Sargassum fusiforme low molecular weight fucoidan and application thereof in resisting pm2.5 lung injury

Abstract

The application discloses a kind of Sargassum fusiforme low molecular weight fucoidan, and its preparation method is as follows: S1, defatting treatment;S2, preparation of crude polysaccharide;S3, degradation of crude polysaccharide: Sargassum fusiforme fucoidan crude product is dissolved into polysaccharide solution with water, H2O2 is added, and degradation is carried out under ultraviolet light, to obtain Sargassum fusiforme low molecular weight fucoidan DSFF;S4, purification: Sargassum fusiforme low molecular weight fucoidan DSFF is dissolved into polysaccharide solution with water, loaded on DEAE-52 cellulose anion exchange column and eluted with NaCl solution, the eluate is concentrated and dialyzed, and freeze-drying is obtained.The application of the Sargassum fusiforme low molecular weight fucoidan in the preparation of drugs with anti-PM2.5 lung injury effect.The Sargassum fusiforme low molecular weight fucoidan of the application has low molecular weight, is easy to absorb, and has high bioavailability.The research of the application lays a foundation for the development of Sargassum fusiforme fucoidan related lung alveolus protection products.

Description

Technical Field

[0001] This invention relates to a low molecular weight fucoidan from Sargassum fusiforme and its application in combating PM2.5 lung injury, belonging to the field of polysaccharide extraction technology. Background Technology

[0002] Sargassum fusiforme ( Sargassum fusiforme Sargassum fusiforme, also known as sea barley, deer antler tip, or longevity vegetable, belongs to the phylum Phaeophyta, order Fucus, and family Sargassum. It is a perennial warm-temperate algae. It is mainly distributed in the southeastern coastal areas of China, as well as Japan, North Korea, and South Korea. Sargassum fusiforme is a plant with both medicinal and edible uses, and is considered a marine vegetable and a health supplement for extending life expectancy.

[0003] Fucoidan, also known as fucoidan sulfate, is a complex sulfated polysaccharide. Its main component is fucoidan, and it also contains small amounts of neutral sugars (such as glucose, rhamnose, and galactose), uronic acid, and trace amounts of proteins and metal ions. Related studies have shown that fucoidan possesses significant pharmacological activities, including anti-tumor activity, cardiovascular protection, improvement of renal function, and antioxidant effects, demonstrating promising potential in disease treatment.

[0004] CN 120118213 A discloses a method for preparing low molecular weight fucoidan from Sargassum fusiforme that has an auxiliary protective effect against chemically induced liver injury. Three low molecular weight fucoidan were extracted from Sargassum fusiforme (molecular weights of 78.86 kDa, 64.95 kDa, and 46.37 kDa, respectively), and experimental studies demonstrated that the fucoidan with a molecular weight of 64.95 kDa has an auxiliary protective effect against chemically induced liver injury. However, the molecular weight of the prepared fucoidan is still not low enough, and its application in combating PM2.5 lung injury is not discussed. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a low molecular weight fucoidan from Sargassum fusiforme and its application in combating PM2.5 lung injury.

[0006] This invention is achieved through the following technical solution: A method for preparing low molecular weight fucoidan from Sargassum fusiforme includes the following steps: S1. Degreasing treatment: Take dried Sargassum fusiforme and pulverize it to obtain Sargassum fusiforme powder; extract the Sargassum fusiforme powder with ethanol, centrifuge, remove the supernatant, dry it, and obtain defatted Sargassum fusiforme powder. S2. Preparation of crude polysaccharide: Defatted Sargassum powder was added to water and extracted by ultrasonication; then cellulase was added for enzymatic hydrolysis; after enzymatic hydrolysis, calcium chloride or its solution was added, the mixture was allowed to stand, centrifuged, and anhydrous ethanol or ethanol solution was added to the supernatant to make the ethanol concentration 80%–85% (volume percentage), and the mixture was allowed to stand for alcohol precipitation; the precipitate was collected by centrifugation to obtain crude Sargassum fucoidan SFF. S3. Degradation of crude polysaccharide: Take the crude fucoidan from Sargassum fusiforme, dissolve it in water to form a polysaccharide solution; add H2O2 and degrade it under ultraviolet light to obtain Sargassum fusiforme low molecular weight fucoidan DSFF; S4. Purification: Take the low molecular weight fucoidan DSFF from Sargassum fusiforme, dissolve it in water to form a polysaccharide solution, load it onto a DEAE-52 cellulose anion exchange column and elute with NaCl solution. Collect the eluent obtained by elution with 0.8 mol / L NaCl solution; concentrate the eluent, dialyze it, and freeze-dry it to obtain the low molecular weight fucoidan DSFF2 from Sargassum fusiforme.

[0007] Furthermore, in step S1, the specific method of "taking dried Sargassum fusiforme and pulverizing it to obtain Sargassum fusiforme powder" is as follows: take dried Sargassum fusiforme, soak it in water for 12 hours, wash it 3 to 5 times, air dry it at room temperature, place it in an oven at 45°C to dry it, crush it using a high-speed pulverizer, and pass it through an 80-mesh sieve to obtain Sargassum fusiforme powder.

[0008] Further, in step S1, the specific method of "extracting Sargassum fusiforme powder with ethanol, centrifuging, removing supernatant, drying, and obtaining defatted Sargassum fusiforme powder" is as follows: Sargassum fusiforme powder is added to a 95% ethanol solution (volume percentage) at a material-to-liquid ratio of 1:50 (w / v, g:ml), and extracted three times at 40°C, with extraction times of 3 h, 2 h, and 1 h respectively; after extraction, it is centrifuged (4000 rpm, 20 min), the supernatant is removed, and the solids are dried in an oven at 45°C to obtain defatted Sargassum fusiforme powder.

[0009] Further, the specific method of step S2 is as follows: Add defatted Sargassum fusiforme powder to water at a material-to-liquid ratio of 1:70 (g:ml), and extract using ultrasonic parameters: ultrasonic time 49 min, ultrasonic power 225 W; adjust pH to 4.5 (adjusted with hydrochloric acid), add cellulase at a concentration of 1.6% of the weight of the defatted Sargassum fusiforme powder, and hydrolyze for 19 h; after hydrolysis, filter the hydrolysate, concentrate to 1 / 10 of the original volume, add calcium chloride solution to make the calcium chloride concentration 20 mg / mL, allow to stand, centrifuge (4000 rpm, 20 min), and collect the supernatant; add 4 times the volume of anhydrous ethanol, allow to stand overnight (12 h) at 4℃, centrifuge (4000 rpm, 20 min), reconstitute the solids, freeze-dry, and obtain crude Sargassum fusiforme fucoidan.

