Low molecular weight polysaccharide prepared from sarcodia montagneana and having anti-Alzheimer's disease activity

Abstract

The invention provides the low-molecular-weight polysaccharide which is prepared from the sarcodia montagneana and has anti-Alzheimer's disease activity, so that the application prospect of the sarcodia montagneana is effectively improved, and a new component choice is provided for treatment of Alzheimer's disease. The low molecular weight polysaccharide provided by the invention is prepared by carrying out fermentation on sarcodia montagneana by using an Alternania sp. Fungus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.15181. The invention provides a method for preparing low-molecular-weight polysaccharide with anti-Alzheimer's disease activity from sarcodia montagneana, and the prepared low-molecular-weight polysaccharide shows an obvious A beta aggregation inhibition effect in an in-vitro experiment and also shows a protection effect on an SH-SY5Y cell injury model induced by A beta 1-42. The prepared low-molecular-weight polysaccharide can be used as a functional component in the field of food nutrition or health.

Description

Technical Field

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

[0002] As the global population ages, the incidence of Alzheimer's disease (AD), a common neurodegenerative disease, is increasing year by year, placing a heavy burden on patients and their families. The main features of AD include memory loss, cognitive dysfunction, and behavioral abnormalities. Its pathological mechanism is complex and involves multiple factors, including β-amyloid protein (Aβ) deposition, neurofibrillary tangles, neuroinflammation, and oxidative stress.

[0003] In recent years, more and more studies have shown that active ingredients in natural products have shown great potential in the prevention and treatment of Alzheimer's disease. Auricularia auricula is a wild algae plant found in the deep sea, belonging to the genus Auricularia in the red algae family. Existing studies have shown that Auricularia auricula polysaccharides have multiple biological activities such as antioxidant and anti-tumor activities, which make them a candidate substance for the study of Alzheimer's disease treatment.

[0004] The present invention utilizes the fungus Alternaria sp. W-1 isolated in the laboratory in the early stage to degrade the Auricularia auricularia raw material to obtain low molecular weight Auricularia auricularia polysaccharides, and explores the potential protective effect of the low molecular weight Auricularia auricularia polysaccharides 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 present invention aims to provide a kind of medicine with anti-Alzheimer's function prepared from Auricularia auricula Low molecular weight polysaccharides with anti-dementia activity can be produced, thereby effectively improving the application prospects of Auricularia auricularia and providing new component options for the treatment of Alzheimer's disease.

[0006] The low molecular weight polysaccharide provided by the present invention is a polysaccharide with a deposit number of CGMCC No.15181. Alternaria sp . Prepared by fermenting sea fungus with fungi; Furthermore, the preparation method of the low molecular weight polysaccharide 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; The ethanol solution has a concentration of 85%; 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 (SLP); As a specific record of the embodiment, the fermentation is cultured at 37°C for 24h; The centrifugation is performed at 8000 r / min for 15 min.

[0007] The present invention also provides a use of the low molecular weight polysaccharide in the preparation of a product for preventing or treating Alzheimer's disease; The product is a functional food or medicine; The present invention also provides a product for preventing or treating Alzheimer's disease, which contains the low molecular weight polysaccharide in a pharmacologically effective concentration.

[0008] Furthermore, the product comprises the low molecular weight polysaccharide and a pharmaceutically acceptable carrier. The dosage form of the drug is tablets, granules, powders, capsules or oral liquids.

[0009] The present invention provides a method for preparing a low molecular weight polysaccharide with anti-Alzheimer's disease activity from Auricularia auricula. The prepared low molecular weight polysaccharide not only shows an obvious inhibitory effect on Aβ aggregation in in vitro experiments, but also has an anti-Alzheimer's disease activity. 1-42 The induced SH-SY5Y cell injury model also showed a protective effect. The prepared low molecular weight polysaccharide can be used as a functional component in the field of food nutrition or health. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 :The effect of Auricularia auriculariae low molecular weight polysaccharide on Aβ aggregation detected by ThT fluorescence; Figure 2 :Survival rate of SH-SY5Y cells; Figure 3 : Effects of low molecular weight polysaccharides on SH-SY5Y cell morphology, where A is the blank group, B is the injury group, and C is the sample group; Figure 4 :The effect of low molecular weight polysaccharide on LDH leakage rate of SH-SY5Y cells, Figure 5: Effects of low molecular weight polysaccharides on the protein expression levels of p-Akt, p-GSK-3β, and β-catenin, where A is the effect of low molecular weight polysaccharides (SLP) from Auricularia auricula on the relative expression of p-Akt protein in SH-SY5Y cells; B is the effect of low molecular weight polysaccharides (SLP) from Auricularia auricula on the relative expression of p-GSK-3β protein in SH-SY5Y cells; C is the effect of low molecular weight polysaccharides (SLP) from Auricularia auricula on the relative expression of β-catenin protein in SH-SY5Y cells. DETAILED DESCRIPTION

