A low-molecular-weight polysaccharide prepared from Sargassum fusiforme with anti-Alzheimer's disease activity

Abstract

The present invention provides a low-molecular-weight polysaccharide prepared from Sargassum fusiforme, which effectively improves the application prospect of Sargassum fusiforme and provides a new component option for the treatment of Alzheimer's disease. The low-molecular-weight polysaccharide provided by the present invention is prepared by fermenting Sargassum fusiforme with a Alternaria sp . fungus with the preservation number of CGMCC No. 15181. The present invention provides a method for preparing a low-molecular-weight polysaccharide with anti-Alzheimer's disease activity from Sargassum fusiforme. The prepared low-molecular-weight polysaccharide shows an obvious effect of inhibiting Aβ aggregation in in vitro experiments, and it also shows a protective effect on the SH-SY5Y cell damage model induced by Aβ 1‑42 . The prepared low-molecular-weight polysaccharide can be used as a functional component in the fields of food nutrition or health.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of bioactive products, and particularly relates to a low-molecular-weight polysaccharide with anti-Alzheimer's disease activity prepared from Sargassum fusiforme. Background Art

[0002] With the aggravation of the aging of the global population, Alzheimer's disease (AD), as a common neurodegenerative disease, has an increasing incidence rate year by year, bringing a heavy burden to patients and their families. The main characteristics of Alzheimer's disease include memory loss, cognitive dysfunction, and behavioral abnormalities. Its pathological mechanism is complex and involves multiple factors, including β-amyloid (Aβ) deposition, neurofibrillary tangles, neuroinflammation, and oxidative stress, etc.

[0003] In recent years, more and more studies have shown that the active ingredients in natural products have shown great potential in the prevention and treatment of Alzheimer's disease. Sargassum fusiforme is a wild algae plant produced in the deep sea and belongs to the genus Sargassum in the Rhodophyta. Existing studies have shown that Sargassum fusiforme polysaccharide has various biological activities such as antioxidant and anti-tumor activities, and these characteristics make it a candidate substance for studying the treatment of Alzheimer's disease.

[0004] The present invention uses the fungus Alternaria sp. W-1 isolated in the early stage of the laboratory to degrade the Sargassum fusiforme raw material to obtain a low-molecular-weight Sargassum fusiforme polysaccharide, and explores the potential protective effect of the low-molecular-weight Sargassum fusiforme polysaccharide on the Alzheimer's disease model, in order to provide a new strategy for the prevention and treatment of Alzheimer's disease. Summary of the Invention

[0005] The purpose of the present invention is to provide a low-molecular-weight polysaccharide with anti-Alzheimer

[0006] disease activity prepared from Sargassum fusiforme, so as to effectively improve the application prospect of Sargassum fusiforme and provide a new component selection for the treatment of Alzheimer's disease.

[0007] The low-molecular-weight polysaccharide provided by the present invention is prepared by fermenting Sargassum fusiforme with the fungus Alternaria sp. with the preservation number of CGMCC No. 15181;

[0008] Furthermore, the preparation method of the low-molecular-weight polysaccharide includes the following steps:

[0009] 1) After crushing Sargassum fusiforme, soak it with an ethanol solution, filter and collect the filter residue and dry it to obtain Sargassum fusiforme powder;

[0010] The ethanol solution has a concentration of 85%;

[0011] 2) After dissolving the Sargassum fusiforme powder prepared in step 1) in water, it is fermented with the fungus Alternaria sp. with the preservation number CGMCC No. 15181. After the fermentation is completed, it is boiled and centrifuged, the supernatant is collected, rotary evaporated and concentrated, dialyzed and purified, and freeze-dried to obtain the low molecular weight polysaccharide (SLP) of Sargassum fusiforme;

[0012] As a specific record of the embodiment, the fermentation is carried out at 37 °C for 24 h;

[0013] The centrifugation is carried out at 8000 r / min for 15 min.

[0014] The present invention also provides a use of the low molecular weight polysaccharide, which is an application in the preparation of products for preventing or treating Alzheimer's disease;

[0015] The product is a functional medicine;

[0016] The present invention also provides a product for preventing or treating Alzheimer's disease, which contains the low molecular weight polysaccharide at a pharmacologically effective concentration.

[0017] Further, the product contains the above-mentioned low molecular weight polysaccharide and a pharmaceutically acceptable carrier. The dosage form of the drug is tablets, granules, powders, capsules or oral liquids.