[0010] Further, step S3 is specifically performed as follows: Take crude fucoidan from Sargassum fusiforme, dissolve it in water to prepare a 5 mg / mL polysaccharide solution, add an equal volume of 300 mM H2O2 solution, and mix well; then perform ultraviolet treatment with the following parameters: wavelength 254 nm, irradiation power 7200 μW / cm². 2 Irradiation time was 90 min; after degradation, manganese dioxide was added to remove excess H2O2, centrifuged (8000 rpm, 20 min), the supernatant was collected, 4 times the volume of anhydrous ethanol was added, and the mixture was left to stand overnight (12 h) at 4℃, centrifuged (4000 rpm, 20 min), the solids were reconstituted, and freeze-dried to obtain Sargassum fusiforme low molecular weight fucoidan DSFF.

[0011] Further, step S4 is specifically performed as follows: Low molecular weight fucoidan DSFF from Sargassum fusiforme is dissolved in water to prepare a 12.5 mg / mL polysaccharide solution, which is then loaded onto a DEAE-52 cellulose anion exchange column. The column is eluted sequentially with pure water, 0.4 mol / L, 0.8 mol / L, 1.2 mol / L, 1.6 mol / L, and 2.0 mol / L sodium chloride solutions, with a flow rate controlled at 1.0 mL / min. The eluent is collected in 10 mL tubes, and the eluent eluted with 0.8 mol / L NaCl solution is also collected. The eluent is concentrated and then dialyzed for desalting. Finally, it is freeze-dried to obtain low molecular weight fucoidan DSFF2 from Sargassum fusiforme.

[0012] The low molecular weight fucoidan obtained by extracting and preparing Sargassum fusiforme using the above method has a low molecular weight (22.8 kDa) and has an auxiliary protective effect against PM2.5-induced alveolar damage. It can be used to prepare drugs with anti-PM2.5 lung damage effects.

[0013] The low molecular weight fucoidan from Sargassum fusiforme of this invention has low molecular weight, is easily absorbed, and has high bioavailability. In vitro cell experiments have shown that it can act as a regulator and protectant against oxidative stress and apoptosis, exerting protective properties against lung cells. Furthermore, in vivo zebrafish experiments have found that it can promote the excretion of particulate matter and reduce swim bladder damage. This research provides new ideas and references for the intensive processing and high-value utilization of Sargassum fusiforme, and lays the foundation for the development of Sargassum fusiforme fucoidan-related alveolar protective products.

[0014] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0015] Figure 1 DEAE-52 cellulose column chromatography elution curve of low molecular weight fucoidan from Sargassum fusiforme.

[0016] Figure 2Photograph of a low molecular weight fucoidan sample from Sargassum fusiforme.

[0017] Figure 3 A comparative schematic diagram showing the ABTS radical scavenging capacity (a), DPPH radical scavenging capacity (b), hydroxyl radical scavenging capacity (c), superoxide anion scavenging capacity (d), and ferric ion reducing capacity (e) of different Sargassum fusiforme polysaccharides. Fucoidan represents commercial fucoidan; the significance of differences is indicated by letter notation; the same applies below.

[0018] Figure 4 Schematic diagram of the effects of different concentrations of PM2.5 (a), DSFF2 (b), and FUC (c) on MH-S cell viability.

[0019] Figure 5 Schematic diagram of the effect of different concentrations of DSFF2 on the viability of MH-S cells exposed to PM2.5.

[0020] Figure 6 Schematic diagram of the effects of different concentrations of DSFF2 on ROS production (a), MDA content (b), SOD activity (c), CAT activity (d), and GSH-Px activity (e) in MH-S cells.

[0021] Figure 7 Schematic diagram of the effect of DSFF2 on PM2.5-induced apoptosis levels in MH-S cells.

[0022] Figure 8 Schematic diagram of Western blot bands for Caspase-3, Bax, and Bcl-2 proteins.

[0023] Figure 9 Schematic diagram of the effects of DSFF2 on Caspase-3 (a), Bax (b), Bcl-2 (c), Keap1, and Nrf2 (d) protein levels induced by PM2.5 in MH-S cells.

[0024] Figure 10 Schematic diagram of Western blot bands for Keap1, Nrf2, NQO1 and HO-1 proteins.

[0025] Figure 11 Schematic diagram of the effects of DSFF2 on the levels of Keap1 (a), Nrf2 (b), NQO1 (c), and HO-1 (d) proteins induced by PM2.5 in MH-S cells.

[0026] Figure 12Schematic diagram of the effects of different concentrations of PM2.5 (a), DSFF2 (b), and FUC (c) on the survival rate of zebrafish embryos.

[0027] Figure 13 Schematic diagram of the effect of DSFF2 on the survival rate of PM2.5-induced zebrafish embryos.

[0028] Figure 14 Morphological photographs of zebrafish at 24, 48 and 168 hpf after induction treatment with different concentrations of PM2.5.

[0029] Figure 15 Schematic diagram of the effect of different concentrations of PM2.5 on the swim bladder area of ​​zebrafish.

[0030] Figure 16 Schematic diagram of ROS fluorescence staining in zebrafish.

[0031] Figure 17 Schematic diagram of the effect of DSFF2 on ROS content in zebrafish induced by PM2.5.

[0032] Figure 18 Schematic diagram of fluorescent staining for zebrafish cell death.

[0033] Figure 19 Schematic diagram of the effect of DSFF2 on PM2.5-induced cell mortality in zebrafish.

[0034] Figure 20 Schematic diagram of fluorescent staining for lipid peroxidation in zebrafish.

[0035] Figure 21 Schematic diagram of the effect of DSFF2 on the production of lipid peroxides induced by PM2.5 in zebrafish.

[0036] Figure 22 Photograph of zebrafish swim bladder damage.

[0037] Figure 23 Schematic diagram of the effect of DSFF2 on PM2.5-induced swim bladder defects in zebrafish.

[0038] Figure 24 Photograph of fluorescent particles inside a zebrafish.

[0039] Figure 25 Schematic diagram of the effect of DSFF2 on particulate matter removal in zebrafish induced by PM2.5. Detailed Implementation

[0040] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0041] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0042] Example 1: Preparation of low molecular weight fucoidan from Sargassum fusiforme The steps are as follows: S1. Degreasing treatment: Take dried Sargassum fusiforme (harvested in Dongtou City, Wenzhou, Zhejiang Province), soak it in water for 12 hours, wash it 3 times, air dry it at room temperature, dry it in a 45℃ oven, crush it using a high-speed pulverizer, and pass it through an 80-mesh sieve to obtain Sargassum fusiforme powder; add 95% ethanol solution at a material-to-liquid ratio of 1:50 (g:ml), and extract it 3 times at 40℃, with extraction times of 3 hours, 2 hours, and 1 hour respectively; after extraction, centrifuge (4000 rpm, 20 min), remove the supernatant, and dry the solids in a 45℃ oven to obtain defatted Sargassum fusiforme powder.

