sulfate (catalog number S11142), glucuronic acid standard (catalog number B25302), MTT (thiazolyl blue) (catalog number S19063), DEAE-cellulose DE-52 (catalog number S14024), and Sepharose were also used. CL-6B (catalog number S14087) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; DMSO (dimethyl sulfoxide, catalog number R012316) and 5% phenol solution (catalog number R094001) were purchased from Shanghai Yien Chemical Technology Co., Ltd.; Bradford protein assay kit (catalog number P0006), BCA protein assay kit (catalog number P0012), Caspase 3 activity assay kit (catalog number C1116), Caspase 8 activity assay kit (catalog number C1151), Caspase 9 activity assay kit (catalog number C1157), reactive oxygen species assay kit (catalog number S0033S), and ultrasensitive ECL chemiluminescence assay kit (catalog number P0018FS) were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0034] 1.2 Instruments and Equipment

[0035] AUW120 electronic balance, Shimadzu Corporation, Japan; XRDKQ-500DB CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; Varioskan Flash multi-functional microplate reader, Thermo Fisher Scientific, USA; Dual-beam UV-Vis spectrophotometer, Beijing Spectrometry General Instrument Co., Ltd.; DHG-9070A forced-air drying oven, Shanghai Yiheng Science Co., Ltd.; HL-6 digital display constant temperature water bath, Changzhou Aohua Instrument Co., Ltd.; Eppendorf 5810R refrigerated centrifuge, Eppendorf GmbH, Germany; FD8508 vacuum freeze dryer, Ilshin GmbH, South Korea; EYELA rotary evaporator, EYELA Corporation, Japan. Bruker Tensor-27 Fourier Transform Infrared Spectrometer (Bruker GmbH, Germany); Agilent 1100 High Performance Liquid Chromatograph (Agilent Technologies, USA); Leica DMI4000B Intelligent Inverted Fluorescence Microscope (Ernst Leitz GmbH, Germany); Cyto FLEX Flow Cytometer (Beckman Coulter GmbH, Germany); Tanon 5200 Fully Automated Chemiluminescence Imaging Analysis System (Shanghai Tanon Technology Co., Ltd.)

[0036] 1.3 Experimental Methods

[0037] 1.3.1 Preparation of crude polysaccharide

[0038] 1.3.1.1 Preparation of Heinz and Sargassum hemifolia polysaccharides

[0039] Sargassum fusiforme and Sargassum henneriifolium were pulverized and sieved (0.18 mm pore size) to prepare an aqueous solution of Sargassum fusiforme at a mass-to-volume ratio of 1 g:30 mL. The solution was then extracted in an 80°C water bath for 3.5 h. The extract was ultrasonically treated (60°C, 350 W) for 50 min, centrifuged (4000 r / min) for 20 min, and concentrated to obtain a concentrated sample solution. Anhydrous ethanol was added to the concentrated solution at a ratio of v(concentrate):v(anhydrous ethanol) = 7:3. After 24 h, the solution was centrifuged to obtain the supernatant. Anhydrous ethanol was then added again until the volume fraction reached 80%, and after another 24 h, the solution was centrifuged to obtain the desired precipitate. The precipitate was washed twice each with alternating acetone and anhydrous ethanol. Then, the precipitate was dissolved in a small amount of water. Sevage reagent [v(n-butanol):v(chloroform) = 1:4] was added at a volume ratio of v(polysaccharide solution):v(Sevage reagent) = 5:1. The mixture was vigorously shaken for 1 hour. After separation in a separatory funnel, the precipitate was removed. This process of adding Sevage reagent was repeated 6 times until no denatured proteins appeared. The precipitate was dialyzed for 48 hours and then freeze-dried under vacuum to obtain Sargassum fusiforme fucoidan (Fh) and Sargassum hemifolia fucoidan (Fb).

[0040] 1.3.1.2 Preparation of Sargassum fusiforme polysaccharide SFPS

[0041] SFPS polysaccharide was extracted using hot water extraction. Dried leaves of *Sargassum fusiforme* were ground into powder and sieved through a 40-mesh sieve. The powder was mixed with 95% ethanol at a ratio of 1:4, and the mixture was refluxed for 3 hours. The precipitate was then dissolved in hot water to prepare a 1:50 solution. The solution was treated in a boiling water bath at 100°C for 4 hours, centrifuged, and the supernatant was collected and rotary evaporated. Alcohol precipitation was performed overnight at 4°C. Protein was removed using the Sevage method, followed by dialyzing and freeze-drying to obtain *Sargassum fusiforme* polysaccharide (SFPS).

