Preparation method of sargassum fusiforme low-molecular-weight fucoidin with auxiliary protection effect on chemical liver injury

Abstract

The invention discloses a preparation method of sargassum fusiforme low-molecular-weight fucoidin with an auxiliary protection effect on chemical liver injury. The preparation method comprises the following steps: S1, degreasing treatment; s2, preparing crude polysaccharide; s3, degradation of crude polysaccharide: dissolving the sargassum fucoidin crude product with water to obtain a polysaccharide solution, and performing ultrasonic treatment; then, H2O2 is added for degradation, and the sargassum fusiforme low-molecular-weight fucoidin DSFF is obtained; and S4, purification: dissolving the sargassum fusiforme low-molecular-weight fucoidin DSFF in water to obtain a polysaccharide solution, loading the polysaccharide solution on a DEAE-52 cellulose anion exchange column, eluting with a NaCl-phosphate buffer solution, dialyzing and desalting the eluent, and freeze-drying to obtain the sargassum fusiforme low-molecular-weight fucoidin DSFF2. The sargassum fusiforme low-molecular-weight fucoidin prepared by the method can be used for preparing medicines with an auxiliary protection effect on chemical liver injury. The research of the invention lays a foundation for the development of liver protection products related to sargassum fucoidin.

Description

Technical Field

[0001] The invention relates to a method for preparing low-molecular-weight fucoidan from Sargassum fusiformis which has an auxiliary protective effect on chemical liver damage, and belongs to the technical field of polysaccharide extraction. Background Art

[0002] Sargassum fusiforme, also known as antler tip, seaweed sprout, barley, etc., is a brown algae belonging to the class Pilostomata, the order Fucosales, and the family Sargassum. It is widely distributed in the northwest Pacific Ocean and is found in my country, South Korea, and Japan. Sargassum fusiforme is thick and juicy, rich in nutrition, and has a good flavor. It can be eaten and used as medicine. It has high nutritional and medicinal value. It is a traditional delicacy and Chinese herbal medicine for the coastal people of my country, Japan, and South Korea, and is highly regarded as a marine vegetable and a health and longevity product.

[0003] Fucoidan, also known as fucoidan, fucoidan sulfate, fucoidan, brown algae polysaccharide, etc., is mainly derived from brown algae and is a type of polysaccharide containing fucose and sulfate groups. Fucoidan has a variety of biological functions, such as anti-coagulation, anti-tumor, anti-thrombosis, anti-virus, antioxidant and enhancing the body's immune function. In recent years, the structure, biological function and relationship between structure and function of fucoidan have attracted the attention of more and more experts and scholars. Relevant studies have shown that the biological activity of fucoidan is correlated with its molecular weight. The smaller the molecular weight, the better its biological activity. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a method for preparing low-molecular-weight fucoidan from Sargassum fusiformis which has an auxiliary protective effect on chemical liver damage.

[0005] The present invention is achieved through the following technical solutions: A method for preparing low molecular weight fucoidan from Sargassum fusiformis having an auxiliary protective effect on chemical liver damage comprises the following steps: S1. Defatting treatment: taking dried Sargassum fusiformis and crushing it to obtain Sargassum fusiformis powder; extracting the Sargassum fusiformis powder with ethanol for 1 to 2 times, centrifuging, removing the supernatant, and drying to obtain Sargassum fusiformis defatted powder; S2. Preparation of crude polysaccharide: adding defatted powder of Sargassum fusiformis to water, and performing microwave extraction; then adding cellulase for enzymolysis; after enzymolysis, adding calcium chloride solution, standing, centrifuging, adding Sevag reagent to the supernatant for deproteinization; centrifuging, adding anhydrous ethanol or ethanol solution to the supernatant to make the ethanol concentration 80% to 85% (volume percentage), standing for alcohol precipitation; centrifuging, collecting the precipitate, which is the crude Sargassum fusiformis fucoidan; the Sevag reagent is composed of chloroform and n-butanol, and the volume ratio of the two is 5:1; S3. Degradation of crude polysaccharides: Take crude Sargassum fusiformis polysaccharide, dissolve it in water to form a polysaccharide solution, and treat it with ultrasound; then, add H 2 O 2 Degradation was performed to obtain low molecular weight fucoidan DSFF from Sargassum fusiformis; S4. Purification: Take the low molecular weight fucoidan DSFF from Sargassum fusiformis, dissolve it in water to form a polysaccharide solution, load it on a DEAE-52 cellulose anion exchange column and elute it with NaCl-phosphate buffer, collect the eluate eluted with 1.0 mol / L NaCl-phosphate buffer; dialyze the eluate for desalination and lyophilize it to obtain the low molecular weight fucoidan DSFF2 from Sargassum fusiformis.

[0006] Furthermore, in the step S1, the specific method of "taking dried Sargassum fusiformis and crushing it to obtain Sargassum fusiformis powder" is: taking dried Sargassum fusiformis, soaking it in water for 12 hours, washing it 3 times, drying it at room temperature, placing it in an oven at 45°C, crushing it with a high-speed grinder, and passing it through a 60-mesh sieve to obtain Sargassum fusiformis powder.

[0007] Furthermore, in the step S1, the specific method of "extracting the Sargassum powder with ethanol for 1 to 2 times, centrifuging, removing the supernatant, and drying to obtain the Sargassum defatted powder" is: adding the Sargassum powder to a 95% ethanol solution (volume percentage), with a solid-liquid ratio of 1:20 (g:ml), and extracting it twice by reflux at 85°C using a condensing reflux device, each time for 2 hours; centrifuging after extraction (3800 rpm, 20min), removing the supernatant, and placing the solid in an oven at 45°C to dry, to obtain the Sargassum defatted powder.