[0043] S2. Preparation of crude polysaccharide: Pure water was added to the defatted Sargassum powder at a material-to-liquid ratio of 1:70 (g:ml), and ultrasonic extraction was performed at an ultrasonic power of 225 W for 49 min. After extraction, the pH was adjusted to 4.5 (adjusted with hydrochloric acid), and 1.6% cellulase was added for enzymatic hydrolysis for 19 h. After enzymatic hydrolysis, the hydrolysate was filtered, concentrated to 1 / 10 of the original volume, and calcium chloride solution was added to make the calcium chloride concentration 20 mg / ml. The mixture was allowed to stand overnight at 4℃ (12 h), centrifuged (4000 rpm, 20 min), and the supernatant was collected. Four times the volume of anhydrous ethanol was added, and the mixture was allowed to stand overnight at 4℃ (12 h), centrifuged (4000 rpm, 20 min), and the solid was collected. The solid was reconstituted to remove residual ethanol, and then freeze-dried to obtain crude Sargassum fucoidan SFF.

[0044] S3. Degradation of crude polysaccharide: The crude fucoidan SFF from Sargassum fusiforme was prepared into a 5 mg / mL polysaccharide solution with pure water. An equal volume of 300 mM H2O2 solution was added, mixed well, and irradiated under ultraviolet light at a wavelength of 254 nm and a power of 7200 μW / cm². 2Under the conditions of irradiation degradation for 90 min; after degradation, manganese dioxide was added to remove excess H2O2, centrifuged (8000 rpm, 20 min), the supernatant was collected, 4 volumes of anhydrous ethanol were added, and the mixture was left to stand overnight (12 h) at 4℃, centrifuged (4000 rpm, 20 min), the solids were reconstituted, and freeze-dried to obtain Sargassum fusiforme low molecular weight fucoidan DSFF.

[0045] S4. Purification: The above-mentioned low molecular weight fucoidan DSFF from Sargassum fusiforme was prepared into a 12.5 mg / mL polysaccharide solution using pure water and purified by DEAE-52 cellulose anion exchange column chromatography. The solution was eluted sequentially with pure water, 0.4 mol / L, 0.8 mol / L, 1.2 mol / L, 1.6 mol / L, and 2.0 mol / L sodium chloride solutions. The eluent was collected in 10 mL tubes. The distribution of the low molecular weight fucoidan from Sargassum fusiforme was detected by absorbance measurement at 490 nm using the phenol-sulfuric acid method. The results are as follows: Figure 1 As shown. Gradient elution with NaCl solution yielded three peak components (corresponding to eluents from 0.4 mol / L NaCl solution, 0.8 mol / L NaCl solution, and 1.2 mol / L NaCl solution, respectively). The eluents were concentrated and desalted by dialysis; after dialysis, they were lyophilized and temporarily named DSFF1, DSFF2, and DSFF3, respectively. Photos of the low molecular weight fucoidan samples from *Sargassum fusiforme* are shown below. Figure 2 As shown, the molecular weights of DSFF1, DSFF2, and DSFF3 are 12.5 kDa, 22.8 kDa, and 41.8 kDa, respectively. It can be seen that the molecular weights are extremely low, even lower than the low molecular weight fucoidan extracted from Sargassum fusiforme (78.86 kDa, 64.95 kDa, and 46.37 kDa, respectively) obtained in CN 120118213 A.

[0046] Experiment 1: Screening for the antioxidant activity of low molecular weight fucoidan from Sargassum fusiforme (a) Detection of antioxidant activity Each sample (SFF, DSFF1, DSFF2, DSFF3 prepared in Example 1, and commercial fucoidan) (the commercial fucoidan was purchased from Maclean's, trade number F889413, with a purity of 98%) was prepared into polysaccharide solutions of different concentrations (0.5, 1.0, and 2.0 mg / mL) and used as sample solutions for the following tests.

[0047] (1) Determination of ABTS free radical scavenging ability A 7 mM ABTS stock solution was prepared using 2.45 mM K₂S₂O₈ and reacted at room temperature in the dark for 16 h. The solution was diluted 30-fold before use. 200 μL of the sample solution was mixed with 800 μL of ABTS and reacted at room temperature in the dark for 10 min. The absorbance value (A) at 734 nm was measured. Ultrapure water was used instead of ABTS as sample blank (A0), ultrapure water was used instead of sample solution as blank (A1), and ultrapure water alone was used as control blank (A2).

[0048] ABTS free radical scavenging rate (%) = ×100%.

[0049] (2) Determination of DPPH free radical scavenging ability A 0.1 mg / mL DPPH working solution was prepared using methanol. 100 μL of the sample solution was mixed with 100 μL of the working solution, and the mixture was reacted in the dark for 30 min. The absorbance value A at 515 nm was then measured. Methanol was used instead of DPPH as the sample blank group A0, methanol was used instead of the sample as the control group A1, and methanol alone was used as the control blank group A2.

[0050] DPPH free radical scavenging rate (%) = [1-(A-A0) / (A1-A2)]×100%.

[0051] (3) Determination of hydroxyl radical scavenging ability Take 100 μL of sample, add 100 μL of FeSO4 solution (9 M) and 100 μL of salicylic acid-ethanol solution (9 M) sequentially, mix, then add 100 μL of H2O2 solution (9 M) and shake well. React at 37℃ in the dark for 30 min, and measure the absorbance value A at 510 nm. Use water instead of the sample as the blank group A0, and use water instead of H2O2 as the control group A1.

[0052] Hydroxyl radical scavenging rate (%) = [1-(A-A1) / A0]×100%.

[0053] (4) Determination of superoxide anion free radical scavenging ability Take 100 μL of sample solution and mix it with 4.5 mL of Tris-HCl buffer (50 mM, pH 8.2). React at 25 °C for 20 min, then quickly add 0.4 mL of pyrogallol solution (3 mM) and react precisely at 25 °C for 5 min. Finally, add 1 mL of hydrochloric acid (8 mM) to terminate the reaction. Measure the absorbance value A at 320 nm. Use water instead of the sample as the sample blank A0, and use water instead of the pyrogallol solution as the control group A1.

[0054] Superoxide anion radical scavenging rate (%) = [1-(A-A1) / A0]×100%.