[0011] The sea fungus used in the embodiment of the present invention is collected from the sea area of ​​Weihai, my country. Alternaria sp . Alternaria W-1 is a fungus isolated from kelp by the Fermented Functional Food Research and Development Laboratory of Qingdao Agricultural University. It was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on January 2, 2018, with the address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 15181. The yeast and Aspergillus niger used were purchased from the strain library, with strain numbers CICC 1300 and CICC 40273 respectively; SH-SY5Y cells were purchased from Pronocell Life Science Technology Co., Ltd.

[0012] The culture medium used for the cells in the specific examples was purchased from Wuhan Punosai Life Science Co., Ltd., and the AChE inhibition rate-related reagents were purchased from Solebao Technology Co., Ltd. 1-42 Inhibition rate-related reagents were purchased from Bio-Technology Co., Ltd.

[0013] The present invention is described in detail below in conjunction with embodiments and drawings.

[0014] Example 1: Preparation of low molecular weight polysaccharide from Auricularia auricula of the present invention First, soak the sea fungus in water for 24 hours, drain the water, dry it, and crush it. Add 85% ethanol at a solid-liquid ratio of 1:30 (g / mL) and soak it for 24 hours, filter it, collect the filter residue and dry it to obtain sea fungus powder for later use.

[0015] Add the pre-treated Auricularia auricula powder to deionized water at a solid-liquid ratio of 1:30 (g / mL), adjust the pH to 4.5, and sterilize for later use. Alternaria sp. W-1, yeast and Aspergillus niger were added to the Auricularia auricula solution at a 5% inoculation rate, cultured at 37°C for 24 h, boiled for 15 min, centrifuged at 8000 r / min for 10 min, the supernatant was collected, concentrated to 1 / 2 of the original volume at 55°C, dialyzed and purified with a MW500 dialysis bag for 48 h, and freeze-dried to obtain Alternaria spFermented Auricularia auricularia low molecular weight polysaccharide (SLP), yeast fermented Auricularia auricularia low molecular weight polysaccharide (SSLP) and Aspergillus niger fermented Auricularia auricularia low molecular weight polysaccharide (ASLP).

[0016] Example 2: Molecular weight determination of three low molecular weight polysaccharides of Auricularia auricula described in the present invention Will Alternaria sp The molecular weights of fermented Auricularia auricularia low molecular weight polysaccharides (SLP), yeast fermented Auricularia auricularia low molecular weight polysaccharides (SSLP) and Aspergillus niger fermented Auricularia auricularia low molecular weight polysaccharides (ASLP) were determined using an 18-angle laser scattering instrument. Specifically, three Auricularia auricularia low molecular weight polysaccharide solutions with a sample concentration of 0.1 mg / mL were prepared, filtered through a 0.22μm filter membrane, and injected into the instrument.

[0017] Chromatographic conditions: PSS GRAM 3000Å ​​column; flow rate 0.75 mL / min; injection volume 100 μL; column temperature 35°C; mobile phase 0.02% NaN2 solution. Data were analyzed using Astra software (Wyatt Technology, Santa Barbara, CA, USA), and the refractive index increment (dn / dc) was 0.138 mL / g.

[0018] The molecular weight determination results of three low molecular weight polysaccharides of Auricularia auricula are shown in Table 1.