[0008] Furthermore, the specific method of step S2 is: adding the defatted powder of Sargassum fusiformis to water, with a solid-liquid ratio of 1:80 (g:ml), microwave extraction, and the parameters are: temperature 70°C, power 450 W, time 50 min; adjusting the pH to 4.5 (hydrochloric acid adjustment), adding cellulase (the added amount is 1.0% of the weight of the defatted powder of Sargassum fusiformis), and enzymolysis for 3 h; centrifugation (3800 rpm, 20 min), removing solids, adding calcium chloride solution to the supernatant to make the final concentration of calcium chloride 20 mg / ml, standing at 4°C for 12 h, centrifuging (3800 rpm, 20 min), taking the supernatant, adding Sevag reagent, shaking thoroughly for 30 min, centrifuging (8000 rpm, 20 min), taking the supernatant, adding 4 times the volume of anhydrous ethanol, standing at 4°C for 12 h, centrifuging (3800 rpm, 20 min), re-dissolving the solids, and freeze-drying to obtain the crude Sargassum fusiformis fucoidan.

[0009] Furthermore, the specific method of step S3 is as follows: taking crude Sargassum fusiformis polysaccharide, dissolving it in water to prepare a 5 mg / mL polysaccharide solution, and ultrasonically treating it at the following parameters: temperature 25°C, ultrasonic frequency 40 kHz, time 2 h; then, adding an equal volume of 1 mol / L H 2 O 2 solution, and degraded for 2 h; after degradation, manganese dioxide was added to remove excess H 2 O 2 , centrifuge (8000 rpm, 20 min), take the supernatant, add 4 times the volume of anhydrous ethanol, let stand at 4°C for 12 h, centrifuge (3800 rpm, 20 min), re-dissolve the solid, and freeze-dry to obtain the low molecular weight fucoidan DSFF from Sargassum fusiformis.

[0010] Furthermore, the specific method of step S4 is: take the low molecular weight fucoidan DSFF from Sargassum fusiformis, dissolve it in water to prepare a 5 mg / mL polysaccharide solution, load it on a DEAE-52 cellulose anion exchange column, and elute it with phosphate buffer, 0.5 mol / L, 1.0 mol / L, 1.5 mol / L, 2.0 mol / L, and 2.5 mol / L sodium chloride-phosphate buffer solution in sequence, and collect the eluate eluted with 1.0 mol / L NaCl-phosphate buffer; the eluate is placed in a 500 kDa dialysis bag for dialyzed desalination, the water is changed every 4 hours, and the dialysis is performed for 3 days; after dialysis, it is freeze-dried to obtain the low molecular weight fucoidan DSFF2 from Sargassum fusiformis.

[0011] The low molecular weight fucoidan of Sargassum fusiformis prepared by the above method has a low molecular weight (64.95 kDa) and has an auxiliary protective effect on chemical liver damage, and can be used to prepare drugs with an auxiliary protective effect on chemical liver damage.

[0012] The low molecular weight fucoidan of Sargassum fusiformis of the present invention has low molecular weight, is easy to absorb, and has high bioavailability. In vitro cell experimental studies have shown that it can be used as a regulator and protector of oxidative stress and cell apoptosis, and exerts liver cell protective properties. The research of the present invention provides new ideas and references for the deep processing and high-value utilization of Sargassum fusiformis, and lays a foundation for the development of liver protection products related to Sargassum fusiformis fucoidan.

[0013] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Schematic diagram of the absorbance detection results of low molecular weight Fucus polysaccharides from Sargassum fusiformis.

[0015] Figure 2: Photos of DSFF1, DSFF2, and DSFF3.

[0016] Figure 3 : Schematic diagram comparing the hydroxyl radical scavenging abilities of different Sargassum fusiformis fucoidans.

[0017] Figure 4 : Schematic diagram comparing the DPPH radical scavenging abilities of different Sargassum fusiformis fucoidans.

[0018] Figure 5 : Schematic diagram comparing the superoxide anion radical scavenging abilities of different Sargassum fucoidans.

[0019] Figure 6 : Schematic diagram comparing the iron ion reducing capacity of different Sargassum fucoidans.

[0020] Figure 7 : Schematic diagram of the effect of different concentrations of DSFF2 on the survival rate of BRL 3A cells, where the significance of the difference is indicated by letters, the same below.

[0021] Figure 8 :Schematic diagram of the effect of different concentrations of Vc on the survival rate of BRL 3A cells.

[0022] Fig. 9 : Different concentrations of H 2 O 2 Schematic diagram of the effect on the survival rate of BRL 3A cells.

[0023] Fig.10 :Effects of different concentrations of DSFF2 pretreatment on H 2 O 2 Schematic diagram of the effect of induced injury on the survival rate of BRL 3A cells.

[0024] Fig.11 : Schematic diagram of the effect of DSFF2 on ROS production in BRL 3A cells.

[0025] Fig.12 :DSFF2 vs H 2 O 2 Schematic diagram of the effect of induced MDA content in BRL 3A cells.

[0026] Fig.13 :DSFF2 vs H 2 O 2 Schematic representation of the effect of induced SOD activity in BRL 3A cells.

[0027] Fig.14 :DSFF2 vs H 2 O 2Schematic representation of the effect of induced CAT activity in BRL 3A cells.

[0028] Fig.15 :DSFF2 vs H 2 O 2 Schematic representation of the effect of induced GSH-Px activity in BRL 3A cells.

[0029] Fig.16 :DSFF2 vs H 2 O 2 Schematic diagram of the effect of induced apoptosis levels on BRL 3A cells.

[0030] Fig.17 : Schematic diagram of Western blot bands of Caspase-3, Bax, and Bcl-2 proteins.

[0031] Fig.18 :DSFF2 vs H 2 O 2 Schematic diagram of the effect of induced Caspase-3 protein levels in BRL 3A cells.