[0055] (5) Determination of the reducing power of iron ions Prepare a 0.1 mg / mL Trolox standard solution. Take 900 μL of FRAP working solution and mix it with 100 μL of standard solutions of different concentration gradients. Incubate at 37℃ in the dark for 10 min and measure the absorbance value A at 593 nm. Plot a standard curve with the Trolox standard solution concentration on the x-axis and the absorbance value on the y-axis.

[0056] Take 900 μL of FRAP working solution, mix it with 100 μL of sample solution, and react at 37 °C in the dark for 10 min. Measure the absorbance value A at 593 nm. Substitute the absorbance value into the standard curve to calculate the corresponding Trolox equivalent.

[0057] (II) Results Comparison of ABTS free radical scavenging abilities of different Sargassum fucoidan species Figure 3 As shown in (a). Comparison of DPPH free radical scavenging abilities of different Sargassum fusiforme polysaccharides. Figure 3 As shown in (b). Comparison of the hydroxyl radical scavenging abilities of different Sargassum fusiforme polysaccharides. Figure 3 As shown in (c). Comparison of superoxide anion scavenging abilities of different Sargassum fusiforme polysaccharides. Figure 3 As shown in (d). Comparison of the iron ion reducing power of different Sargassum fusiforme polysaccharides. Figure 3 As shown in (e).

[0058] Depend on Figure 3 It was observed that the ABTS, DPPH, hydroxyl radical, superoxide anion, and ferric ion reducing abilities of various Sargassum fusiforme polysaccharides exhibited concentration-dependent scavenging capabilities. Among them, DSFF2 showed the strongest free radical scavenging effect at all tested concentrations. At a concentration of 2 mg / mL, its scavenging rates for ABTS, DPPH, hydroxyl radicals, and superoxide anion were 78.80±0.38%, 57.77±1.74%, 82.67±1.00%, and 55.60±1.25%, respectively. Furthermore, a consistent trend was observed in the ferric ion reducing ability experiment; the antioxidant capacity of DSFF2 at a concentration of 2 mg / mL was equivalent to 14.81±0.35 μg / mL Trolox, significantly superior to other Sargassum fusiforme polysaccharides.

[0059] In summary, DSFF2 exhibits the best antioxidant capacity, significantly superior to DSFF1, DSFF3, and commercial fucoidan. Therefore, DSFF2 was selected for subsequent experimental processing and analysis.

[0060] Experiment 2: In vitro cell experiments on low molecular weight fucoidan from Sargassum fusiforme (a) Detection of MH-S cell viability under PM2.5 and DSFF2 treatment using the MTT assay (1) Collect MH-S cells (a mouse alveolar macrophage cell line obtained by transformation with SV40 virus, which can be purchased from the market) at a density of 4 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated at 37°C for 24 h. The supernatant was discarded from each well, and three experimental groups were set up: One group was incubated with different concentrations of PM2.5 solution (prepared in serum-free medium with concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 μg / mL, filtered through a 0.22 μm membrane), and the cells were incubated for 24 h. The cell viability was then detected using the MTT assay.

[0061] One group was incubated with different concentrations of DSFF2 solution (prepared in serum-free medium at concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 μg / mL, filtered through a 0.22 μm membrane), and the cells were incubated for 24 h. Cell viability was then detected using the MTT assay.

[0062] One group was incubated with different concentrations of commercial fucoidan (FUC) solutions (same as in Experiment 1) (prepared in serum-free medium with concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 μg / mL, filtered through a 0.22 μm membrane), and the cells were incubated for 24 h. The cell viability was then detected using the MTT assay.

[0063] The method for measuring cell viability using the MTT assay is as follows: After incubation, add 50 μL of MTT solution (2 mg / mL) to each well and incubate at 37°C for 4 h; discard the supernatant, add 150 μL of DMSO, shake at room temperature for 30 min, and measure the absorbance at a wavelength of 490 nm to calculate the cell viability.

[0064] Cell viability (%) = Absorbance of sample group / Absorbance of blank group × 100%.

[0065] The PM2.5 used in this invention is provided and certified by the National Institute of Environmental Studies (NIES), and its composition is shown in Tables 1 and 2.

[0066] Table 1 Certified values ​​of PM2.5 components

[0067] AAS: Atomic Absorption Spectrometry; HG-AAS: Hydride Generation-Atomic Absorption Spectrometry; HG-ICP-AES: Hydride Generation-Inductively Coupled Plasma Atomic Emission Spectrometry; ICP-AES: Inductively Coupled Plasma Atomic Emission Spectrometry; ICP-MS: Inductively Coupled Plasma Mass Spectrometry; INAA: Instrumented Neutron Activation Analysis; PIXE: Proton-Induced X-ray Emission Spectrometry; XRF: X-ray Fluorescence Spectrometry.

[0068] Table 2 Reference values ​​for PM2.5 components

[0069] HG-AAS: Hydride generation-atomic absorption spectrometry; HG-ICP-AES: Hydride generation-inductively coupled plasma atomic emission spectrometry; HR-ICP-MS: High-resolution inductively coupled plasma mass spectrometry; ICP-AES: Inductively coupled plasma atomic emission spectrometry; ICP-MS: Inductively coupled plasma mass spectrometry; INAA: Instrumental neutron activation analysis; PIXE: Proton-induced X-ray emission spectrometry; XRF: X-ray fluorescence spectrometry.

[0070] The effects of different concentrations of PM2.5 treatment on MH-S cell viability, such as Figure 4 As shown in (a), the effects of different concentrations of DSFF2 treatment on MH-S cell viability are as follows: Figure 4 As shown in (b), the effects of different concentrations of FUC treatment on MH-S cell viability are as follows: Figure 4 As shown in (c), the survival rate of MH-S cells decreased significantly with increasing PM2.5 concentration (p < 0.05). After exposure to 200, 400, and 800 μg / mL PM2.5, the cell survival rates decreased to 53.24%, 41.43%, and 35.55%, respectively. In this study, MH-S cells were exposed to 200 μg / mL PM2.5 for 24 h to establish an in vitro lung injury model. Furthermore, when the DSFF2 concentration was within the range of 0–50 μg / mL, cell viability remained above 80%, indicating minimal cytotoxicity. The results of FUC were similar to those of DSFF2.

[0071] (2) Collect MH-S cells at a density of 4 × 10⁻⁶ cells per well. 4Cells were seeded at a density of 100 cells / mL in 96-well plates and incubated at 37°C for 24 h. The supernatant was discarded, and different concentrations of DSFF2 solution (prepared in serum-free medium at concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 μg / mL, filtered through a 0.22 μm membrane) were added for 2 h. Then, 200 μg / mL PM2.5 solution was added and incubated for 24 h. Cell viability was detected using the MTT assay.