[0019] Table 1: Molecular weight distribution of three polysaccharides Sample name peak Weight average molecular weight (Da) Number average molecular weight (Da) Polydispersity Index Yeast fermentation of low molecular weight polysaccharides from Auricularia auricula Peak 1 <![CDATA[6.141×10 5 (±4.329%)]]> <![CDATA[9.391×10 5 (±4.649%)]]> 6.539（±66.352%） Low molecular weight polysaccharide from Auricularia auricula fermented by Aspergillus niger Peak 1 <![CDATA[5.461×10 5 (±4.631%)]]> <![CDATA[1.444×10 5 (±4.093%)]]> 3.781（±6.180%） Fermented Auricularia auricula low molecular weight polysaccharide Peak 1 <![CDATA[7.172×10 3 (±20.466%)]]> <![CDATA[1.197×10 3 (±23.404%)]]> 5.993（±31.090%） Example 3: In vitro inhibition of Aβ by three low molecular weight polysaccharides from Auricularia auricula 1-42 Aggregation ability The aggregation of β-amyloid protein (Aβ) is one of the important pathogenic factors of Alzheimer's disease. The abnormal increase of β-folded structure in Aβ aggregates can lead to neurotoxicity, which in turn triggers a series of pathological changes in Alzheimer's disease. ThT is a benzothiazole dye that can specifically bind to the β-folded structure in Aβ aggregates. The bound ThT will emit strong fluorescence at a wavelength of 480 nm, and the fluorescence intensity will increase with the increase of β-folded structure, and is proportional to the concentration of Aβ aggregates within a certain range. Therefore, ThT fluorescence experiments are widely used for experimental characterization in Aβ aggregation research. The low molecular weight polysaccharide of Auricularia auricula was mixed with 10 μM pretreated Aβ 1-42 After co-incubation, 20 μL of the mixture was mixed with 180 μL of 5 μM ThT solution and sampled for detection after 36 hours of incubation. 1-42The blank control and EGCG were used as the positive control. The fluorescence intensity was measured by a microplate reader at an excitation wavelength of λex = 450 nm and an emission wavelength of λem = 480 nm to further calculate the Aβ 1-42 Inhibition rate. In vitro Aβ 1-42 The inhibition rate is calculated according to the following formula:

[0020] Where: Fa is the fluorescence intensity after mixing the sample, Fb is the fluorescence intensity of the sample solution alone, and Fo is the fluorescence intensity of spontaneous aggregation when Aβ is alone.

[0021] As can be seen from Figure 1, when Aβ was incubated alone at 37°C 1 - 42 When the monomer solution (i.e. blank control) was used, the fluorescence intensity of ThT showed a gradual increase. This phenomenon indicates that Aβ1-42 monomers self-assembled and aggregated over time. 1 - 42 After the monomers were incubated with Auricularia auriculariae low molecular weight polysaccharides and EGCG, although the fluorescence intensity also increased with time, its fluorescence intensity was significantly lower than that of Aβ 1 - 42 Fluorescence intensity of monomers when incubated alone. Among the three low molecular weight polysaccharides from Auricularia auricula, SLP had the best inhibitory effect, followed by SSLP, and the effect of ASLP was relatively poor.

[0022] Example 4: Neuroprotective effect and anti-Alzheimer's disease activity of the Auricularia auricula low molecular weight polysaccharide SLP of the present invention 1) Detection of SLP on Aβ using CCK-8 method 1-42 Preventive and protective effects of induced SH-SY5Y cell injury model Using cell survival rate as an indicator, oligomeric Aβ 1-42 Induce SH-SY5Y cells to establish a cell injury model. The experiment set up four groups: blank control group, Aβ injury group (50 μM), positive control group and sample group (1 μg / mL, 5 μg / mL, 25 μg / mL, 50μg / mL, 100 μg / mL, 200 μg / mL). Each group was set up with 6 parallel wells, each well volume was 100 μL, and the edge wells were filled with sterile DPBS. After 24 hours of culture, the control group was replaced with DMEM / F-12 complete medium; after 24 hours of culture, oligomeric Aβ was added to the Aβ injury group. 1-42 (50 μM), and then cultured for 48 hours; after 24 hours of culture, different concentrations of samples were added to the sample group, and the supernatant was discarded after 4 hours of pre-protection, and then oligomeric Aβ was added 1-42(50 μM) for 48 hours. During the injury period, take photos to observe and record the injury. Afterwards, discard the supernatant in the 96-well plate, add CCK-8 solution to each well according to the kit instructions, and incubate in an incubator at 37°C and 5% CO2 for 4 hours. Oscillate on a microplate reader for 10 minutes, and measure the absorbance of each well at a wavelength of 490 nm to calculate the cell survival rate.