[0032] Fig.19 :DSFF2 vs H 2 O 2 Schematic diagram of the effect of induced Bax protein levels in BRL 3A cells.

[0033] Fig. 20 :DSFF2 vs H 2 O 2 Schematic representation of the effect of induced Bcl-2 protein levels in BRL 3A cells.

[0034] Fig.21 :DSFF2 vs H 2 O 2 Schematic representation of the effects of Keap1 and Nrf2 in induced BRL 3A cells.

[0035] Fig. 22 : Schematic diagram of Western blot bands of Keap1, Nrf2, NQO1 and HO-1 proteins.

[0036] Fig.23 :DSFF2 vs H 2 O 2 Schematic representation of the effect of induced Keap1 protein levels in BRL 3A cells.

[0037] Fig.24 :DSFF2 vs H 2 O 2 Schematic representation of the effect of induced Nrf2 protein levels in BRL 3A cells.

[0038] Fig.25 :DSFF2 vs H 2 O 2 Schematic representation of the effect of induced NQO1 protein levels in BRL 3A cells.

[0039] Fig.26 :DSFF2 vs H 2 O 2 Schematic representation of the effect of induced HO-1 protein levels in BRL 3A cells.

[0040] Fig. 27 :Schematic diagram of the effect of different concentrations of DSFF2 on the survival rate of zebrafish embryos.

[0041] Fig.28 :DSFF2 vs H 2 O 2 Schematic diagram of the effects of induction on the survival rate of zebrafish embryos.

[0042] Fig.29 : Schematic diagram of ROS fluorescence staining in zebrafish.

[0043] Fig.30 :DSFF2 vs H 2 O 2 Schematic diagram of the effects of induced ROS content in zebrafish.

[0044] Fig.31 : Schematic diagram of fluorescent staining of zebrafish cell death.

[0045] Fig.32 :DSFF2 vs H 2 O 2 Schematic representation of the effects of induced cell death in zebrafish.

[0046] Fig.33 : Schematic diagram of zebrafish lipid peroxidation fluorescence staining.

[0047] Fig.34 :DSFF2 vs H 2 O 2 Schematic diagram of the effects of induced lipid peroxide production in zebrafish. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0049] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0050] Example 1 Preparation of low molecular weight fucoidan from Sargassum fusiformis Here are the steps: S1. Defatting: Soak dried Sargassum fusiformis (harvested in Dongtou City, Wenzhou, Zhejiang) for 12 h, wash 3 times, dry at room temperature, dry in a 45°C oven, crush with a high-speed grinder, pass through a 60-mesh sieve to obtain Sargassum fusiformis powder, add 95% ethanol at a solid-liquid ratio of 1:20 (w / v), reflux twice at 85°C using a condensation reflux device, each time for 2 h, centrifuge after defatting (3800 rpm, 20 min), remove the supernatant, and dry the solid in a 45°C oven to obtain the defatted Sargassum fusiformis powder.

[0051] S2. Preparation of crude polysaccharide: pure water was added to the defatted powder of Sargassum fusiformis at a solid-liquid ratio of 1:80 (w / v), and the mixture was extracted for 50 min at a microwave temperature of 70°C and a microwave power of 450 W, and the pH was adjusted to 4.5 (adjusted by hydrochloric acid), and 1.0% cellulase was added for extraction for 3 h. After the enzymatic hydrolysis, the mixture was centrifuged (3800 rpm, 20 min) to remove the solids, and calcium chloride solution was added to the supernatant to make the concentration of calcium chloride 20 mg / ml, and the mixture was allowed to stand at 4°C for 12 h, centrifuged (3800 rpm, 20 min), the supernatant was taken, chloroform-n-butanol mixed solution was added to remove protein (shake well for 30 min), centrifuged (8000 rpm, 20 min), the supernatant was taken, 4 times the volume of anhydrous ethanol was added, the mixture was allowed to stand at 4°C for 12 h, centrifuged (3800 rpm, 20 min), the solids were taken for re-dissolution, and freeze-dried to obtain the crude Sargassum fusiformis polysaccharide SFF.

[0052] S3. Degradation of crude polysaccharides: The crude Sargassum fusiformis polysaccharide was prepared with pure water to form a 5 mg / mL polysaccharide solution, which was degraded at 25°C and an ultrasonic frequency of 40 kHz for 2 h. An equal volume of 1 mol / L H 2 O 2 Solution, degradation for 2 h, MnO2 removes excess H 2 O 2 , centrifuge (8000 rpm, 20 min), take the supernatant, add 4 times the volume of anhydrous ethanol, let stand at 4°C for 12 h, centrifuge (3800 rpm, 20 min), re-dissolve the solid, and freeze-dry to obtain the low molecular weight fucoidan DSFF from Sargassum fusiformis.

[0053] S4. Purification: The above-mentioned low molecular weight fucoidan from Sargassum fusiformis was prepared into a 5 mg / mL polysaccharide solution with pure water, and the obtained polysaccharide solution was purified by DEAE-52 cellulose anion exchange column chromatography, and eluted with phosphate buffer, 0.5 mol / L, 1.0 mol / L, 1.5 mol / L, 2.0 mol / L, and 2.5 mol / L NaCl-phosphate buffer solution in sequence, and the distribution of low molecular weight fucoidan from Sargassum fusiformis was detected by measuring the absorbance at a wavelength of 490 nm using the phenol-sulfuric acid method. The results are as follows: Figure 1 As shown. The eluates after elution with 0.5 mol / L, 1.0 mol / L, and 1.5 mol / L NaCl-phosphate buffer were collected, and the same polysaccharide components were combined and dialyzed with ultrapure water (500 kDa). The water was changed every 4 hours for 3 days. The dialyzed samples were freeze-dried to obtain three types of low molecular weight fucoidan from Sargassum fusiformis, which were named DSFF1, DSFF2, and DSFF3. The photos of each low molecular weight fucoidan sample from Sargassum fusiformis are shown in the figure. Figure 2 As shown. Among them, the molecular weights of DSFF1, DSFF2, and DSFF3 are 78.86 kDa, 64.95 kDa, and 46.37 kDa, respectively. It can be seen that the molecular weight of the fucoidan prepared by the present invention is extremely low, which is significantly better than the prior art. For example, the molecular weight of the fucoidan used in the literature "Protective Effect and Mechanism of Fucoidan on Liver Damage in Alcohol-Exposed Mice" (Yang Jia et al., Food Science, Vol. 44, No. 137-145, 2023) is 675.6 kDa.