[0042] 1.3.2 Physicochemical properties and structural characterization of crude polysaccharides

[0043] 1.3.2.1 Chemical Composition Analysis

[0044] Total sugar content was determined using the phenol-sulfuric acid method, with fucose as the standard sugar; L-fucose content was determined using the Dische colorimetric method; uronic acid content was determined using the carbazole colorimetric method, calculated as glucuronic acid; and sulfate content was determined using the gelatin turbidimetric method.

[0045] 1.3.2.2 Infrared Spectroscopic Analysis

[0046] First, potassium bromide was ground into a finer powder using an agate mortar and dried under an infrared lamp for 4 hours. A semi-transparent sheet about 1 mm thick was pressed into potassium bromide as a blank. The sample was mixed with potassium bromide and ground to form a pellet, which was then scanned in the range of 500–4000 nm.

[0047] 1.3.3 Determination of in vitro antitumor activity of crude polysaccharides

[0048] 1.3.3.1 Cell Culture

[0049] After resuscitation, HepG2 and LO2 cells were placed in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin antibiotic solution. The cell culture flasks were then placed in a 37°C, 5% CO2 incubator for static culture. When the cell confluence reached 70%–80%, the cells were passaged. Cells that were growing well and in the logarithmic growth phase were used for the following experiments.

[0050] 1.3.3.2 MTT assay to detect the effect of crude polysaccharide on inhibiting the growth of human hepatocellular carcinoma HepG2 and normal human hepatocytes LO2.

[0051] 100 μL of HepG2 cells (2 × 10⁻⁶) were added. 4 (cells / mL) and LO2 cells (5×10⁻⁶) 4Cells (cells / mL) were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. 100 μL of different concentrations of Sargassum fucoidan were added to each well to achieve final fucoidan concentrations of 2, 4, and 8 mg / mL. A blank group (culture medium only) and a control group (cell suspension only) were also established. After culturing for another 72 h, 20 μL of 5 mg / mL MTT was added to each well in the dark. The plates were then incubated at 37°C with 5% CO2 for 4 h in the dark. After removing the supernatant from each well with a syringe, 150 μL of DMSO was added, and the plates were shaken for 10 min. The D value was measured at 570 nm. The D value of the control group was taken as 100%, and the cell viability was calculated using the following formula: Cell viability = (Di - D0) / (Dj - D0); where D0 is the optical density of the blank group; Di is the optical density of different treatment groups; and Dj is the optical density of the control group.

[0052] 1.3.4 Preparation of fucoidan (sulfate ester) DF1 and DF2 from Sargassum hemifolia

[0053] 1.3.4.1 Purification of crude polysaccharide from Sargassum hemifolia

[0054] 0.1 g Fb was dissolved in 100 mL of distilled water and packed onto a DEAE-52 cellulose column (2.6 × 50 cm, Cl- form). Elution was first performed with distilled water, followed by a linear gradient elution with 0.2–1.8 M sodium chloride aqueous solution at a flow rate of 1 mL / min. The eluent was collected in test tubes using an automatic collector. The polysaccharide content in each tube was determined at 490 nm using the phenylsulfuric acid colorimetric method until complete elution. Figure 1 As shown, the first peak is a neutral sugar. The elution fractions from 0.6M and 1.5M sodium chloride aqueous solutions were collected and named F1 and F2, respectively, with yields of 38.57% and 23.61%, respectively. They were then dialyzed, concentrated, and lyophilized.

[0055] 1.3.4.2 Oxidative degradation of fucoidan from Sargassum hemifolia

[0056] F1 and F2 were degraded using hydrogen peroxide and ascorbic acid. The raw material (0.3 g) was dissolved in distilled water to prepare a 15 g / L aqueous solution, which was then placed in a 30°C water bath. Ascorbic acid (0.2590 g) was added, and hydrogen peroxide (5%, 1 mL) was added while stirring. After stirring for 2 hours, the mixture was obtained by centrifugation. The supernatant was dialyzed, concentrated, and lyophilized. Finally, it was purified using a Separose CL-6B gel column (1.6 × 80 cm).

