Ascophyllum nodosum fucoidan ANFS, a preparation method thereof and application thereof in preparing preparations for preventing and treating alzheimer's disease

Abstract

The application discloses a kind of Ascophyllum nodosum fucoidan ANFS and its preparation method and application in preparation for preventing and treating alzheimer's disease preparation, belong to marine biological medicine technical field.The main chain of the Ascophyllum nodosum fucoidan ANFS is composed of (1→3) connected α-L fucose residue, and branch is monosaccharide or sulfate group.Sulfated modification occurs at C-2 and C-4 of fucose residue, and its molecular weight is 15-30 kDa.The application proves by experiment, ANFS can significantly improve the motion behavior defect of zebra fish, inhibit acetylcholinesterase activity, improve catalase and glutathione peroxidase activity, reduce intracellular reactive oxygen species level, reduce brain neuron apoptosis, and effectively protect the integrity of blood-brain barrier.The polysaccharide is rich in source, and preparation process is simple, and safety is high, has wide application prospect in the development of marine medicine and functional product for treating alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of marine organisms and medicines, and particularly relates to a Cystoseira barbata fucoidan ANFS, a preparation method thereof and application of the Cystoseira barbata fucoidan ANFS in preparation of a preparation for preventing and treating Alzheimer's disease. BACKGROUND

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, and its main pathological features include β-amyloid deposition, Tau protein hyperphosphorylation, cholinergic system dysfunction, oxidative stress and neuronal apoptosis. At present, the drugs used in clinical practice such as cholinesterase inhibitors (donepezil, etc.) and NMDA receptor antagonists (memantine, etc.) can only alleviate symptoms and cannot stop the disease progression, and have problems such as large side effects and single target. Therefore, finding new therapeutic drugs with multiple targets, high safety and the ability to delay disease progression has become a research hotspot.

[0003] Fucoidan is a sulfated polysaccharide derived from brown algae, and has various biological activities such as anti-tumor, anti-virus, immune regulation, anti-oxidation and neuroprotection. In recent years, studies have shown that fucoidan with certain structures can penetrate the blood-brain barrier and exhibit neuroprotective potential, such as improving Aβ-induced cognitive impairment and inhibiting acetylcholinesterase activity. However, fucoidans obtained from different algae and by different extraction methods differ significantly in structure and have different biological activities.

[0004] Cystoseira barbata is an important raw material for industrial production of algal glue, and a large amount of soaking liquid will be produced during its processing into algal powder, which is rich in water-soluble fucoidan. At present, this part of by-product is mostly discarded, which not only causes resource waste but also brings environmental pressure. It is of great significance to realize high-value utilization of seaweed resources and develop new marine drugs to recover high-activity fucoidan from Cystoseira barbata processing waste liquid and further explore the relationship between its structure and anti-Alzheimer's disease activity. SUMMARY

[0005] In view of the above status in the prior art, the purpose of the present application is to provide a Cystoseira barbata fucoidan ANFS, a preparation method thereof and application of the Cystoseira barbata fucoidan ANFS in preparation of a preparation for preventing and treating Alzheimer's disease. The Cystoseira barbata fucoidan ANFS with a clear structure is separated from Cystoseira barbata processing waste liquid, the preparation method thereof is simple, and the zebrafish in vivo experiment proves that the Cystoseira barbata fucoidan ANFS has anti-Alzheimer's disease activity.

[0006] To achieve the above-mentioned purpose, the technical scheme is adopted as follows:

[0007] The present application provides a kind of Ascophyllum nodosum fucoidan ANFS, the main chain of the Ascophyllum nodosum fucoidan ANFS is composed of (1→3) linked α-L fucose residue, branch is monosaccharide or sulfate group, and sulfation modification occurs at C-2 and C-4 of fucose residue.

[0008] Further, the molecular weight of the Ascophyllum nodosum fucoidan ANFS is 15-30 kDa, and the monosaccharide composition is mainly fucose, containing glucosamine, glucose and galactose.