[0054] Experiment 1 Screening of Antioxidant Activity of Low Molecular Weight Fucoidan from Sargassum fusiformis (1) Determination of hydroxyl radical scavenging ability Each sample (SFF, DSFF1, DSFF2, DSFF3 prepared in Example 1; the same below) was prepared into polysaccharide solutions of different concentrations (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg / mL). Take 100 μL of sample and add 100 μL of FeSO 4 solution (9 M), 100 μL salicylic acid-ethanol solution (9 M), and after mixing, 100 μL H 2 O 2 The solution (9 M) was shaken and reacted at 37°C for 30 min, and the absorbance value A at 510 nm was measured. Vitamin C (Vc) was used as the positive control, and water was used instead of the sample as the blank group A. 0 , replace H with water 2 O 2 As control group A 1 .

[0055] Hydroxyl free radical scavenging rate (%) = [1-(AA 1 ) / A 0 ]×100%.

[0056] (2) Determination of DPPH free radical scavenging ability Each sample was prepared into polysaccharide solutions of different concentrations (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg / mL), and 0.1 mg / mL DPPH working solution was prepared using methanol. 100 μL of sample solution was mixed with 100 μL of working solution, reacted in the dark for 30 min, and the absorbance value A at 515 nm was detected. Vc was used as the positive control, and methanol was used instead of DPPH as the sample blank group A. 0 , methanol replaces the sample as control group A 1 , methanol alone was used as the control blank group A 2 .

[0057] DPPH free radical scavenging rate (%) = [1-(AA 0 ) / (A 1 -A 2 )]×100%.

[0058] (3) Determination of superoxide anion radical scavenging ability Prepare each sample into polysaccharide solution of different concentrations (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg / mL). Take 100 μL of sample, mix with 4.5 mL Tris-HCl buffer (50 mM, pH 8.2), react at 25 °C for 20 min, then quickly add 0.4 mL pyrogallol solution (3 mM), react at 25 °C for 5 min. Finally, add 1 mL hydrochloric acid (8 mM) to terminate the reaction. Determine the absorbance value A at 320 nm. Use Vc as the positive control and use water instead of the sample as the sample blank group A. 0 , water was used instead of pyrogallol solution as control group A 1 .

[0059] Superoxide anion free radical scavenging rate (%) = [1-(AA 1 ) / A 0 ]×100%.

[0060] (4) Determination of iron ion reducing ability Prepare 0.1 mg / mL Trolox standard solution, take 900 μL FRAP working solution, mix with 100 μL standard solution of different concentration gradients, react at 37℃ in the dark for 10 min, and measure the absorbance value A at 593 nm. Draw a standard curve, the horizontal axis is the concentration of Trolox standard solution, and the vertical axis is the absorbance value. Prepare each sample into polysaccharide solution of different concentrations (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg / mL), take 900 μL FRAP working solution, mix with 100 μL sample solution, react at 37℃ in the dark for 10 min, and measure the absorbance value A at 593 nm. Substitute the absorbance value into the standard curve to calculate the corresponding Trolox equivalent.

[0061] Results: Comparison of the hydroxyl radical scavenging ability of different Sargassum fucoidan Figure 3 As shown in Figure 2, the DPPH free radical scavenging ability of different Sargassum fucoidan is compared. Figure 4 As shown in Figure 2, the superoxide anion radical scavenging ability of different Sargassum fucoidan is compared. Figure 5 As shown in the figure, the comparison of the iron ion reducing capacity of different Sargassum fucoidan is as follows Figure 6 As shown. Figure 3 It can be seen that in the hydroxyl free radical scavenging experiment, the scavenging ability of DSFF1 and DSFF2 for hydroxyl free radicals is not much different, and the scavenging ability of DSFF3 for hydroxyl free radicals is poor. 50 The values ​​were 0.33, 0.32 and 0.45 mg / mL, respectively. The IC 50 The value was 6.54 mg / mL, and the scavenging ability of the three components was significantly higher than that of SFF. Figure 4 It can be seen that in the DPPH free radical scavenging experiment, the scavenging abilities of DSFF1 and DSFF3 were similar, and IC 50 DSFF2 had the best scavenging ability (IC 50 is 1.63 mg / mL). Figure 5 It can be seen that in the superoxide anion free radical scavenging experiment, the scavenging ability of DSFF2 exceeded that of DSFF1 and DSFF3, and its IC 50 is 1.74 mg / mL. Figure 6 It can be seen that in the iron ion reducing ability determination experiment, the iron ion reducing ability of DSFF2 is better than that of crude fucoidan. When the concentration is 8 mg / mL, its iron reducing ability is 0.21 mmol TE / g.

[0062] In summary, DSFF2 has the best antioxidant capacity, which is significantly better than DSFF1 and DSFF3. Therefore, DSFF2 was selected for subsequent experimental processing and analysis.