[0057] 1.3.4.3 Molecular weight determination

[0058] High-performance liquid chromatography (HPLC) was used with an Ultrahydrogel Column 500 (7.8 mm × 300 mm) and a 0.2 mol / L sodium sulfate aqueous solution as the mobile phase. The elution rate was 0.6 mL / min, and the column temperature was maintained at 35 °C. A differential detector (Agilent 1200, USA) was used, and the injection volume was 10 μL. Standard curves were prepared using 200,000, 100,000, 40,000, 20,000, 10,000, and 4,000 kDa dextran as standards, and the relative molecular mass and retention time of the polysaccharides were calculated based on the standard curves.

[0059] 1.3.5 Analysis of the antitumor mechanism of fucoidan from Sargassum hemifolia

[0060] 1.3.5.1 Flow cytometry detection of apoptosis

[0061] HepG2 cells in the logarithmic growth phase were used to prepare a cell suspension in culture medium, counted, and seeded into 6-well plates (6 × 10⁻⁶). 4 Cells / mL (2 mL per well). After culturing for 24 h, cells were cultured for another 48 h in DF1 and DF2 polysaccharide medium at concentrations of 1, 2, and 4 mg / mL, respectively. The supernatant was collected, washed twice with PBS, and the adherent cells were digested with trypsin. The mixture was then centrifuged (1000 rpm, 5 min), the supernatant was discarded, and 100 μL of 1× Binding Buffer, 5 μL of Annexin V-FITC, and 5 μL of PI were added. The mixture was incubated in the dark for 10 min, and then 400 μL of Binding Buffer was added. After mixing, the mixture was analyzed by flow cytometry.

[0062] 1.3.5.1 Determination of Caspase Enzyme Activity

[0063] The steps for detecting caspase activity are as follows: 1×10 5 2 mL of HepG2 cells were seeded per well in 6-well plates and pre-incubated for 24 h. Then, 1, 2, and 4 mg / mL of DF1 and DF2 were added for 48 h. The supernatant was collected, and the cells were washed twice with pre-cooled PBS. The adherent cells were then digested with trypsin and centrifuged (4℃, 2700 rpm, 5 min). Cells were then divided into two groups of 2 × 10⁻⁶ cells per well. 6Cell lysis buffer was added at a ratio of 100 μL per cell, incubated on ice for 15 min, and then centrifuged (4°C, 12000 rpm, 15 min). The supernatant was the protein extract. The protein concentration in the protein extract was determined using a Bradford protein assay kit, following the kit instructions. To detect caspase activity, 40 μL of assay buffer and 50 μL of protein were added to a 96-well plate, and the volume was brought up to 90 μL with lysis buffer. After mixing, 10 μL of the enzyme substrate Ac-DEVD-pNA (2 mM) was added. The 96-well plate was incubated overnight at 37°C. The A405 concentration was measured using a microplate reader, with the control group as 100%, and the relative caspase activity of the treatment group was calculated.

[0064] 1.3.5.2 Western blot detection of Caspase family protein activity expression

[0065] HepG2 cells in the logarithmic growth phase were counted at a ratio of 5 × 10⁻⁶. 5 HepG2 cells were seeded at a density of 5 mL cells / mL in 10 cm culture dishes and cultured for 24 h. Then, HepG2 cells were treated with 2 and 4 mg / mL DF1 and DF2 for 48 h. The supernatant was collected, and the supernatant and adherent cells were washed with pre-cooled PBS. Cells were then lysed with 50 μL RIPA cell lysis buffer (1% containing pmsf) and incubated at 4 °C for 30 min. Adherent cells were collected using a cell scraper, mixed, and centrifuged at 12000 rpm for 30 min. The supernatant was collected, and the protein content was determined using a BCA protein assay kit. An equal amount of protein sample was added to each well, and the mixture was subjected to 4%–20% SDS-PAGE electrophoresis. The sample was then transferred to a PVDF membrane, blocked with rapid blocking buffer for 15 min, washed with TBST, and incubated overnight at 4 °C with primary antibody. After washing with TBST, the membrane was incubated with secondary antibody at room temperature for 2 h. After washing, ECL luminescence solution was added, and the membrane was visualized using a chemiluminescence imaging system.