[0009] Further, the monosaccharide composition of the Ascophyllum nodosum fucoidan ANFS is: fucose is 90%-96% in molar percentage, glucosamine is 1%-3%, glucose is 2%-4%, and galactose is 1%-3%.

[0010] Further, the sulfate group content of the Ascophyllum nodosum fucoidan ANFS is 25-35%.

[0011] The present application also provides a preparation method of the Ascophyllum nodosum fucoidan ANFS, comprising the following steps:

[0012] (1) filtering and concentrating under reduced pressure to obtain a concentrated solution after soaking the soaked solution of Ascophyllum nodosum processing;

[0013] (2) adding calcium chloride solution to the concentrated solution to precipitate alginate, and collecting the supernatant by centrifugation;

[0014] (3) dialysis desalination is carried out on the supernatant to obtain a dialysate;

[0015] (4) after concentrating the dialysate, adding ethanol for alcohol precipitation, collecting the precipitate and freeze-drying to obtain crude polysaccharide;

[0016] (5) the crude polysaccharide is separated and purified by anion exchange column and gel column in turn, and the target component is collected, desalted and freeze-dried to obtain purified Ascophyllum nodosum fucoidan ANFS.

[0017] Further, the molecular weight of the Ascophyllum nodosum fucoidan ANFS is 15-30 kDa, and the monosaccharide composition is mainly fucose, containing glucosamine, glucose and galactose.

[0018] Further, in step (5), Q-Sepharose Fast Flow anion exchange column is used for elution, 0, 0.5, 1.0, 1.5, 2 and 2.5 mol / L NaCl solution are used for stepwise elution, and the elution component of 2 mol / L NaCl is collected as the target component.

[0019] Further, the gel column in the step (5) is Sephacryl S-400 / HR gel column, and the elution condition is 0.2 mol / L NH4HCO3 solution, and the flow rate is 0.3 mL / min.

[0020] The application further provides application of the fucoidan ANFS of Ascophyllum nodosum in preparation of a preparation for preventing and / or treating Alzheimer's disease.

[0021] Further, the application is embodied in that the ANFS can play an anti-Alzheimer's disease role through one or more of the following mechanisms:

[0022] a) inhibiting acetylcholinesterase activity and improving cholinergic system function;

[0023] b) improving antioxidant enzyme activity, eliminating active oxygen and relieving oxidative stress;

[0024] c) reducing cell apoptosis;

[0025] d) protecting the integrity of the blood-brain barrier.

[0026] Compared with the prior art, the application has the following advantages and beneficial technical effects:

[0027] 1. The application uses the soaking liquid, a processing by-product of Ascophyllum nodosum, as a raw material, realizes waste utilization, has high resource utilization rate, low cost and environmental friendliness.

[0028] 2. The application first separates the fucoidan ANFS with a unique structure from the raw material, and systematically clarifies the fine structure of the main chain, the side chain and the sulfation site.

[0029] 3. The application first systematically proves that the fucoidan ANFS derived from Ascophyllum nodosum has multi-target anti-Alzheimer's disease activity through a zebrafish model, the action mechanism is clear, and a solid scientific foundation is laid for development of the fucoidan ANFS into a marine innovative drug or a functional health product. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a Q-Sepharose Fast Flow separation diagram of crude polysaccharide of Ascophyllum nodosum;

[0031] Figure 2 is high-performance gel permeation chromatography of fucoidan of Ascophyllum nodosum on a Shodex OHpak SB-804HQ chromatographic column; wherein a is a polysaccharide molecular weight standard curve; and b is a high-performance gel permeation liquid chromatogram of ANFS;

[0032] Figure 3is a single sugar composition analysis chart of Ascophyllum nodosum fucoidan; wherein, a is a high performance liquid chromatogram of 10 kinds of single sugar standard; b is a high performance liquid chromatogram of single sugar composition of ANFS;

[0033] Figure 4 is a zebrafish behavior detection chart; wherein, a is a zebrafish motion trajectory chart representative chart; b is a zebrafish swimming distance; c is a zebrafish swimming speed;

[0034] Figure 5 is a zebrafish enzyme activity detection chart; wherein, a is an ANFS improved AChE enzyme activity level; b is an ANFS improved CAT enzyme activity level; c is an ANFS improved GPx enzyme activity level;