[0063] Experiment 2 In vitro cell experiment on low molecular weight fucoidan from Sargassum fusiformis 1. MTT assay to detect the survival rate of BRL 3A cells under DSFF2 treatment (1) Collect BRL 3A cells and plate 1×10 cells per well. 5 The cells were inoculated at a density of 10 cells / mL in a 96-well plate 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 added with different concentrations of DSFF2 samples (prepared with serum-free medium at 0, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 μg / mL, filtered through a 0.22 μm filter membrane), and one group was added with different concentrations of H 2 O 2 (prepared with serum-free medium at 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mM, filtered through 0.22μm membrane), one group was added with different concentrations of Vc (prepared with serum-free medium at 0, 3.125, 6.25, 12.5, 25, 50 and 100μg / mL, filtered through 0.22μm membrane), incubated for 24 h, and the cell survival rate was detected by MTT method.

[0064] The method of measuring cell viability by MTT method is as follows: after the incubation, add 50 μL of MTT solution (prepared in serum-free medium at a concentration of 2 mg / mL) to each well and incubate in the incubator for 4 h; remove the supernatant, add 150 μL of DMSO, shake and react at room temperature for 30 min, detect the absorbance at 490 nm, and calculate the cell survival rate. Cell survival rate (%) = absorbance value of sample group / absorbance value of blank group × 100%.

[0065] Schematic diagram of the effect of different concentrations of DSFF2 on cell survival rate Figure 7 As shown, DSFF2 at a concentration of 25 μg mL -1 It was basically non-toxic to BRL 3A cells at a concentration of 50 μg mL -1 Therefore, the maximum concentration of DSFF2 was set at 25 μg mL -1 .

[0066] Vc has a good antioxidant effect, which can inhibit the generation of oxidative stress products and alleviate oxidative damage, thereby reducing liver damage. Therefore, Vc was selected as the positive control group in this study. The effect of different concentrations of Vc on cell survival rate is shown in the figure. Figure 8As shown in the figure, when the concentration of Vc is between 0 and 25 μg / mL, the cell survival rate can reach more than 80%. When the concentration exceeds 6.25 μg / mL, the cell viability begins to decrease significantly (p < 0.05). After treatment with 50 and 100 μg / mL of Vc, the cell viability decreases to 78.34% and 65.26%, respectively. Therefore, in subsequent experiments, a concentration of 6.25 μg / mL of Vc was selected as the positive control of the experimental group.

[0067] Different concentrations of H 2 O 2 Schematic diagram of the effect on cell survival rate Fig. 9 As shown, it can be seen that when H 2 O 2 At a concentration of 0.4 mM, the survival rate of BRL 3A cells was 67%, while at a concentration of 0.5 mM, the survival rate of BRL 3A cells was 35%. 2 O 2 The concentration was set to 0.4 mM.

[0068] (2) Collect BRL 3A cells and plate 1×10 cells per well. 5 The cells were inoculated at a density of 10 cells / mL in a 96-well plate and incubated at 37°C for 24 h. The supernatant was discarded from each well and different concentrations of DSFF2 samples were added for 2 h. The supernatant was removed and 0.4 mM HCl was added to each well. 2 O 2 After 24 h of treatment, the supernatant was removed and the cell survival rate was detected using the MTT method.

[0069] Effect of different concentrations of DSFF2 pretreatment on H 2 O 2 Schematic diagram of the effect of induced injury on cell survival rate Fig.10 As shown, compared with the control group (set to 100%), H 2 O 2 The survival rate of cells treated with DSFF2 was significantly reduced, but after DSFF2 pretreatment, the 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 25-400 μg / mL. This indicates that DSFF2 pretreatment can effectively inhibit H 2 O 2 It has a damaging effect on BRL 3A cells and improves the survival rate of cells, but too high a concentration will affect the protective effect.

[0070] 2. Determination of ROS generation in BRL 3A cells The method was the same as that of Experiment 1. After the experiment, 50 μL of 2 mg / mL DCFH-DA probe was added to each well and incubated in a light-proof incubator 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.

[0071] Schematic diagram of the effect of DSFF2 on ROS production in BRL 3A cells Fig.11 As shown, by H 2 O 2 Induction, the amount of ROS produced in BRL 3A increased significantly, while it was significantly reduced in a dose-dependent manner after DSFF2 treatment. This indicates that after pretreatment with DSFF2, ROS generation can be effectively inhibited, cell oxidative damage caused by ROS can be reduced, and liver protection properties can be exerted.

[0072] 3. Determination of MDA content and SOD, CAT and GSH-Px activities in BRL 3A cells (1) Collect BRL 3A cells and plate 2.5 × 10 cells per well. 5 The cells were inoculated into 6-well plates at a density of 10 cells / mL and pre-incubated in an incubator for 24 h. Different concentrations of DSFF2 were added for pre-treatment for 2 h. The supernatant was removed and 0.4 mM H 2 O 2 Treat for 24 h; discard the cell supernatant, wash twice with pre-cooled 1X PBS, add 150 μL of cell lysis buffer to each well, lyse on ice for 5 min, collect in a centrifuge tube and lyse on ice for 30 min, and collect the supernatant after centrifugation.

[0073] (2) Follow the instructions of the commercial kit to determine the MDA content and the activities of SOD, CAT, and GSH-Px.

[0074] DSFF2 to H 2 O 2 Schematic diagram of the effect of induced MDA content and SOD, CAT, and GSH-Px activities in BRL 3A cells Fig.12 , Fig.13 , Fig.14 , Fig.15 As shown, H 2 O 2After stimulation, the MDA content in BRL 3A cells increased, and the activities of SOD, CAT, and GSH-Px decreased. After treatment with DSFF2, the changes were reversed. When the concentration was 25 μg / mL, compared with the model control group, the MDA content decreased by 138.89%, and the activities of SOD, CAT, and GSH-Px increased by 30.15%, 31.36%, and 19.81%, respectively. In summary, DSFF2 pretreatment can effectively inhibit the generation of oxidative stress products and increase the activity of antioxidant enzymes, thereby playing a protective role in liver cells.