[0018] The present invention provides a method for preparing a low molecular weight polysaccharide with anti-Alzheimer's disease activity from Sargassum fusiforme. The prepared low molecular weight polysaccharide shows an obvious inhibitory effect on Aβ aggregation in in vitro experiments, and it also shows a protective effect on the SH-SY5Y cell injury model induced by Aβ 1-42 induction. Description of the Drawings

[0019] Figure 1 : ThT fluorescence detection of the effect of the low molecular weight polysaccharide of Sargassum fusiforme on Aβ aggregation;

[0020] Figure 2 : Survival rate graph of SH-SY5Y cells;

[0021] Figure 3 : Effect of low molecular weight polysaccharide on the morphology of SH-SY5Y cells, where A is the blank group, B is the injury group, and C is the sample group;

[0022] Figure 4 : Effect of low molecular weight polysaccharide on the LDH leakage rate of SH-SY5Y cells,

[0023] Figure 5 : Effect of low molecular weight polysaccharide on the protein expression levels of p-Akt, p-GSK-3β, and β-catenin. Detailed implementation manners

[0024] In the embodiments of the present invention, Sargassum fusiforme is collected from the sea area of Weihai, China. Alternaria sp. W-1 used is a fungus previously isolated from kelp by the Research and Development Laboratory of Fermented Functional Foods, Qingdao Agricultural University, and was deposited in the China General Microbiological Culture Collection Center on January 2, 2018, with the deposit number CGMCC No. 15181. The yeasts and Aspergillus niger used are purchased from the strain bank, and the strain numbers are CICC 1300 and CICC40273 respectively; SH-SY5Y cells are purchased from Punosai Life Technology Co., Ltd.

[0025] In the specific embodiments, the cell culture medium is purchased from Punosai Life Technology Co., Ltd. in Wuhan, and the reagents related to the AChE inhibition rate are purchased from Solarbio Science & Technology Co., Ltd., and the reagents related to the Aβ 1-42 inhibition rate are purchased from Beyotime Institute of Biotechnology.

[0026] The present invention will be described in detail below in conjunction with the embodiments and the drawings.

[0027] Example 1: Preparation of low molecular weight polysaccharides from Sargassum fusiforme of the present invention

[0028] First, soak Sargassum fusiforme in clear water for 24 h, drain the water, dry it, and crush it. Add 85% ethanol at a solid-liquid ratio of 1:30 (g / mL) and soak for 24 h, filter, collect the filter residue and dry it to obtain Sargassum fusiforme powder for standby.

[0029] Add the pretreated Sargassum fusiforme powder to deionized water at a solid-liquid ratio of 1:30 (g / mL), adjust the pH to 4.5, and sterilize for standby. Add the activated Alternaria sp. W-1, yeasts and Aspergillus niger to the Sargassum fusiforme solution at an inoculation amount of 5% respectively, culture at 37 °C for 24 h, boil for 15 min, centrifuge at 8000 r / min for 10 min, collect the supernatant, rotary evaporate and concentrate to 1 / 2 of the original volume at 55 °C, dialyze and purify with a MW500 dialysis bag for 48 h, and freeze-dry to obtain Alternaria sp-fermented low molecular weight polysaccharides from Sargassum fusiforme (SLP), yeast-fermented low molecular weight polysaccharides from Sargassum fusiforme (SSLP) and Aspergillus niger-fermented low molecular weight polysaccharides from Sargassum fusiforme (ASLP).

[0030] Example 2: Determination of the molecular weights of the three low molecular weight polysaccharides from Sargassum fusiforme of the present invention

[0031] The molecular weights of Alternaria sp-fermented Sargassum fusiforme low molecular weight polysaccharide (SLP), yeast-fermented Sargassum fusiforme low molecular weight polysaccharide (SSLP), and Aspergillus niger-fermented Sargassum fusiforme low molecular weight polysaccharide (ASLP) were determined using a multi-angle laser light scattering instrument. Specifically, three kinds of Sargassum fusiforme low molecular weight polysaccharide solutions with a sample concentration of 0.1 mg / mL were prepared, filtered through a 0.22 μm filter membrane, and injected into the instrument.

[0032] Chromatographic conditions: PSS GRAM Chromatographic column; flow rate 0.75 mL / min; injection volume 100 μL; column temperature 35 °C; mobile phase 0.02% NaN2 solution. The data was analyzed using Astra software (Wyatt Technology, Santa Barbara, CA, USA), and the refractive index increment (dn / dc) was 0.138 mL / g.