[0035] Figure 6 is a zebrafish ROS staining chart; wherein, a is a zebrafish ROS staining; b is a zebrafish fluorescence intensity contrast;

[0036] Figure 7 is a zebrafish apoptosis staining chart; wherein, a is a zebrafish larva brain overhead view and side view; b is a zebrafish brain apoptosis cell number statistics;

[0037] Figure 8 is a zebrafish blood-brain barrier staining chart. DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be further described in detail below in combination with specific embodiments and drawings, but the protection scope of the present application is not limited thereto.

[0039] Example 1: Preparation of Ascophyllum nodosum fucoidan ANFS

[0040] The preparation method of the Ascophyllum nodosum fucoidan ANFS of the present application comprises the following steps:

[0041] 1. Take 25 L of the soaking liquid after processing Ascophyllum nodosum, and concentrate under reduced pressure at 55-58℃ to obtain 5 L of concentrated liquid.

[0042] 2. Slowly add 3 mol / L CaCl2 solution to the concentrated liquid until no white flocculent is produced, centrifuge at 5000 rpm for 15 min, and collect the supernatant.

[0043] 3. Desalt the supernatant by dialysis with a dialysis bag with a molecular weight cut-off of 3500 Da.

[0044] 4. Concentrate the dialysate, add 4 volumes of 95% ethanol, and stand overnight at 4℃. Centrifuge to collect the precipitate, and freeze-dry to obtain crude polysaccharide.

[0045] 5. The crude polysaccharide was dissolved in water and loaded onto a Q-Sepharose Fast Flow anion exchange column, which was eluted with 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mol / L NaCl solutions, respectively, and the elution peak of 2 mol / L NaCl solution was collected.

[0046] 6. The above component was further purified by a Sephacryl S-400 / HR gel column (eluent: 0.2 M NH4HCO3; flow rate: 0.3 mL / min), and the main peak was collected, desalted, and lyophilized to obtain pure ANFS.

[0047] Example 2: Structural characterization of the fucoidan ANFS from Ascophyllum nodosum

[0048] 1. Purity and molecular weight determination by high-performance gel permeation liquid chromatography (HPGPC)

[0049] (1) Preparation of sample and standard:

[0050] Polysaccharide sample determination: 2 mg of the fucoidan ANFS prepared in Example 1 was accurately weighed, 400 μL of 0.1 mol / L Na2SO4 solution was added, and vortexed until completely dissolved to prepare a 5 mg / mL polysaccharide sample solution. The solution was filtered through a 0.22 μm water-based microporous filter for standby use.

[0051] Standard preparation: 2 mg of dextran standard with different molecular weights (weight average molecular weight Mw: 5.9, 9.6, 21.1, 47.7, 107, 200, 344, and 708 kDa) was added to 400 μL of 0.1 mol / L Na2SO4 solution, vortexed and dissolved to prepare a series of 5 mg / mL molecular weight standard solutions. The solutions were filtered through a 0.22 μm water-based microporous filter for standby use.

[0052] (2) Chromatographic conditions:

[0053] Analytical column: Shodex Ohpak SB-804HQ;

[0054] Mobile phase: 0.1 mol / L Na2SO4 solution (ultrasonic degassing for 30 min);

[0055] Flow rate: 0.5 mL / min;

[0056] Column temperature: 35°C;

[0057] Injection volume: 20 μL;

[0058] Detector: differential refractive index detector.

[0059] (3) Standard curve drawing and data calculation: The logarithm value of the weight average molecular weight of the dextran standard (log Mw) was used as the vertical coordinate (Y axis), and the retention time (RT, unit: min) was used as the horizontal coordinate (X axis) to draw a standard curve (a in Figure 2 The linear regression equation was obtained by linear regression analysis. The retention time of the sample was substituted into the linear regression equation to calculate the weight average molecular weight (Mw) of the sample.