[0075] Effect of DSFF2 on apoptosis in BRL 3A cells (1) Place the cell slides in a 24-well plate at 1.5 × 10 5 BRL 3A cells were inoculated at a density of 10 cells / mL and pre-incubated in an incubator for 24 h. Different concentrations of DSFF2 were added for pre-treatment for 2 h. The supernatant was removed and 0.4 mM H 2 O 2 Treat for 24 h; discard the cell supernatant, wash three times with 1X PBS, add an appropriate amount of anti-fluorescence quenching sealing solution containing Hoechst 33342 and PI for staining at room temperature for 15 min, observe the cell morphology and take pictures under an inverted fluorescence microscope.

[0076] (2) BRL 3A cells were collected at 2.5×10 per well. 5 The cells were inoculated into 6-well plates at a density of 10 cells / mL and pre-incubated in an incubator for 24 h. Different concentrations of DSFF2 were added for pre-treatment for 2 h. The supernatant was removed and 0.4 mM H 2 O 2 Treat for 24 h; discard the cell supernatant, wash the well plate 3 times with pre-cooled 1X PBS, add 150 μL cell lysis buffer to each well and lyse on ice for 5 min, collect in centrifuge tube and lyse on ice for 30 min, take part of the supernatant after centrifugation to determine the protein content according to the instructions of the BCA kit, add 4X loading buffer to the other part, heat denature at 100℃, and store at -20℃.

[0077] (3) Prepare SDS-PAGE gel according to the instructions. Add 30-50 μg sample protein to each well and start electrophoresis, then transfer to the membrane. After blocking at room temperature, incubate with primary and secondary antibodies to label the target protein. After sealing, the slides can be developed and the grayscale value can be analyzed using Image J software.

[0078] DSFF2 to H 2 O 2 Schematic diagram of the effect of induced apoptosis level of BRL 3A cells Fig.16 As shown. Fig.16 It can be seen that after H2 O 2 After stimulation, apoptosis is activated, the nucleus divides, and the Hoechst 33342 dye binds to the lysed apoptotic bodies to form bright blue fluorescent bodies; in addition, the cell membrane ruptures, and the PI dye can enter the cell, making the cytoplasm present red fluorescence. After DSFF2 pretreatment, the number of apoptotic bodies in the image is significantly reduced, and the PI staining area is also significantly reduced. When the DSFF2 concentration reaches 25 μg / mL, the Hoechst 33342 and PI staining effects are not significantly different from the positive control Vc staining effect, and are close to the control group.

[0079] DSFF2 to H 2 O 2 Schematic diagram of the effect of induced Caspase-3, Bax, and Bcl-2 protein levels in BRL 3A cells Fig.17 , Fig.18 , Fig.19 , Fig. 20 As shown. Through quantitative analysis of the level of cell apoptosis, it was found that compared with the control group, H 2 O 2 Treatment with DSFF2 significantly increased the levels of Caspase-3 and Bax in BRL 3A cells (p < 0.05), increasing by 1.55-fold and 2.18-fold, respectively. However, after pretreatment with DSFF2, the levels of Caspase-3 and Bax proteins decreased in a dose-dependent manner with the concentration of DSFF2 ( Fig.18 , Fig.19 ). When the concentration of DSFF2 was 25 μg / mL, Caspase-3 and Bax proteins were downregulated to the same level as the blank group. Fig. 20 The level of Bcl-2 protein in cells is affected by H 2 O 2 However, after DSFF2 pretreatment, the Bcl-2 level in the sample group increased significantly with the increase of sample concentration (p < 0.05). When the DSFF2 concentration reached 25 μg / mL, the Bcl-2 level in the sample group reached H 2 O 2 In summary, H 2 O 2 Stimulation can activate BRL 3A cell apoptosis; DSFF2 treatment can downregulate the levels of Caspase-3 and Bax proteins in cells, upregulate the level of Bcl-2 protein, inhibit cell apoptosis, and thus exert liver-protective properties.

[0080] 5. Effect of DSFF2 on Keap1 / Nrf2 signaling pathway in BRL 3A cells The method was the same as Experiment 4.

[0081] DSFF2 to H 2 O 2 Schematic diagram of the effects of Keap1 and Nrf2 in induced BRL 3A cells Fig.21 As shown in the blank group, Keap1 showed almost no green fluorescence, but after H 2 O 2 After stimulation, the fluorescence intensity of Keap1 was significantly enhanced, while that of Nrf2 was weakened. This phenomenon was reversed after pretreatment with DSFF2. When the concentration of DSFF2 was 25 μg / mL, the fluorescence quantity of Keap1 and Nrf2 in the cells showed almost the same green fluorescence intensity as in the blank group.

[0082] DSFF2 to H 2 O 2 Schematic diagram of the effect of induced Keap1, Nrf2, NQO1 and HO-1 protein levels in BRL 3A cells Fig. 22 , Fig.23 , Fig.24 , Fig.25 , Fig.26 When BRL 3A cells were exposed to H 2 O 2 During stimulation, compared with the blank group, the Keap1 protein content in the cells increased significantly, while the Nrf2, NQO1 and HO-1 protein contents decreased sharply, falling to 0.55, 0.28 and 0.24 times of β-actin protein, 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 reached 25 μg / mL, the Keap1 protein level dropped to 0.79 times that of the blank group, while Nrf2, NQO1 and HO-1 increased to 0.87, 0.82 and 0.70 times, respectively ( Fig.19 , Fig. 20 , Fig.21 The above results further prove that DSFF2 can reduce the level of Keap1 protein, thereby weakening Keap1-induced Nrf2 ubiquitination degradation, and activated Nrf2 nuclear translocation into the cell nucleus to recognize and bind to ARE to form a dimer, thereby activating the expression of antioxidant enzyme genes, thereby playing a role in protecting liver cells.