[0033] The results of the molecular weight determination of the three low molecular weight Sargassum fusiforme polysaccharides are shown in Table 1.

[0034] Table 1: Molecular weight distribution table of three polysaccharides

[0035]

[0036] Example 3: In vitro inhibition of Aβ aggregation by three low molecular weight Sargassum fusiforme polysaccharides 1-42 The aggregation of β-amyloid (Aβ) is one of the important pathogenic factors of Alzheimer's disease. The abnormal increase in the β-sheet structure in Aβ aggregates leads to neurotoxicity and then triggers a series of pathological changes in Alzheimer's disease. ThT is a benzothiazole dye that can specifically bind to the β-sheet structure in Aβ aggregates. After binding, ThT emits strong fluorescence at a wavelength of 480 nm, and the fluorescence intensity increases with the increase in the β-sheet structure and is proportional to the concentration of Aβ aggregates within a certain range. Therefore, the ThT fluorescence experiment is widely used for experimental characterization in Aβ aggregation research. After co-incubating the low molecular weight Sargassum fusiforme polysaccharide with pre-treated 10 μM Aβ, 20 μL of the mixed solution was taken and mixed with 180 μL of 5 μM ThT solution. After incubating for 36 hours, samples were taken for detection. Using the separately incubated Aβ as a blank control and EGCG as a positive control, the fluorescence intensity was measured by a microplate reader at an excitation wavelength λex = 450 nm and an emission wavelength λem = 480 nm, and the Aβ inhibition rate was further calculated. The in vitro Aβ inhibition rate is calculated according to the following formula: 1-42 After co-incubating the low molecular weight Sargassum fusiforme polysaccharide with pre-treated 10 μM Aβ, 20 μL of the mixed solution was taken and mixed with 180 μL of 5 μM ThT solution. After incubating for 36 hours, samples were taken for detection. Using the separately incubated Aβ 1-42 as a blank control and EGCG as a positive control, the fluorescence intensity was measured by a microplate reader at an excitation wavelength λex = 450 nm and an emission wavelength λem = 480 nm, and the Aβ 1-42 inhibition rate was further calculated. The in vitro Aβ 1-42 inhibition rate is calculated according to the following formula:

[0037]

[0038] Wherein: Fa is the fluorescence intensity after mixing the samples. Fb is the fluorescence intensity of only the sample solution, and Fo is the fluorescence intensity of the spontaneous aggregation of Aβ alone.

[0039] From Figure 1 It can be observed that when Aβ 1-42 monomer solution (i.e., blank control) is incubated alone at 37 °C, the fluorescence intensity of ThT shows a gradually increasing trend. This phenomenon indicates that Aβ1-42 monomers undergo self-assembly aggregation over time. However, when Aβ 1-42 monomers are co-incubated with low molecular weight polysaccharides from Sargassum fusiforme and EGCG respectively, although the fluorescence intensity also increases with the extension of time, its fluorescence intensity is significantly lower than that of Aβ 1-42 monomers incubated alone. Among the three low molecular weight polysaccharides from Sargassum fusiforme, SLP has the best inhibitory effect, SSLP is the second, and ASLP has a relatively poor effect.

[0040] Example 4: Neuroprotective effect and anti-Alzheimer's disease activity of the low molecular weight polysaccharide SLP from Sargassum fusiforme of the present invention

[0041] 1) Use the CCK-8 method to detect the preventive and protective effect of SLP on the Aβ 1-42 induced SH-SY5Y cell injury model

[0042] Taking cell viability as an index, oligomeric Aβ 1-42 is used to induce SH-SY5Y cells to establish a cell injury model. The experiment is set up with four groups: a blank control group, an Aβ injury group (50 μM), a positive control group, and a sample group (1 μg / mL, 5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL). Each group has 6 parallel wells, with a volume of 100 μL per well, and the edge wells are filled with sterile DPBS. After culturing for 24 hours, the control group is replaced with DMEM / F-12 complete medium; the Aβ injury group is added with oligomeric Aβ 1-42 (50 μM) after culturing for 24 hours and continues to be cultured for 48 hours; the sample group is added with different concentrations of the sample after culturing for 24 hours, and after 4 hours of pre-protection, the supernatant is discarded, and then oligomeric Aβ 1-42 (50 μM) is added to damage for 48 hours. During the damage period, photos are taken to observe and record the damage situation. Then, the supernatant in the 96-well plate is discarded, and CCK-8 solution is added to each well according to the kit instructions and incubated in an incubator at 37 °C and 5% CO 2 for 4 hours. Shake for 10 minutes on an enzyme-linked immunosorbent assay (ELISA) reader, and measure the absorbance value of each well at a wavelength of 490 nm to calculate the cell viability.