[0060] The results are shown in b in Figure 2 The HPGPC chromatogram of Ascophyllum nodosum fucoidan ANFS showed a single symmetrical peak, and the peak shape had no tailing and no impurity peak, indicating that it had high purity and uniform molecular weight distribution; the weight average molecular weight (Mw) was calculated to be 20.725 kDa.

[0061] 2. Determination of monosaccharide composition of Ascophyllum nodosum fucoidan ANFS by high performance liquid chromatography

[0062] (1) Accurately weigh 3 mg of the Ascophyllum nodosum fucoidan prepared in Example 1, add 400 μL of 2 mol / L trifluoroacetic acid (TFA) solution in an ampoule, and hydrolyze at 105°C under sealed conditions for 6 h. After hydrolysis, repeatedly add methanol and remove excess TFA by rotary evaporation under reduced pressure.

[0063] (2) Dissolve the Ascophyllum nodosum fucoidan hydrolysate and the monosaccharide standard in 100 μL of distilled water, add 100 μL of 0.3 mol / L NaOH solution and 120 μL of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution, respectively, and react in a 70°C water bath for 60 min. After cooling to room temperature, add 100 μL of 0.3 mol / L HCl solution for neutralization reaction; then extract with dichloromethane for 3 times, discard the organic phase to remove unreacted PMP; take the upper aqueous phase and filter through a 0.22 μm organic phase microporous filter for standby.

[0064] (3) Determine the monosaccharide composition of Ascophyllum nodosum fucoidan by HPLC, and the chromatographic conditions are as follows:

[0065] Chromatographic column: Eclipse XDB-C18 (5 μm, 4.6 mm x 250 mm);

[0066] Mobile phase: acetonitrile: phosphate buffer (pH 6.7) = 17:83 (by volume);

[0067] Injection volume: 10 μL;

[0068] Column temperature: 35°C;

[0069] Detector: UV detector (detection wavelength 254 nm);

[0070] Flow rate: 1.0 mL / min.

[0071] The results are shown in a and b in Table 1, wherein the fucoidan in Ascophyllum nodosum mainly contains fucose, accounting for 94.48%, and a small amount of glucosamine (1.12%), glucose (2.65%) and galactose (1.75%). Figure 3 Figure 3 The results are shown in a and b in Table 1, wherein the fucoidan in Ascophyllum nodosum mainly contains fucose, accounting for 94.48%, and a small amount of glucosamine (1.12%), glucose (2.65%) and galactose (1.75%).

[0072] Example 3: Protective effect of Ascophyllum nodosum fucoidan ANFS on AlCl3-induced zebrafish AD model

[0073] 1. Solution preparation

[0074] (1) ANFS solution: accurately weigh 50 mg of ANFS powder, dissolve in 1 mL of pure water to obtain an ANFS stock solution with a concentration of 50 mg / mL, and store in a -20°C refrigerator for standby.

[0075] (2) Aluminum chloride solution: accurately weigh 33.8 mg of aluminum chloride powder, dissolve in 1 mL of deionized water, mix well to obtain a 140 mM aluminum chloride stock solution, and store in a 4°C refrigerator for standby.

[0076] (3) Donepezil solution: accurately weigh 25 mg of donepezil powder, dissolve in 10 mL of deionized water to prepare a 25 mg / mL donepezil solution, mix well and store in a -20°C freezer for standby.

[0077] (4) Methyl cellulose: 3 g of methyl cellulose powder is added to 70°C 120 mL of water and placed in an ultrasonic wave for dissolution, mixed thoroughly and stored at 4°C for standby.

[0078] (5) Methylene blue: accurately weigh 10 mg of powder, dissolve in 10 mL of pure water to prepare a 0.1% concentration, and store at 4°C for standby.

[0079] (6) Anesthetic: accurately weigh 10 mg of tricaine powder, dissolve in 10 mL of pure water, and store in a 4°C refrigerator for standby.

[0080] (7) PTU: accurately weigh 20 mg of phenylthiourea powder, dissolve in 20 mL of pure water, then shake and mix well, and store at 4°C for standby.

[0081] (8) PBS buffer: dilute 100X PBS buffer and use. Store at 4°C for standby.