[0083] Experiment 3 In vitro zebrafish experiment on low molecular weight fucoidan from Sargassum fusiformis 1. DSFF2 concentration screening (1) Successfully fertilized zebrafish embryos that grew and developed well at 7-9 h after fertilization were selected and transferred into 12-well culture plates, with 20 embryos per well. After removing the residual culture medium, different groups were set up according to the experiment. The blank group was treated with the same volume of 1X PBS buffer. Different concentrations of DSFF2 (12.5, 20, 50, 100, 200 μg / mL) were added as needed, and the embryo survival rate was measured for 7 consecutive days in a suitable environment.

[0084] (2) According to different experimental groups, different concentrations of DSFF2 were added as needed and incubated for 2 h. Then, H 2 O 2 Oxidative stress was induced for 24 h, and the survival rate of zebrafish embryos was measured.

[0085] Effects of different concentrations of DSFF2 on the survival rate of zebrafish embryos Fig. 27 As shown in the figure, when the concentration of DSFF2 was between 12.5 and 50 μg / mL and cultured for 7 days, there was no significant change in the survival rate of zebrafish embryos. When the concentration of DSFF2 reached 100 μg / mL, zebrafish embryos began to die on the fourth day, and the embryo survival rate dropped to 86.67% on the seventh day. Therefore, a concentration range of DSFF2 of 12.5 to 50 μg / mL was selected for subsequent experiments.

[0086] DSFF2 to H 2 O 2 Effects of induced cytokines on the survival rate of zebrafish embryos Fig.28 As shown, it can be seen that after H 2 O 2 After induction stimulation, the survival rate of zebrafish embryos decreased significantly (p < 0.05). Compared with the 100% survival rate of the blank group, the survival rate of the control group decreased to 57.5%. After pre-incubation with different concentrations of DSFF2 (12.5-50 μg / mL), the embryo survival rate was significantly improved, and at a concentration of 50 μg / mL, it rose to 80%. This shows that low molecular weight fucoidan has a certain preventive and protective effect on oxidative stress damage in zebrafish embryos.

[0087] 2. DSFF2 vs H 2 O 2 Effects of induced ROS levels in zebrafish (1) Successfully fertilized zebrafish embryos that grew and developed well at 7 to 9 hours after fertilization were selected and transferred into 12-well cell culture plates, with 20 embryos per well. The culture medium was discarded and different concentrations of DSFF2 were added as needed according to different experimental groups and incubated for 2 hours. Then, H 2 O 2 Oxidative stress was induced for 24 h.

[0088] (2) To study H 2 O 2 Stimulation of ROS production in zebrafish with H 2 O 2 After 48 h of treatment, zebrafish larvae were stained with DCFH-DA for 1 h. After incubation, zebrafish larvae were washed with culture medium, and single zebrafish were photographed under a fluorescence microscope after anesthesia, and the fluorescence intensity of the images was quantified using ImageJ.

[0089] Zebrafish ROS fluorescence staining Fig.29 As shown, DSFF2 has an effect on H 2 O 2 Effects of induced ROS levels in zebrafish Fig.30 As shown. It can be seen that compared with the blank group, H 2 O 2 The amount of ROS produced in zebrafish was increased to 214.86%. DSFF2 reduced the ROS level by 25.87% and 59.19% at the doses of 12.5μg / mL and 25μg / mL, respectively. When the concentration of DSFF2 reached 50μg / mL, the ROS level dropped from 214.86% to 127.84%, and the ROS level was significantly improved (p < 0.05). The above results show that DSFF2 can effectively inhibit the production of oxidative stress products in zebrafish.

[0090] 3. DSFF2 vs H 2 O 2 Effects of induced cell death in zebrafish (1) The method is the same as Experiment 2.

[0091] (2) To study H 2 O 2 Stimulated cell death rate of zebrafish. At 3 days post fertilization, zebrafish larvae were stained with acridine orange for 30 min and washed with culture medium. After anesthesia, single zebrafish were photographed under a fluorescence microscope, and the fluorescence intensity of the images was quantified using ImageJ.

[0092] Zebrafish cell death fluorescent staining Fig.31 As shown, DSFF2 has an effect on H 2 O 2 Effects of induced cell death in zebrafish Fig.32 As shown. It can be seen that compared with the control group, H 2 O 2The cell death rate of zebrafish treated with DSFF2 increased from 100% to 300.41%. However, DSFF2 at concentrations of 12.5, 25, and 50 μg / mL reduced the death rate of zebrafish to 279.59%, 221.12%, and 170.00%, respectively. These results indicate that H 2 O 2 The survival rate of zebrafish was significantly reduced by DSFF2, while DSFF2 could reduce H 2 O 2 Treated zebrafish mortality.

[0093] 4. DSFF2 vs H 2 O 2 Effects of induced lipid peroxide production in zebrafish (1) The method is the same as Experiment 2.

[0094] (2) To study H 2 O 2 Stimulated lipid peroxidation in zebrafish. At 3 days after fertilization, zebrafish larvae were stained with DPPP for 3 h. The zebrafish larvae were then washed with culture medium, and a single zebrafish was photographed under a fluorescence microscope after anesthesia. The fluorescence intensity of the images was quantified using ImageJ.

[0095] Zebrafish lipid peroxidation fluorescence staining Fig.33 As shown, DSFF2 has an effect on H 2 O 2 Effects of induced lipid peroxide production in zebrafish Fig.34 As shown in Figure 2, compared with the blank group (100%), H 2 O 2 The lipid peroxidation level of zebrafish treated with 12.5, 25 and 50 μg / mL DSFF2 increased to 325.57%. 2 O 2 The induced lipid peroxidation levels of zebrafish were reduced to 278.91%, 203.41% and 153.29%, respectively. These results showed that DSFF2 significantly inhibited H 2 O 2 The above results indicate that low molecular weight fucoidan from Sargassum fusiformis can effectively inhibit oxidative damage in zebrafish, exert antioxidant effects, and thus play a hepatoprotective role.