[0072] The effects of different concentrations of DSFF2 on the viability of PM2.5-exposed MH-S cells, such as Figure 5 As shown, compared with the control group, the cell survival rate was significantly reduced after PM2.5 exposure. However, after DSFF2 pretreatment, cell survival rate increased in a dose-dependent manner within the range of 3.125–25 μg / mL, but decreased in a dose-dependent manner within the range of 50–800 μg / mL. This indicates that DSFF2 pretreatment can effectively inhibit the damaging effect of PM2.5 exposure on MH-S cells and improve cell survival rate, but excessively high concentrations can affect this protective effect. Therefore, in subsequent experimental treatments and analyses, the concentrations of DSFF2 were set at 3.125, 6.25, 12.5, and 25 μg / mL, and correspondingly, the concentrations of FUC as controls were set at 6.25, 12.5, and 25 μg / mL.

[0073] (II) Determination of ROS production in MH-S cells MH-S cells were treated with different concentrations of DSFF2 and FUC solutions, using the same method as in experiment (I) (2) above. After treatment, 50 μL of a 2 mg / mL DCFH-DA probe was added to each well, and the cells were incubated in the dark for 30 min. The absorbance was measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 516 nm.

[0074] The effect of DSFF2 on ROS production in MH-S cells is as follows: Figure 6 As shown in (a), PM2.5 exposure significantly increased ROS production in MH-S cells, while DSFF2 treatment significantly reduced ROS production in a dose-dependent manner, and was significantly better than FUC. This indicates that DSFF2 pretreatment can effectively inhibit ROS generation and reduce ROS-induced cell damage.

[0075] (III) Determination of MDA content and SOD, CAT and GSH-Px activities in MH-S cells (1) Collect MH-S cells at a density of 2.5 × 10⁶ cells per well. 5Cells were seeded at a density of 100 cells / mL in 6-well plates and pre-incubated in an incubator for 24 h. Then, different concentrations of DSFF2 solution and FUC solution were added for pretreatment for 2 h. Then, 200 μg / mL PM2.5 was added and incubated for 24 h. The cell supernatant was discarded, and the cells were washed twice with pre-cooled 1X PBS. 150 μL of cell lysis buffer was added to each well, and the cells were lysed on ice for 5 min. The cells were collected in centrifuge tubes and lysed on ice for 30 min. The supernatant was collected after centrifugation.

[0076] (2) Perform subsequent operations according to the instructions of the commercial reagent kit to determine the MDA content and the activities of SOD, CAT and GSH-Px.

[0077] Results: The effects of different concentrations of DSFF2 on MDA content in MH-S cells were as follows: Figure 6 As shown in (b), the effects of different concentrations of DSFF2 on SOD activity in MH-S cells are as follows: Figure 6 As shown in (c), the effects of different concentrations of DSFF2 on CAT activity in MH-S cells are as follows: Figure 6 As shown in (d), the effects of different concentrations of DSFF2 on GSH-Px activity in MH-S cells are as follows: Figure 6 As shown in (e), PM2.5 exposure increased MDA content and decreased SOD, CAT, and GSH-Px activities in MH-S cells. This reversal occurred after DSFF2 treatment. When the DSFF2 concentration was 25 μg / mL, the MDA content decreased to 165.74% of the control group, while the activities of SOD, CAT, and GSH-Px increased to 79.05%, 81.15%, and 80.06% of the control group, respectively. These results indicate that DSFF2 pretreatment effectively inhibited the formation of PM2.5-induced oxidative stress products and increased the activity of antioxidant enzymes, with significantly better effects than FUC.

[0078] (iv) Effects of DSFF2 on apoptosis in MH-S cells (1) In a 24-well plate with cell slides placed on it, use 1.5 × 10⁶ cells per well. 5 MH-S cells were seeded at a density of 10 cells / mL and pre-incubated in an incubator for 24 h. Then, different concentrations of DSFF2 solution and FUC solution (12.5 μg / mL) were added for pretreatment for 2 h, followed by incubation with 200 μg / mL PM2.5 for 24 h. The cell supernatant was discarded, and the cells were washed three times with 1X PBS. The cells were then stained at room temperature for 15 min with an anti-fluorescence quenching mounting medium containing Hoechst 33342 and PI. Cell morphology was observed and photographed under an inverted fluorescence microscope.

[0079] (2) Collect MH-S cells at a density of 2.5 × 10⁶ cells per well. 5Cells were seeded at a density of 100 cells / mL in 6-well plates and pre-incubated in an incubator for 24 h. Then, different concentrations of DSFF2 solution and FUC solution (12.5 μg / mL) were added for 2 h of pretreatment, followed by incubation with 200 μg / mL PM2.5 for 24 h. The cell supernatant was discarded, and the plates were washed three times with pre-cooled 1X PBS. 150 μL of cell lysis buffer was added to each well and the plates were lysed on ice for 5 min. The cells were collected in centrifuge tubes and lysed on ice for 30 min. After centrifugation, a portion of the supernatant was used to determine the protein content, and the other portion was added to 4X loading buffer and denatured at 95°C for 30 min. The plates were then stored at -20°C.

[0080] (3) Prepare SDS-PAGE gels according to the instructions. Add 20-30 μg of sample protein to each well and start electrophoresis, then transfer the gel to a membrane. After blocking at room temperature, incubate with primary and secondary antibodies to label the target protein. After mounting, develop the gel and analyze the grayscale values ​​using ImageJ software.

[0081] Results: The effect of DSFF2 on PM2.5-induced apoptosis levels in MH-S cells was as follows: Figure 7 As shown, after PM2.5 exposure, cells suffer severe oxidative damage, leading to increased apoptosis levels, cell membrane rupture, and the entry of PI dye into the cells, causing the cytoplasm to exhibit red fluorescence. After DSFF2 pretreatment, the PI-stained area was significantly reduced. When the DSFF2 concentration was 25 μg / mL, the PI staining effect was close to that of the control group, indicating that DSFF2 can inhibit PM2.5-induced apoptosis and has a good protective effect.