[0082] ​(9) Juvenile fish water configuration: 1 mM MgS04, 0.5 mM KCl, 15 mM NaCl, 0.05 mM (NH4)3P04, 0.15 mM KH2P04, 0.7 mM NaHC03, and 1 mM CaCl2, with a volume of 1 L.

[0083] 2. Zebrafish AD model establishment

[0084] The 3-day post-fertilization (3 dpf) zebrafish larvae were randomly transferred to a six-well plate, about 30 larvae per well. Then, the zebrafish AD model was established by continuous treatment with 80 μΜ AlCl3 from 3 dpf to 6 dpf for 3 days.

[0085] 3. Grouping and administration method

[0086] Healthy 3 dpf zebrafish larvae from the same batch were selected for the experiment and randomly divided into a control group (E3 fish water), a model group (80 μΜ AlCl3 solution), a positive drug group (80 μΜ AlCl3 solution + 4 μΜ Donepezil solution), and low, medium, and high concentration ANFS groups (80 μΜ AlCl3 solution + 10, 20, and 30 μg / mL ANFS), with about 30 fish in each group. The fish were placed in a six-well culture plate, and an appropriate amount of E3 fish water and drug solution was added to each well to maintain a volume of 5 mL. The blank control group was added with E3 fish water, and the other groups were added with the corresponding drug solution after the addition of E3 fish water. Fresh E3 fish water and drug solution were replaced every 24 h, and the administration was continuous (3 dpf~5 dpf). After each replacement of the drug solution, the six-well culture plate was placed in a constant temperature incubator with an environmental temperature of 28±0.5℃, maintained ventilation, and always kept in a 14 h light: 10 h dark environment to simulate day-night alternation.

[0087] 4. Behavioral detection

[0088] (1) Light-dark alternation behavior: 6 dpf zebrafish larvae from each group after drug addition were randomly selected and washed 3 times with E3 fish water. They were placed in a 48-well plate, and 1 mL of clean E3 fish water was added to each well. The flow water temperature device was turned on in advance to ensure that the experimental environment was 28±0.5℃. The zebrafish behavior analysis multi-well plate was placed in the flow water tank, and the infrared imaging trajectory capture plate was used to record the speed interval, speed change, and trajectory movement direction of each larva. After 20 min of adaptation, the experimental program was set to 10 min light and 10 min dark for one cycle, and three alternating light-dark cycles were performed, with a total experimental time of 60 min. Zeblab software was used to record and analyze the movement distance and reaction speed change of zebrafish from light and dark cycles.

[0089] (2) Tactile Behavior Analysis: Six-day-fly (dpf) zebrafish juveniles were randomly selected from each group after medication and rinsed three times with E3 fish-raising water. They were then placed sequentially into each well of a 24-well plate, with 1 mL of clean E3 fish-raising water added to each well. The flow water temperature control was turned on beforehand to ensure the experimental environment was 28±0.5℃. The zebrafish behavior analysis multi-well plate was placed in flowing water to maintain a constant temperature throughout the experiment, avoiding the influence of temperature changes on behavioral differences. The movement trajectory, direction, and distance of each zebrafish juvenile were recorded and captured with the assistance of an infrared imaging trajectory capture plate. After acclimatization for 20 minutes, the experiment was set to a cycle of 12 minutes of light and 10 minutes of darkness. Two observation areas, one large and one small, were set in the wells of the 24-well plate using Zeblab software. The centers of the large and small circles overlapped, and the diameter of the large circle was twice that of the small circle. Zeblab software was used to record and analyze the changes in movement distance and speed of the zebrafish during the light and dark cycles.

[0090] (3) Activity Behavior Analysis: Six-day-fly (dpf) zebrafish fry were randomly selected from each group after medication and rinsed three times with E3 fish-raising water. They were placed in 48-well plates, with 1 mL of clean E3 fish-raising water added to each well. The flow water temperature control was turned on beforehand to ensure the experimental environment was 28±0.5℃. The zebrafish behavior analysis multi-well plate was placed in flowing water to maintain a constant temperature throughout the experiment, avoiding the influence of temperature changes on behavioral differences. The movement trajectory, direction, and distance of each zebrafish fry were recorded and captured with the assistance of an infrared imaging trajectory capture plate. The experimental program was set to 20 minutes, and Zeblab software was used to record and analyze the activity level of the zebrafish.