[0096] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A method for preparing low molecular weight fucoidan from Sargassum fusiformis having auxiliary protective effect on chemical liver damage, characterized in that: The following steps are involved: S1. Defatting treatment: taking dried Sargassum fusiformis and crushing it to obtain Sargassum fusiformis powder; extracting the Sargassum fusiformis powder with ethanol for 1 to 2 times, centrifuging, removing the supernatant, and drying to obtain Sargassum fusiformis defatted powder; S2. Preparation of crude polysaccharide: adding defatted powder of Sargassum fusiformis to water and performing microwave extraction; then adding cellulase for enzymolysis; after enzymolysis, adding calcium chloride solution, standing and centrifuging, and adding Sevag reagent to the supernatant for deproteinization; Centrifuge, add ethanol to the supernatant, let stand and precipitate; centrifuge, collect the precipitate, which is the crude product of Sargassum fusiformis fucoidan; S3. Degradation of crude polysaccharides: Take crude Sargassum fusiformis fucoidan, dissolve it in water to form a polysaccharide solution, and treat it with ultrasound; then, add H2O2 to degrade it to obtain Sargassum fusiformis low molecular weight fucoidan DSFF; S4. Purification: Take the low molecular weight fucoidan DSFF from Sargassum fusiformis, dissolve it in water to form a polysaccharide solution, load it on a DEAE-52 cellulose anion exchange column and elute it with NaCl-phosphate buffer, collect the eluate eluted with 1.0 mol / L NaCl-phosphate buffer; dialyze the eluate for desalination and lyophilize it to obtain the low molecular weight fucoidan DSFF2 from Sargassum fusiformis.

2. The method for preparing the low molecular weight fucoidan from Sargassum fusiformis having auxiliary protective effect on chemical liver damage according to claim 1, characterized in that: In the step S1, the specific method of "taking dried Sargassum fusiformis and crushing it to obtain Sargassum fusiformis powder" is: taking dried Sargassum fusiformis, soaking it in water for 12 hours, washing it three times, drying it at room temperature, placing it in an oven to dry at 45° C., crushing it with a high-speed grinder, and passing it through a 60-mesh sieve to obtain Sargassum fusiformis powder.

3. The method for preparing the low molecular weight fucoidan from Sargassum fusiformis having auxiliary protective effect on chemical liver damage according to claim 1, characterized in that: In the step S1, the specific method of "extracting the Sargassum powder with ethanol for 1 to 2 times, centrifuging, removing the supernatant, and drying to obtain the Sargassum defatted powder" is: adding the Sargassum powder to a 95% ethanol solution with a solid-liquid ratio of 1:20, and extracting it twice by reflux at 85°C using a condensing reflux device, each time for 2 hours; centrifuging after extraction, removing the supernatant, and placing the solid in an oven at 45°C to dry to obtain the Sargassum defatted powder.

4. The method for preparing the low molecular weight fucoidan from Sargassum fusiformis having auxiliary protective effect on chemical liver damage according to claim 1, characterized in that: The specific method of step S2 is: adding defatted Sargassum fusiformis powder into water, with a solid-liquid ratio of 1:80, and performing microwave extraction with the following parameters: temperature 70° C., power 450 W, and time 50 min; adjusting the pH to 4.5, adding cellulase, and performing enzymolysis for 3 h; Centrifuge to remove solids, add calcium chloride solution to the supernatant to make the final concentration of calcium chloride 20 mg / ml, let stand at 4°C for 12 h, centrifuge, take the supernatant, add Sevag reagent, shake thoroughly for 30 min, centrifuge, take the supernatant, add 4 times the volume of anhydrous ethanol, let stand at 4°C for 12 h, centrifuge, re-dissolve the solids, and lyophilize to obtain the crude product of Sargassum fusiformis fucoidan.

5. The method for preparing the low molecular weight fucoidan from Sargassum fusiformis having auxiliary protective effect on chemical liver damage according to claim 1, characterized in that: The specific method of step S3 is as follows: taking crude Sargassum fusiformis fucoidan, dissolving it in water to prepare a 5 mg / mL polysaccharide solution, and subjecting it to ultrasonic treatment with the following parameters: temperature 25°C, ultrasonic frequency 40 kHz, and time 2 h; then, adding an equal volume of 1 mol / L H2O2 solution and degrading it for 2 h; after degradation, adding manganese dioxide to remove excess H2O2, centrifuging, taking the supernatant, adding 4 times the volume of anhydrous ethanol, standing at 4°C for 12 h, centrifuging, re-dissolving the solid, and freeze-drying to obtain Sargassum fusiformis low molecular weight fucoidan DSFF.

6. The method for preparing the low molecular weight fucoidan from Sargassum fusiformis having auxiliary protective effect on chemical liver damage according to claim 1, characterized in that: The specific method of step S4 is as follows: taking the low molecular weight fucoidan DSFF from Sargassum fusiformis, dissolving it in water to prepare a 5 mg / mL polysaccharide solution, loading it on a DEAE-52 cellulose anion exchange column, eluting it with phosphate buffer, 0.5 mol / L, and 1.0 mol / L sodium chloride-phosphate buffer solutions in sequence, and collecting the eluate eluted with 1.0 mol / L NaCl-phosphate buffer; placing the eluate in a 500 kDa dialysis bag for dialyzed desalination, changing the water every 4 hours, and dialyzing for 3 days; and freeze-drying after dialysis to obtain the low molecular weight fucoidan DSFF2 from Sargassum fusiformis.

7. The low molecular weight fucoidan of Sargassum fusiformis prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the low molecular weight fucoidan of Sargassum fusiformis according to claim 7 in the preparation of a drug having an auxiliary protective effect on chemical liver damage.

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