[0066] 1.3.5.3 Detection of intracellular ROS levels using the DCFH-DA probe method

[0067] Prepare a 10 μmol / L DCFH-DA solution using serum-free culture medium. HepG2 cells in logarithmic growth phase were digested, counted, and seeded into 96-well plates (2 × 10⁶ cells / well). 4 100 μL of DF1 and DF2 at concentrations of 1, 2, and 4 mg / mL were added to each well for treatment. After 48 h, the culture medium was removed, and the cells were washed once with serum-free culture medium. 30 μL of DCFH-DA solution was added to each well, and the cells were incubated at 37 °C for 20 min. The cells were then washed three times with PBS. Fluorescence intensity (excitation wavelength 488 nm, emission wavelength 525 nm) was measured, with the control group as 100%. The ROS content of the treatment groups was calculated.

[0068] 1.3.6 Data Statistics and Analysis

[0069] Each experiment was repeated three times, and experimental data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0 software for significance analysis, and the LSD method was used for multiple comparisons. The significance level was α = 0.05, and P < 0.05 was considered significant. Figures and tables were generated using Origin 2021 software.

[0070] 2 Results Analysis

[0071] 2.1 Chemical composition of Fh, Fb and SFPS

[0072] As shown in Table 1, Fh, Fb, and SFPS are all heteropolysaccharides containing sulfate groups and some uronic acid. Moreover, Fb has the highest total sugar content (75.33±3.99)%, and its L-fucose content (15.63±3.37)% and sulfate group content (29.16±0.86)% are both higher than those of Fh, while the uronic acid content of SFPS (16.96±2.85)% is higher than that of Fh and Fb.

[0073] Table 1 Chemical composition of Fh, Fb and SFPS

[0074]

[0075] Note: Different letters in the same column represent significant differences (P<0.05) between groups of the same nutrient component, n=3.

[0076] 2.2 Infrared spectra of Fh, Fb and SFPS

[0077] Depend on Figure 1It is evident that Fh, Fb, and SFPS exhibit absorption peaks at 3600–3200 nm (hydroxyl O-H stretching vibration) and weaker peaks at 3000–2800 nm (carbohydrate methyl C-H stretching vibration), indicating their polysaccharide content. Furthermore, the absorption peaks at 1740–1680 nm for Fh, Fb, and SFPS correspond to the asymmetric C=O stretching vibration of the carboxyl group, suggesting the presence of carboxyl groups and potentially a certain amount of uronic acid. The presence of C-H stretching and deformation vibrations at 1450–1420 nm and the S=O stretching vibration of the sulfate group at 1250–1200 nm indicates that they are all sulfated polysaccharides. A strong absorption peak at 833 nm represents both the characteristic absorption of α-glycosidic bonds and the characteristic absorption of C-O-S vibrations. A small peak at 906 nm represents the characteristic absorption of β-glycosidic bonds, but the absorption at this position is not significant. The results indicate that the glycosidic bonds of Fh, Fb, SFPS, and fucoidan are predominantly α-glycosidic bonds, with a small number of β-glycosidic bonds. The fucoidan standard shows an absorption peak at 850 nm, indicating that its sulfate group is attached at the C4 position in an axial bond position. In contrast, Fh, Fb, and SFPS show absorption peaks at 825 nm, 819 nm, and 817 nm, respectively, indicating that their sulfate groups are all attached at the C2 or C3 positions in a flat bond position.

[0078] 2.3 Effects of Fh, Fb and SFPS on the survival rate of HepG2 and LO2 cells

[0079] Depend on Figure 2 It was found that, compared with the blank control group, Fh, Fb, and SFPS all had inhibitory effects on human hepatocellular carcinoma HepG2 cells and normal human liver LO2 cells within the concentration range of 2–8 mg / mL. However, the inhibitory effect on HepG2 cells was stronger than that on LO2 cells, indicating that Fh, Fb, and SFPS have cell selectivity. Simultaneously, with the increase of the mass concentration of Fh, Fb, and SFPS, the cell survival rate of HepG2 cells showed a decreasing trend, indicating that Fh, Fb, and SFPS all had a good dose-response effect in inhibiting the growth of HepG2 cells. When the mass concentration reached 8 mg / mL, the survival rate (%) of HepG2 cells for Fh, Fb, and SFPS decreased from 100% in the control group to (27.21±3.69)%, (21.17±2.54)%, and (34.40±0.77)%, respectively, indicating that Fb had a stronger inhibitory effect on HepG2 cells than Fh and SFPS.