[0043] Figure 2 The results show the low molecular weight polysaccharide from Sargassum fusiforme on Aβ1-42 Effect on the survival rate of SH-SY5Y cells induced to be damaged. As Figure 2 shown, when the sample concentrations were 1 μg / mL and 5 μg / mL, the low molecular weight polysaccharide from Sargassum fusiforme had a promoting effect on cell proliferation, and the proliferation effect was the best, with the proliferation rates being 107.9% and 103.39% respectively.

[0044] Figure 3 The morphological changes of cells under different treatment conditions were shown. In the blank group (A), the axons of SH-SY5Y cells extended significantly, and the morphology was round and shiny; after adding Aβ 1-42 alone for damage (B), the axons of the cells disappeared, the morphology was round, and the volume decreased significantly. While in the cells pre-protected with the low molecular weight polysaccharide from Sargassum fusiforme after Aβ 1-42 damage (C), the degree of damage was significantly reduced, and the cell morphology was relatively intact. The results showed that this sample could reduce the damage to cell morphology by inhibiting Aβ 1-42 .

[0045] 2) Effect of the low molecular weight polysaccharide SLP from Sargassum fusiforme on the LDH leakage rate

[0046] LDH is a glycolytic enzyme that converts lactic acid to pyruvate in the microbial lactic acid synthesis pathway. When the cell membrane is damaged, lactate dehydrogenase will be released into the culture medium, so the degree of cell damage can be evaluated by detecting its leakage rate in the culture medium. Vigorously growing SH-SY5Y cells were seeded in 96-well plates at 1×10 4 cells per well, and after culturing for 24 hours, they were divided into four groups: blank control group, Aβ damage group (50 μM), positive control group, and sample groups (1, 5, 10, 15, 25 μg / mL). Each group had 6 parallel wells, 100 μL per well, and the edge wells were filled with sterile DPBS. After 24 hours, the following treatments were carried out: the control group was replaced with DMEM / F-12 complete medium; the Aβ damage group was treated with oligomeric Aβ 1-42 (50 μM) for 48 hours; the sample groups were added with samples at doses of 1, 5, 10, 15, 25 μg / mL respectively, and after pre-protecting for 4 hours, the supernatant was removed, and then Aβ 1-42 (50 μM) was added for treatment for 48 hours. Each group was washed 1 - 2 times with sterile D-Hanks to remove the supernatant; according to the instructions of the LDH kit, the LDH release reagent was diluted 10 times with DPBS, and after mixing, it was added to each well (150 μL) and incubated at 37 °C for 1 hour. 120 μL of the supernatant was taken from each well to a new 96-well plate, and the absorbance was measured at 490 nm.

[0047] As Figure 4 shown, Aβ 1-42Incubating SH-SY5Y cells alone for 48 h led to a significant exacerbation of cell damage and a significant increase in the LDH release level (p < 0.05). Compared with the damage group, after pre-protection with low molecular weight polysaccharides from Sargassum fusiforme at different concentrations, the LDH release level decreased to varying degrees. Among them, the samples at concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 25 μg / mL significantly inhibited the increase in LDH (p < 0.05).

[0048] 3) Detect the protein expression levels of p-Akt, p-GSK-3β, and β-catenin by enzyme-linked immunosorbent assay

[0049] The PI3K / Akt pathway is closely related to the formation of Aβ. As a downstream target of PI3K, Akt plays a neuroprotective role through phosphorylation. Inhibiting Akt signal transduction will enhance the activity of GSK-3β, while phosphorylation of GSK-3β will activate γ-secretase, thereby increasing Aβ 1-42 synthesis and exacerbating the condition of AD. By regulating the cleavage of amyloid precursor protein (APP) by γ-secretase, GSK-3β can also reduce Aβ production and delay the occurrence of AD. In addition, the Wnt / β-catenin signaling pathway also plays an important role in neuroregulation in the pathogenesis of AD. Establish a cell damage model, lyse the cells according to the instructions of RIPA lysis buffer, centrifuge at 15000 r / min at 4 °C for 15 minutes, and take the supernatant. Determine the protein contents of p-Akt, p-GSK-3β, and β-catenin according to the instructions of the ELISA kit, and measure the total protein amount of each group by the BCA method to correct the experimental results.