[0091] The results of the behavioral analysis are as follows Figure 4 As shown, Figure 4 Figure a shows the swimming trajectories of zebrafish in each group after treatment with the drug for 96 hpf in a 60-minute alternating light and dark environment. Within the box, red lines represent fast-moving zebrafish (v ≥ 6 cm / s), green lines represent medium-speed swimming (3 cm / s ≤ v < 6 cm / s), and black lines represent slow-moving zebrafish (v < 2 cm / s). The swimming distance and speed of the zebrafish were statistically analyzed in the dark environment, the light-stimulated environment, and throughout the entire experimental period. The results are shown below. Figure 4 b in Figure 4As shown in c, the swimming distance and speed of the blank control group were significantly higher than those of the experimental control group. The zebrafish in the experimental control group exhibited obvious kinetic impairment, indicating successful modeling. Compared with the experimental control group, the swimming distance of the positive drug group and the ANFS group increased significantly during the experiment, indicating that ANFS can improve behavioral impairment. After 96 h of drug treatment, the swimming speed of zebrafish in each group per unit time under cyclical light and dark environments is shown in Figure c. Figure 4 As shown in c, fish prefer dark environments and swim faster in darkness than in light. Compared to the blank control group, the zebrafish in the experimental control group swam slowly throughout the experiment, and their recovery to normal speed was very slow after the light stimulation ended, indicating behavioral disorders and slow responses in the experimental model group. Compared to the experimental control group, the zebrafish in the positive drug group and the ANFS group swam faster on average, showing some recovery and improved responsiveness to sudden light stimulation. These results demonstrate that, compared to the model group, ANFS treatment (10, 20, 30 µg / mL) dose-dependently improves behavioral disorders and slow responses in zebrafish, increasing their total swimming distance and swimming speed.

[0092] 5. Enzyme activity detection

[0093] Juvenile zebrafish of strain AB were treated with the drug continuously from 3 days post-fertilization to 5 days post-fertilization (dpf). After treatment, the juveniles were washed three times with pre-cooled (4°C) phosphate-buffered saline (PBS, pH 7.4) to thoroughly remove residual drug. Then, the juveniles were transferred to 1.5 mL sterile enzyme-free centrifuge tubes, with 100 juveniles per group, and 200 μL of physiological saline was added for low-temperature mechanical homogenization. The homogenate was centrifuged at 4°C and 12000 × g for 10 minutes, and the supernatant was collected for subsequent protein concentration and enzyme activity assays. The total protein concentration in the tissue homogenate supernatant was determined using a BCA protein concentration assay kit. The activities of acetylcholinesterase (AChE), catalase (CAT), and glutathione peroxidase (GPx) were measured strictly according to the kit instructions.

[0094] The enzyme activity assay results are as follows: Figure 5 As shown, AChE plays a crucial role in neurotransmission and is closely related to the pathogenesis and progression of Alzheimer's disease (AD). AChE is an enzyme that degrades acetylcholine. Studies have shown that low AChE activity can combat learning and memory decline and behavioral disorders. Currently, the mainstream clinical treatment for AD is AChE inhibitors. Figure 5As shown in figure a, compared with the blank control group, the AChE enzyme activity in the AD model group was significantly increased; compared with the AD model group, the donepezil treatment group and ANFS showed the same trend, with significantly decreased AChE activity. The experimental results indicate that ANFS can inhibit AChE activity in a dose-dependent manner, and has the potential to become an effective drug for treating AD. CAT is a hallmark peroxidase in vivo, and the level of CAT enzyme activity is generally considered to be a direct reflection of antioxidant capacity. Figure 5 As shown in b, compared to the blank control group, the CAT enzyme activity level in the experimental control group was significantly reduced. Compared to the experimental control group, the CAT enzyme activity level in the ANFS group was significantly increased. This improved the abnormal expression of CAT enzyme activity. Therefore, the above results suggest that ANFS can have a certain ameliorative effect on the abnormal activity level of CAT enzyme. GPx is a hydrogen peroxide decomposing enzyme in the body, responsible for the decomposition and metabolism of reactive oxygen free radicals such as hydrogen peroxide. Therefore, its enzyme activity level is closely related to antioxidant capacity. Figure 5 As shown in Figure c, within the first 1 minute of the reaction, the GPx enzyme activity level in the model group was significantly lower than that in the blank control group. Compared to the model group, the enzyme activity in the positive control group and the ANFS group was significantly higher, with statistically significant differences. At other times, the GPx enzyme activity levels in the blank control group, experimental control group, and ANFS group all showed a decreasing trend, but were all higher than those in the model group. The experimental results demonstrate that ANFS can significantly inhibit the abnormal increase of AChE in zebrafish and restore the activities of CAT and GPx that were inhibited by AlCl3. Therefore, the above results suggest that ANFS can have a certain ameliorative effect on the abnormal GPx enzyme activity level.