[0082] The effects of DSFF2 on the levels of Caspase-3, Bax, and Bcl-2 proteins induced by PM2.5 in MH-S cells are as follows: Figure 8 , Figure 9 As shown in (ac). Quantitative analysis of apoptosis levels revealed that PM2.5 exposure significantly increased the levels of Caspase-3 and Bax in MH-S cells compared to the control group (p < 0.05), increasing by 4.19-fold and 0.60-fold, respectively. However, after DSFF2 pretreatment, the levels of Caspase-3 and Bax proteins decreased in a dose-dependent manner with increasing DSFF2 concentration. When the DSFF2 concentration was 25 μg / mL, the levels of Caspase-3 and Bax proteins decreased to 0.51-fold and 0.65-fold, respectively, compared to the model group. Furthermore, while the level of Bcl-2 protein in cells decreased significantly after PM2.5 stimulation, after DSFF2 pretreatment, the Bcl-2 level in the sample group increased significantly with increasing sample concentration (p < 0.05). At a DSFF2 concentration of 25 μg / mL, the Bcl-2 level in the sample group reached 1.17-fold that of the PM2.5 group.

[0083] The above results indicate that PM2.5 stimulation can activate apoptosis in MH-S cells; DSFF2 treatment can downregulate the levels of Caspase-3 and Bax proteins and upregulate the level of Bcl-2 protein in cells, thus inhibiting apoptosis, with significantly better effects than FUC.

[0084] (V) Effects of DSFF2 on the Keap1 / Nrf2 signaling pathway in MH-S cells The method is the same as in experiment (IV) above.

[0085] The effects of DSFF2 on Keap1 and Nrf2 in PM2.5-induced MH-S cells are as follows: Figure 9 As shown in (d), Keap1 showed almost no green fluorescence in the control group, but after PM2.5 stimulation, the fluorescence intensity of Keap1 increased significantly, while the fluorescence intensity of Nrf2 decreased. Pretreatment with DSFF2 reversed this phenomenon. When the DSFF2 concentration was 25 μg / mL, the fluorescence levels of Keap1 and Nrf2 in the cells were close to those in the control group.

[0086] The effects of DSFF2 on the levels of Keap1, Nrf2, NQO1, and HO-1 proteins induced by PM2.5 in MH-S cells are as follows: Figure 10 , Figure 11 As shown in the figure, when MH-S cells were stimulated by PM2.5, the Keap1 protein content in the cells increased significantly compared with the control group, while the contents of Nrf2, NQO1, and HO-1 proteins decreased sharply, decreasing to 0.52, 0.19, and 0.45 times that of the control group, respectively. After DSFF2 pretreatment, the Keap1 protein level decreased in a dose-dependent manner (p < 0.05), while the Nrf2, NQO1, and HO-1 protein levels increased in a dose-dependent manner (p < 0.05). When the DSFF2 concentration was 25 μg / mL, the Keap1 protein level decreased to 0.64 times that of the control group, while the Nrf2, NQO1, and HO-1 levels increased to 1.44, 0.85, and 0.84 times, respectively.

[0087] The above results further demonstrate that DSFF2 can reduce Keap1 protein levels, thereby weakening Keap1-induced Nrf2 ubiquitination and degradation. Activated Nrf2 nuclei translocate into the nucleus and bind to ARE to form dimers, initiating the transcription of downstream antioxidant target proteins (NQO1, HO-1) and regulating the activity of antioxidant enzymes (SOD, CAT, and GSH-Px), thereby reducing ROS levels in vivo and inhibiting PM2.5-induced oxidative stress damage, thus playing a protective role for alveoli. Moreover, the effect is significantly better than FUC.

[0088] Experiment 3: In vitro zebrafish experiment with low molecular weight fucoidan from Sargassum fusiforme. (a) Screening of PM2.5, DSFF2 and FUC concentrations (1) Zebrafish embryos that were successfully fertilized and developed well, and were 4–5 h post-fertilization, were transferred into 12-well culture plates, with 15 embryos per well. After removing residual culture medium, the three experimental groups were exposed to different concentrations of PM2.5 solution (100, 200, 400, 600, and 800 μg / mL), DSFF2 solution (6.25, 12.5, 25, 50, 100, and 200 μg / mL), and FUC solution (6.25, 12.5, 25, 50, 100, and 200 μg / mL), respectively. After 24 h of treatment, the culture medium was replaced with fresh embryo culture medium, and the embryos were cultured continuously for 7 days under suitable conditions, with the survival rate recorded daily.

[0089] The effects of different concentrations of PM2.5, DSFF2, and FUC treatments on zebrafish survival rate are as follows: Figure 12 As shown in the figure, when the PM2.5 concentration reached 400 μg / mL, the survival rate of zebrafish decreased to 50% on day 7. Furthermore, with increasing PM2.5 concentration, the toxicity to zebrafish increased, and the survival rate decreased accordingly. Therefore, a PM2.5 concentration of 400 μg / mL was selected for subsequent experimental treatment and analysis. In addition, when the DSFF2 concentration was between 6.25 and 50 μg / mL, the survival rate of zebrafish was consistently above 90% after 7 days of continuous culture. However, when the concentration was 100 μg / mL, the survival rate of zebrafish dropped to 80% on day 7, and when the concentration increased to 200 μg / mL, the survival rate was only 60% on day 7. This indicates that DSFF2 concentrations exceeding 50 μg / mL have a certain toxic effect on zebrafish, and the toxicity gradually increases with increasing sample concentration and culture days. Therefore, DSFF2 solutions with concentrations between 12.5 and 50 μg / mL were selected for subsequent experiments. When the FUC concentration ranged from 12.5 to 50 μg / mL, the survival rate of zebrafish did not change significantly after 7 days of continuous culture. Therefore, a FUC solution with a concentration of 50 μg / mL was selected as a positive control for subsequent studies.

[0090] (2) According to different experimental groups, polysaccharide solutions of different concentrations were added and incubated for 2 h, and then a certain concentration of PM2.5 solution was added and the exposure was continued for 24 h. After that, fresh embryo culture medium was used, and the embryo survival rate of zebrafish was measured 3 days after fertilization.

[0091] The effect of DSFF2 on the survival rate of PM2.5-induced zebrafish embryos, such as Figure 13As shown, the survival rate of zebrafish embryos was significantly reduced after exposure to PM2.5 (p < 0.05), with the survival rate of the control group decreasing to 53.33% compared to the blank group. Pre-incubation with different concentrations of DSFF2 (12.5–50 μg / mL) significantly improved the embryo survival rate, reaching 80% at a concentration of 50 μg / mL. This indicates that low molecular weight fucoidan has a certain protective effect against oxidative damage to zebrafish embryos.