[0080] 2.4 Purification of Fb

[0081] Fb was purified by fractionation using cellulose anion exchange column chromatography, eluting sequentially with distilled water and 0.2–1.8 mol / L NaCl aqueous solutions. The elution curves are shown in the figure. Figure 3The second and third elution peaks were at 0.6 M and 1.5 M, respectively. These peaks were combined and named F1 and F2, with yields of 38.57% and 23.61%, respectively. The resulting elution peaks were then dialyzed, concentrated, and lyophilized. Their molecular weights were determined by high-performance liquid chromatography to be 32.966 kDa and 562.448 kDa, respectively (see...). Figure 4 ).

[0082] 2.5 Oxidative degradation of F1 and F2

[0083] F1 and F2 were degraded using hydrogen peroxide and ascorbic acid, and after purification, as follows: Figure 5 The resulting fractions were relatively simple, DF1 and DF2. Their molecular weights were determined by high-performance liquid chromatography to be 14.893 kDa and 21.292 kDa, respectively (see...). Figure 6 ).

[0084] 2.6 Analysis of the antitumor mechanism of fucoidan from Sargassum hemifolia

[0085] The MTT assay results showed that the survival rate of HepG2 tumor cells significantly decreased after treatment with Sargassum fucoidan (Fb). Studies have shown that inhibiting tumor cell proliferation is often achieved by inducing apoptosis and / or cell cycle arrest. During apoptosis, phosphatidylserine (PS) flips from the cell membrane and is exposed on the outer layer. Since PS has a high affinity for Annexin V, we used the Annexin V / PI double staining method to detect the apoptosis induced by DF1 and DF2 at different concentrations (1, 2, 4 mg / mL) after 48 hours of treatment in HepG2 cells. The experimental results are as follows. Figure 7 As shown. By Figure 7 It can be seen that the relative number of HepG2 cells induced by DF2 (46.25% ± 3.62) was significantly higher than that of DF1 (33.15% ± 1.84), and both DF1 and DF2 could induce significant apoptosis in HepG2 cells with increasing concentration.

[0086] 2.7 Determination of Caspase Enzyme Activity

[0087] The flow cytometry results showed that fucoidan from *Sargassum hemifolia* inhibits HepG2 cell proliferation by inducing apoptosis. Studies have shown that both the exogenous mitochondrial pathway and the endogenous death receptor pathway participate in and jointly regulate apoptosis. Caspase family proteins (a class of cysteine ​​proteases) play important roles in both pathways. Caspase 3 is the final executor of apoptosis in both pathways, while Caspase 8 and Caspase 9 are the initiators of the death receptor and mitochondrial pathways, respectively. We used fluorescence spectroscopy to detect the activity of the key proteins Caspase 3 / 8 / 9 in these two pathways. The experimental results are as follows: Figure 8 As shown in Figure A. Figure 8 As shown in Figure A, treatment of HepG2 cells with Sargassum fucoidan for 48 hours significantly increased the activity of Caspase-3 / 8 / 9 in a concentration-dependent manner, indicating that both the mitochondrial pathway and the death receptor pathway are involved in the Sargassum fucoidan-induced apoptosis process in HepG2 cells. To verify these conclusions, we further used Western blot to detect the protein expression levels of Caspase 3 / 8 / 9. The experimental results are as follows: Figure 8 As shown in B. Figure 8 As shown in Figure B, the protein bands of Caspase-3 / 8 / 9 were significantly lighter after treatment with Sargassum fucoidan, indicating that Caspase-3 / 8 / 9 were cleaved, and the activity of cleaved Caspase-3 / 8 / 9 was enhanced. These results suggest that the death receptor pathway and / or the exogenous mitochondrial pathway are both involved in the apoptosis induced by Sargassum fucoidan in HepG2 cells.

[0088] 2.8 Fucoidan from Sargassum hemifolia induces apoptosis through ROS generation.

[0089] Reactive oxygen species (ROS) are byproducts of aerobic metabolism in cells, including superoxide radicals (·O₂). 2- ROS (Reactive Oxides) include hydrogen peroxide (H₂O₂) and hydroxyl radicals (·OH). Some studies suggest that ROS are harmful substances that can induce oxidative damage to biomolecules, causing membrane lipid peroxidation, leading to the accumulation of lipid peroxides and damage to DNA and proteins, ultimately resulting in disease. On the other hand, some studies also suggest that ROS participate in maintaining intracellular homeostasis and act as second messengers in various signaling pathways. Previous research has also shown that at low concentrations, ROS can promote the growth of various cell types, including cancer cells; conversely, high concentrations of ROS can lead to oxidative stress, inhibit tumor cell proliferation, and induce tumor cell apoptosis. Figure 9A shows that the intracellular ROS fluorescence intensity increases with increasing DF1 and DF2 concentrations. Furthermore, as... Figure 9 As shown in Figure B, compared to the control group, the intracellular ROS content of HepG2 cells treated with DF1 and DF2 increased from 100% in the control group to 327% and 352%, respectively. These experimental results indicate that the intracellular ROS content of HepG2 cells treated with different concentrations of Sargassum fucoidan was significantly increased in a concentration-dependent manner. In conclusion, ROS may be the source of the apoptosis signaling pathway.