[0095] 6. ROS staining

[0096] 3 dpf zebrafish juveniles were randomly selected from each group after drug administration. After washing three times with E3 fish culture water, anesthetic was added, and 30 μM dye solution was added to each group. The fish were then placed in a 28℃ constant temperature incubator for 40 min in the dark. The dye solution was then washed off with E3 fish culture water, and the fluorescence intensity was observed under a stereomicroscope.

[0097] ROS staining results are as follows Figure 6 a and Figure 6 As shown in b, compared with the blank control group, the fluorescence intensity and ROS content of the experimental control group were significantly increased. Compared with the experimental control group, the fluorescence intensity and ROS content of zebrafish in the positive control group and ANFS group were significantly decreased. These experiments demonstrate that ANFS can significantly improve the phenomenon of increased ROS content in zebrafish induced by AlCl3. The results of the DCFH-DA fluorescent probe confirm that ANFS can dose-dependently reduce the increase in ROS levels in zebrafish induced by AlCl3.

[0098] 7. TUNEL apoptosis staining

[0099] Rinse repeatedly with clean E3 fish tank water to ensure that any residual medication is removed. After rinsing, add an appropriate amount of anesthetic to the wells to anesthetize the zebrafish and prevent them from swimming around. For tissue fixation, place the zebrafish and fixative solution in a 4°C environment for 12 hours. On the second day, aspirate 4% formaldehyde from the well plate, add PBS for washing, and gently shake the plate on a shaker for 5 minutes during the washing process. Repeat this operation three times. After washing, add 3% hydrogen peroxide diluted with methanol and seal at room temperature for 15 minutes. Rinse repeatedly three times with clean E3 fish tank water for 5 minutes each time. Then, add 1.5 mL of 0.3% Triton X-100 and sodium citrate antigen retrieval solution (1X) to each well, ensuring thorough mixing with a pipette, and incubate at 4°C for 10 minutes. After incubation, wash with PBS for 5 minutes each time, repeating three times. Collect the zebrafish larvae from each well into a 1.5 mL EP tube. Add TUNEL reaction mixture (Tat enzyme reaction mixture: fluorescent labeling solution = 1:10) to each EP tube. Then incubate at 37°C for 1.5 h (wrap the tube in aluminum foil to protect it from light during incubation). Discard the TUNEL reaction mixture and wash with PBS for 5 min under light-protected conditions. Repeat three times. Observe and acquire images under a fluorescence microscope.

[0100] Apoptosis staining results are as follows Figure 7 a and Figure 7 As shown in b, Aβ deposition can induce oxidative stress in the body, leading to apoptosis. Apoptosis in zebrafish brain cells was detected using TUNEL staining. No significant apoptosis was detected in the blank control group; apoptosis was significantly increased in the AD model group; compared with the experimental control group, apoptosis was significantly reduced in the ANFS group, with the 30 μg / mL group showing the best protective effect and statistical significance. The experimental results demonstrate that ANFS can reduce apoptosis in zebrafish brain cells.