[0092] Morphological photographs of zebrafish at 24, 48, and 168 hpf after PM2.5 induction treatment at different concentrations are shown below. Figure 14 As shown, the effects of different concentrations of PM2.5 on the swim bladder area of ​​zebrafish are as follows: Figure 15 As shown, exposure to high concentrations of PM2.5 resulted in zebrafish exhibiting embryonic encapsulation by PM2.5, scoliosis, and incomplete or absent swim bladder aeration, with the likelihood of these conditions increasing with increasing PM2.5 concentration. These morphological damages in zebrafish demonstrate the toxic effects of PM2.5. Figure 3 Compared with the control group, the relative swim bladder area of ​​zebrafish was significantly reduced after PM2.5 exposure (p < 0.05). When the concentration was 800 μg / mL, the relative swim bladder area decreased to 7.27%, indicating the damaging effect of PM2.5 on the swim bladder.

[0093] (II) Effects of DSFF2 on ROS levels in PM2.5-induced zebrafish (1) Zebrafish embryos that were successfully fertilized and developed well were selected and transferred into 12-well cell culture plates at 4-5 h post-fertilization. Fifteen embryos were placed in each well. The culture medium was discarded, and different concentrations of DSFF2 were added for pretreatment for 2 h according to different experimental groups. Then, PM2.5 with a final concentration of 400 μg / mL was added and incubated for 24 h.

[0094] (2) To study the ROS generation in zebrafish stimulated by PM2.5, zebrafish juveniles were treated with PM2.5 for 3 days post-fertilization and then stained with DCFH-DA for 1 hour. After incubation, the zebrafish juveniles were washed with culture medium, anesthetized, and photographed individually under a fluorescence microscope. The fluorescence intensity of the images was quantified using ImageJ.

[0095] Zebrafish ROS fluorescent staining, such as Figure 16 As shown, the effect of DSFF2 on the ROS content in zebrafish induced by PM2.5 is as follows: Figure 17As shown in the figure, compared with the control group, PM2.5 exposure increased ROS production in zebrafish by 265.56%. DSFF2 at doses of 12.5 μg / mL and 25 μg / mL reduced ROS levels by 41.26% and 76.74%, respectively. When the DSFF2 concentration reached 50 μg / mL, the ROS level decreased to 126.67%, showing a significant improvement in ROS levels (p < 0.05). These results indicate that DSFF2 can effectively scavenge ROS and alleviate oxidative damage.

[0096] (III) Effects of DSFF2 on cell mortality in zebrafish exposed to PM2.5 (1) The method is the same as that of Experiment (II) above.

[0097] (2) To study the cell mortality rate of zebrafish stimulated by PM2.5, zebrafish larvae were stained with acridine orange for 30 min 3 days after fertilization and washed with culture medium. After anesthesia, individual zebrafish were photographed under a fluorescence microscope, and the fluorescence intensity of the images was quantified using ImageJ.

[0098] Zebrafish cell death fluorescent staining, such as Figure 18 As shown, the effect of DSFF2 on PM2.5-induced cell death in zebrafish is as follows: Figure 19 As shown in the figure, the cell mortality rate of zebrafish treated with PM2.5 increased to 285.85% compared with the control group. However, DSFF2 at concentrations of 12.5, 25, and 50 μg / mL reduced the mortality rate of zebrafish to 239.78%, 172.49%, and 120.16%, respectively. These results indicate that PM2.5 significantly reduced the survival rate of zebrafish, while DSFF2 can reduce the mortality rate of zebrafish treated with PM2.5 in a dose-dependent manner.

[0099] (iv) Effect of DSFF2 on the production of lipid peroxides induced by PM2.5 in zebrafish (1) The method is the same as that of Experiment (II) above.

[0100] (2) To study lipid peroxidation in zebrafish stimulated by PM2.5, zebrafish larvae were stained with DPPP for 3 h 3 days after fertilization. The zebrafish larvae were then washed with culture medium, anesthetized, and photographed individually under a fluorescence microscope. The fluorescence intensity of the images was quantified using ImageJ.

[0101] Zebrafish lipid peroxidation fluorescent staining, such as Figure 20 As shown, the effect of DSFF2 on the production of lipid peroxides induced by PM2.5 in zebrafish is as follows: Figure 21As shown in the figure, PM2.5 treatment significantly increased lipid peroxidation levels in zebrafish. However, treatment with DSFF2 at concentrations of 12.5, 25, and 50 μg / mL reduced lipid peroxidation levels in PM2.5-exposed zebrafish to 183.40%, 163.47%, and 133.28% of the control group, respectively. These results indicate that DSFF2 significantly inhibited PM2.5-stimulated lipid peroxidation in a dose-dependent manner.

[0102] The above results indicate that low molecular weight fucoidan from Sargassum fusiforme can effectively inhibit oxidative damage in zebrafish, exert antioxidant effects, and thus protect the alveoli.

[0103] (V) Effects of DSFF2 on PM2.5-induced swim bladder damage in zebrafish (1) The method is the same as that of Experiment (II) above.

[0104] (2) At 168 hpf, the juvenile fish were anesthetized with tricaine and fixed. Their development was observed and photographed under a stereomicroscope. The deformities were recorded, and the swim bladder defect rate was calculated. The swim bladder defect rate was D; the number of zebrafish with swim bladder defects and atrophy was P1; the number of zebrafish with incomplete swim bladder inflation was P2; and the total number of zebrafish was P.

[0105] D = (P1 + P2) / P x 100%.

[0106] Photos of zebrafish swim bladder damage, as shown Figure 22 The effect of DSFF2 on PM2.5-induced swim bladder defects in zebrafish is shown in the figure below. Figure 23 As shown in the figure, compared with the blank group (3.33%), the swim bladder defect rate in the model group (93.33%) was significantly increased (p < 0.05), and the zebrafish showed obvious incomplete swim bladder inflation. However, after DSFF2 pretreatment, the above situation was effectively improved. Furthermore, DSFF2 reduced the swim bladder damage rate in a dose-dependent manner; at a concentration of 50 μg / mL, the swim bladder damage rate decreased to 50.00%. These results indicate that low molecular weight fucoidan from Sargassum fusiforme can reduce swim bladder damage caused by PM2.5 exposure, thereby playing a protective role for the alveoli.

[0107] (vi) Effects of DSFF2 on PM2.5-induced particulate matter removal in zebrafish Zebrafish at 168 hpf were pretreated with different concentrations of DSFF2 for 2 h, then exposed to 400 μg / mL fluorescent microsphere solution and incubated in the dark for 2 h. After washing, they were cultured for another 6 h. Subsequently, they were anesthetized with tricaine and fixed, observed by laser confocal microscopy, and the in vivo fluorescence intensity was quantified using ImageJ software.