Claims

1. The application of fucoidan DF2 from Sargassum hemifolia in the preparation of anti-liver cancer drugs, characterized in that, The preparation method of the fucoidan DF2 from Sargassum fusiforme includes the following steps: S1. Sargassum hemifolia was pulverized and sieved, extracted with water, and then precipitated with alcohol after sonication and concentration to remove fat, removed with Sevage to remove protein, dialyzed and freeze-dried under vacuum to obtain Sargassum hemifolia fucoidan Fb. S2. Dissolve the fucoidan Fb from Sargassum fusiforme in distilled water to prepare a 0.1% (w / w) aqueous solution, pack it onto a DEAE-52 cellulose column, elute with distilled water first, then elute with a linear gradient of 0.2–1.8 M sodium chloride aqueous solution at a flow rate of 1 mL / min. Collect the eluent in test tubes using an automatic collector. Collect the elution fraction of the 1.5 M sodium chloride aqueous solution as F2; ​​then dialyze, concentrate, and lyophilize. S3. F2 was degraded using hydrogen peroxide and ascorbic acid, and the mixture was obtained by centrifugation. The supernatant was dialyzed, concentrated, and lyophilized. Finally, it was purified by Sepharose CL-6B gel column to obtain Sargassum fucosum fucoidan DF2. The degradation of F2 using hydrogen peroxide and ascorbic acid includes the following steps: F2 was prepared into a 15 g / L aqueous solution with distilled water and placed in a 30℃ constant temperature water bath. Based on the mass of F2 raw material used, 0.2590 g of ascorbic acid was added for every 0.3 g of F2 raw material. 1 mL of 5% hydrogen peroxide aqueous solution was added while stirring, and the reaction was stirred for 2 h.

2. The application according to claim 1, characterized in that, In the preparation method described, step S1 is as follows: Sargassum fusiforme is pulverized and passed through a 0.18 mm sieve. Water is added at a ratio of 1 g:30 mL, and the mixture is then extracted in an 80℃ constant temperature water bath for 3.5 h. The mixture is then sonicated at 60℃ and 350 W for 50 min, and concentrated by centrifugation at 4000 r / min for 20 min. Anhydrous ethanol is added to the concentrated solution, with a volume ratio of 7:

3. The mixture is thoroughly mixed, and after 24 h, the supernatant is obtained by centrifugation. Anhydrous ethanol is added again until the volume fraction of the supernatant is 80%, and the mixture is thoroughly mixed. After 24 h, the mixture is centrifuged again to obtain a precipitate. The precipitate is washed twice each with alternating acetone and anhydrous ethanol. A small amount of water is used to dissolve the precipitate into a polysaccharide solution. Sevage is added to the polysaccharide solution, with a volume ratio of 5:1, and the mixture is vigorously shaken for 1 minute. h, after standing in a separatory funnel to separate into layers, remove the precipitate; repeat the above steps 6 times until no denatured protein appears; the Sevage is obtained by mixing n-butanol and chloroform at a volume ratio of 1:4; dialyze for 48 h and freeze dry under vacuum to obtain Sargassum fucoidan Fb.

3. The application according to claim 1, characterized in that, In step S2 of the preparation method, the DEAE-52 cellulose column has a size of 2.6 × 50 cm, Cl - form.

4. The application according to claim 1, characterized in that, The Sepharose CL-6B gel column has a size of 1.6 × 80 cm.

5. The application according to claim 1, characterized in that, This study investigates the application of fucoidan DF2 from Sargassum hemifolia in the preparation of an anti-hepatocellular carcinoma drug that induces tumor cell apoptosis via the death receptor and mitochondrial pathways.

6. An anti-liver cancer drug, characterized in that, It contains the Sargassum fucoidan DF2 as described in claim 1 and a pharmaceutically acceptable carrier.

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