[0101] 8. Evans Blue Staining

[0102] After fish collection, add PTU. The working concentration of EB is 2.5 mg / mL (freshly prepared), and the fish are dyed in the dark for 20 min at 5 dpf. The dye is removed by washing 5-10 times in a 6-well plate. The zebrafish are placed on a cover glass and fixed with a thin layer of methyl cellulose. The position is adjusted so that the abdomen is downward and the back is upward. The zebrafish are placed under a confocal microscope for photography, and the Aleax Fluor 488 (green blood vessels) and Aleax Fluor 594 (EB dye) channels are selected.

[0103] The results of Evans blue staining show that, as shown in Figure 8 compared with the blank control group, the zebrafish blood vessels are significantly reduced under green fluorescence (GFP) observation in the experimental control group, and AlCl3 affects the generation of zebrafish brain blood vessels; after Evans blue staining, the plasma albumin in the blood vessels shows red fluorescence (EB), and the plasma albumin shows leakage, indicating that AlCl3 causes damage to the blood-brain barrier of zebrafish. Compared with the experimental control group, the positive drug group and the ANFS group can improve the inhibition of angiogenesis by AlCl3 under green fluorescence observation, and the overlapping of green fluorescence blood vessels and red fluorescence plasma albumin is observed in the merge. The experimental results prove that ANFS can protect the integrity of the blood-brain barrier of zebrafish.

[0104] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified by those of ordinary skill in the art, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A fucoidan ANFS from Ascophyllum nodosum, characterized in that, The ANFS fucoidan main chain of the Ascophyllum nodosum is composed of (1→3) linked α-L fucose residues, and the branch is a monosaccharide or a sulfate group, and the sulfation modification occurs at C-2 and C-4 positions of the fucose residues; the molecular weight of the ANFS fucoidan of the Ascophyllum nodosum is 15 kDa-30 kDa; the monosaccharide composition is mainly fucose, and also contains glucosamine, glucose and galactose, and the monosaccharide composition of the ANFS fucoidan of the Ascophyllum nodosum is: in terms of molar percentage, fucose is 90%-96%, glucosamine is 1%-3%, glucose is 2%-4%, and galactose is 1%-3%, and the sulfate group content of the ANFS fucoidan of the Ascophyllum nodosum is 25-35%.

2. The method for preparing the Ascophyllum nodosum fucan ANFS according to claim 1, characterized in that, The preparation method comprises the following steps: (1) filtering and reducing pressure concentrating the soaking liquid after processing of the Ascophyllum nodosum to obtain a concentrated liquid; (2) adding a calcium chloride solution to the concentrated liquid to precipitate alginate, and collecting the supernatant by centrifugation; (3) desalting the supernatant by dialysis to obtain a dialysate; (4) concentrating the dialysate, adding ethanol for alcohol precipitation, collecting the precipitate and freeze-drying to obtain a crude polysaccharide; (5) sequentially separating and purifying the crude polysaccharide through an anion exchange column and a gel column, collecting the target component, desalting and freeze-drying to obtain the purified ANFS fucoidan of the Ascophyllum nodosum; the anion exchange column is a Q-Sepharose Fast Flow anion exchange column, and 0, 0.5, 1.0, 1.5, 2 and 2.5 mol / L NaCl solutions are used for stepwise elution, and the 2 mol / L NaCl elution component is collected as the target component.

3. The preparation method according to claim 2, characterized in that, In step (5), the gel column is a Sephacryl S-400 / HR gel column, and the elution condition is 0.2 mol / L NH4HCO3 solution, and the flow rate is 0.3 mL / min.

4. The use of the Ascophyllum nodosum fucan ANFS according to claim 1 for the preparation of a preparation for the prevention and / or treatment of Alzheimer's disease, characterized in that, The ANFS fucoidan of the Ascophyllum nodosum can improve the behavior disorder of zebrafish and inhibit acetylcholinesterase activity.

5. Use according to claim 4, characterized in that, The ANFS fucoidan of the Ascophyllum nodosum can improve the behavior disorder of zebrafish and inhibit acetylcholinesterase activity.

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