[0108] Photographs of fluorescent particles inside zebrafish, as shown Figure 24As shown, the effect of DSFF2 on particulate matter removal in PM2.5-induced zebrafish is as follows: Figure 25 As shown in the figure, compared with the blank control group, the zebrafish in the model group showed obvious red fluorescence in the intestine, indicating that fluorescently labeled particulate matter entered the zebrafish's body. After pretreatment with a low dose of DSFF2 (12.5 μg / mL), the fluorescence intensity decreased to 80.39%. When the concentration of DSFF2 reached 50 μg / mL, the fluorescence intensity decreased significantly (p < 0.05), lower than that of the positive control group (59.00%). Quantitative results showed that the fluorescence intensity under this condition was 49.47%, indicating that some particulate matter was excreted from the zebrafish's body. DSFF2 can promote the excretion of particulate matter to reduce the damaging effects of PM2.5, thereby playing a protective role for the alveoli.

[0109] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for preparing low molecular weight fucoidan from Sargassum fusiforme, characterized in that, Includes the following steps: S1. Degreasing treatment: Take dried Sargassum fusiforme and pulverize it to obtain Sargassum fusiforme powder; extract the Sargassum fusiforme powder with ethanol, centrifuge, remove the supernatant, dry it, and obtain defatted Sargassum fusiforme powder. S2. Preparation of crude polysaccharide: Defatted Sargassum powder was added to water and extracted by ultrasonication; then cellulase was added for enzymatic hydrolysis; after enzymatic hydrolysis, calcium chloride or its solution was added, and the mixture was allowed to stand and centrifuged. Anhydrous ethanol or ethanol solution was added to the supernatant to make the ethanol concentration 80% to 85%, and the mixture was allowed to stand for alcohol precipitation; after centrifugation, the precipitate was collected to obtain crude Sargassum fucoidan SFF. S3. Degradation of crude polysaccharide: Take the crude fucoidan from Sargassum fusiforme, dissolve it in water to form a polysaccharide solution; add H2O2 and degrade it under ultraviolet light to obtain Sargassum fusiforme low molecular weight fucoidan DSFF; S4. Purification: Take the low molecular weight fucoidan DSFF from Sargassum fusiforme, dissolve it in water to form a polysaccharide solution, load it onto a DEAE-52 cellulose anion exchange column and elute with NaCl solution. Collect the eluent obtained by elution with 0.8 mol / L NaCl solution; concentrate the eluent, dialyze it, and freeze-dry it to obtain the low molecular weight fucoidan DSFF2 from Sargassum fusiforme.

2. The method for preparing low molecular weight fucoidan from Sargassum fusiforme according to claim 1, characterized in that, In step S1, the specific method of "taking dried Sargassum fusiforme and crushing it to obtain Sargassum fusiforme powder" is as follows: take dried Sargassum fusiforme, soak it in water for 12 hours, wash it 3 to 5 times, air dry it at room temperature, place it in an oven at 45°C to dry it, crush it using a high-speed pulverizer, and pass it through an 80-mesh sieve to obtain Sargassum fusiforme powder.

3. The method for preparing low molecular weight fucoidan from Sargassum fusiforme according to claim 1, characterized in that, In step S1, the specific method for "extracting Sargassum fusiforme powder with ethanol, centrifuging, removing the supernatant, drying, and obtaining defatted Sargassum fusiforme powder" is as follows: Sargassum fusiforme powder is added to a 95% ethanol solution at a material-to-liquid ratio of 1:50, and extracted three times at 40°C, with extraction times of 3 h, 2 h, and 1 h respectively; after extraction, the mixture is centrifuged to remove the supernatant, and the solids are dried in an oven at 45°C to obtain defatted Sargassum fusiforme powder.

4. The method for preparing low molecular weight fucoidan from Sargassum fusiforme according to claim 1, characterized in that, The specific method of step S2 is as follows: Add defatted Sargassum fusiforme powder to water at a material-to-liquid ratio of 1:70, and extract using ultrasonic parameters: ultrasonic time 49 min, ultrasonic power 225 W; adjust the pH to 4.5, add cellulase at a concentration of 1.6% of the weight of the defatted Sargassum fusiforme powder, and hydrolyze for 19 h; after hydrolysis, filter the hydrolysate, concentrate it to 1 / 10 of the original volume, add calcium chloride solution to make the calcium chloride concentration 20 mg / mL, let stand, centrifuge, and take the supernatant; add 4 times the volume of anhydrous ethanol, let stand overnight at 4℃, centrifuge, reconstitute the solids, freeze-dry, and obtain crude Sargassum fusiforme fucoidan.

5. The method for preparing low molecular weight fucoidan from Sargassum fusiforme according to claim 1, characterized in that, The specific method of step S3 is as follows: Take crude fucoidan from Sargassum fusiforme, dissolve it in water to prepare a 5 mg / mL polysaccharide solution, add an equal volume of 300 mM H2O2 solution, and mix well; perform ultraviolet treatment with the following parameters: wavelength 254 nm, irradiation power 7200 μW / cm². 2 Irradiation time was 90 min; after degradation, manganese dioxide was added to remove excess H2O2, centrifuged, the supernatant was collected, 4 times the volume of anhydrous ethanol was added, and the mixture was left to stand overnight at 4℃, centrifuged, the solid was reconstituted, and freeze-dried to obtain Sargassum fusiforme low molecular weight fucoidan DSFF.

6. The method for preparing low molecular weight fucoidan from Sargassum fusiforme according to claim 1, characterized in that, The specific method of step S4 is as follows: Take Sargassum fusiforme low molecular weight fucoidan DSFF, dissolve it in water to prepare a 12.5 mg / mL polysaccharide solution, load it onto a DEAE-52 cellulose anion exchange column, and elute sequentially with pure water, 0.4 mol / L, 0.8 mol / L, 1.2 mol / L, 1.6 mol / L, and 2.0 mol / L sodium chloride solutions, controlling the flow rate at 1.0 mL / min, and collect the eluent at 10 mL / tube. Collect the eluent eluted with 0.8 mol / L NaCl solution; concentrate the eluent and dialyze to desalt; freeze-dry to obtain Sargassum fusiforme low molecular weight fucoidan DSFF2.

7. The low molecular weight fucoidan from Sargassum fusiforme prepared by the method described in any one of claims 1 to 6.

8. The Sargassum fusiforme low molecular weight fucoidan according to claim 7, characterized in that: The molecular weight of the low molecular weight fucoidan from Sargassum fusiforme is 22.8 kDa.

9. The use of the Sargassum fusiforme low molecular weight fucoidan according to claim 7 or 8 in the preparation of a drug that has an auxiliary protective effect against PM2.5-induced alveolar damage.

10. The use of the Sargassum fusiforme low molecular weight fucoidan according to claim 7 or 8 in the preparation of a drug with anti-PM2.5 lung injury effect.

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