[0050] As Figure 5 (A) shows that compared with the blank group, the relative protein expression of p-Akt in the damage group was significantly down-regulated (p < 0.05), indicating that the modeling method could significantly affect the protein expression level of p-Akt; compared with the damage group, both concentrations in the experimental group significantly up-regulated the protein expression of p-Akt. Among them, the protein expression of p-Akt in the 1 μg / mL sample group was significantly higher than that in the damage group. It shows that this sample can up-regulate the protein expression of p-Akt and play a protective role against Aβ 1-42 induced neurotoxicity in SH-SY5Y cells.

[0051] The protein expression of p-GSK-3β is as Figure 5As shown in (B), compared with the blank group, the protein expression of p-GSK-3β in the injury group was significantly down-regulated (p < 0.05); compared with the injury group, the protein expression levels of p-GSK-3β in the sample groups of 1 μg / mL and 10 μg / mL were significantly up-regulated. Among them, the protein expression in the 1 μg / mL group was significantly higher than that in the blank group (p < 0.05), and there was no significant difference compared with the positive control group (p > 0.05), indicating that the low molecular weight polysaccharide of Sargassum fusiforme at a lower concentration has a certain effect of up-regulating the protein expression of p-GSK-3β, thereby affecting the central nervous system and reducing the toxicity of Aβ.

[0052] As Figure 5 shown in (B), compared with the blank group, the protein expression of β-catenin in the injury group was significantly down-regulated (p < 0.05); compared with the injury group, the protein expression levels of β-catenin in the sample groups were significantly up-regulated, but there was no significant difference between the two sample groups (p > 0.05), indicating that the two components cannot up-regulate the protein expression level of β-catenin in a concentration-dependent manner. However, it can be seen from the figure that there was a significant difference between the groups of 1 μg / mL and 10 μg / mL and the positive control group (p < 0.05), indicating that the sample can up-regulate the protein expression of β-catenin.

[0053] In summary, the present invention provides a low molecular weight polysaccharide with anti-Alzheimer's disease activity prepared from Sargassum fusiforme. The polysaccharide is prepared by fermenting Sargassum fusiforme collected from the Weihai sea area of China with the fungus Alternaria sp. W-1 with the preservation number of CGMCC No. 15181. In vitro experiments show that the low molecular weight polysaccharide of Sargassum fusiforme prepared by it can effectively inhibit Aβ aggregation. Further research found that SLP 1-42 has a preventive and protective effect on the SH-SY5Y cell injury model induced by Aβ, can promote cell proliferation, reduce cell morphological damage, reduce the LDH leakage rate, and can also regulate the key proteins in the PI3K / Akt and Wnt / β-catenin signaling pathways related to Alzheimer's disease (such as up-regulating the protein expressions of p-Akt, p-GSK-3β, and β-catenin). The results show that the low molecular weight polysaccharide of Sargassum fusiforme has significant neuroprotective effects and anti-Alzheimer's disease activity, has great potential in the development of anti-Alzheimer's drugs, and provides a new direction and valuable material basis for the treatment of Alzheimer's disease.

Claims

1. Use of a low molecular weight polysaccharide in the preparation of a drug with anti-Alzheimer's disease activity, characterized in that: The low molecular weight polysaccharide is a polysaccharide with a deposit number of CGMCC No.15181. Alternaria sp . The fungus is fermented to prepare the sea fungus, and the preparation method comprises the following steps: 1) After crushing the sea fungus, soaking it in an ethanol solution, filtering it, collecting the filter residue and drying it to obtain sea fungus powder; 2) Dissolve the fungus powder prepared in step 1) in water, and then Alternaria sp The fungus is fermented, and after the fermentation is completed, it is boiled and centrifuged, the supernatant is collected, concentrated by rotary evaporation, purified by dialysis, and freeze-dried to obtain auricularia auricula low molecular weight polysaccharide; The concentration of the ethanol solution is 85%; The fermentation is carried out by culturing at 37°C for 24 hours; The centrifugation is performed at 8000 r / min for 15 min.

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