[0023] Figure 2 shows the results of the low molecular weight polysaccharide of Auricularia auricula on Aβ 1-42 Effect of induced injury on the survival rate of SH-SY5Y cells. Figure 2 It can be seen that when the sample concentration is 1 µg / mL and 5 µg / mL, the low molecular weight polysaccharide of Auricularia auricula has a promoting effect on cell proliferation, and the proliferation effect is the best, with the proliferation rates being 107.9% and 103.39%, respectively.

[0024] Figure 3 shows the morphological changes of cells under different treatment conditions. In the blank group (A), the axons of SH-SY5Y cells extended significantly, with a rounded and shiny morphology; 1-42 After injury alone (B), the axons of the cells disappeared, the shape became round, and the volume was significantly reduced. 1-42 In cells pre-protected by Auricularia auriculariae low molecular weight polysaccharide (C), the degree of injury was significantly reduced and the cell morphology was relatively intact. The results showed that this sample could inhibit Aβ 1-42 , reducing its damage to cell morphology.

[0025] 2) Effect of Auricularia auriculariae low molecular weight polysaccharide SLP on LDH leakage rate LDH is a glycolytic enzyme that converts lactate into pyruvate in the microbial lactate 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 assessed by detecting its leakage rate in the culture medium. Vigorous SH-SY5Y cells were inoculated in 96-well plates, with 1×10 4 After 24 hours of culture, the cells were divided into four groups: blank control group, Aβ injury group (50 μM), positive control group and sample group (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 performed: the control group was replaced with DMEM / F-12 complete medium; the Aβ injury group was added with oligomeric Aβ 1-42 (50 μM) for 48 hours; 1, 5, 10, 15, and 25 μg / mL samples were added to the sample groups according to the dose. After 4 hours of preprotection, the supernatant was removed and Aβ was added 1-42(50 μM) for 48 hours. Wash each group with sterile D-Hanks 1-2 times, remove the supernatant; dilute the LDH release reagent 10 times with DPBS according to the instructions of the LDH kit, mix well and add to each well (150 μL), incubate at 37℃ for 1 hour. Take 120 μL of supernatant from each well to a new 96-well plate, and measure the absorbance at 490 nm.

[0026] As shown in Figure 4, Aβ 1-42 Incubation of SH-SY5Y cells alone for 48 h resulted in significantly aggravated cell damage and significantly increased LDH release levels (p<0.05). Compared with the injury group, the LDH release levels decreased to varying degrees after pre-protection with different concentrations of Auricularia auricula low molecular weight polysaccharides, and samples with concentrations of 1 µg / mL, 5 µg / mL, 10 µg / mL, 15 µg / mL and 25 µg / mL significantly inhibited the increase of LDH (p<0.05).

[0027] 3) Detection of protein expression levels of p-Akt, p-GSK-3β, and β-catenin by enzyme-linked immunosorbent assay The PI3K / Akt pathway is closely related to the formation of Aβ. Akt, as a downstream target of PI3K, exerts a neuroprotective effect through phosphorylation. Inhibition of Akt signaling enhances the activity of GSK-3β, and phosphorylation of GSK-3β activates γ-secretase, thereby increasing Aβ. 1-42 The synthesis of Aβ can aggravate the AD condition. By regulating the cleavage of Aβ 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 neural regulation in the pathogenesis of AD. To establish a cell injury model, cells were lysed according to the instructions of RIPA lysis buffer, centrifuged at 4℃ and 15000 r / min for 15 minutes, and the supernatant was taken. The p-Akt, p-GSK-3β and β-catenin protein contents were determined according to the instructions of the ELISA kit, and the total protein amount of each group was determined by the BCA method to correct the experimental results.

[0028] like Figure 5 As shown in A, compared with the blank group, the relative protein expression of p-Akt in the injury group was significantly downregulated (p<0.05), indicating that the modeling method can significantly affect the protein expression level of p-Akt; compared with the injury group, both concentrations of the experimental group significantly upregulated the expression of p-Akt protein. Among them, the protein expression of p-Akt in the 1 µg / mL sample group was significantly higher than that in the injury group. This shows that the sample can upregulate the expression of p-Akt protein and has an effect on Aβ 1-42It induces neurotoxicity in SH-SY5Y cells and plays a protective role.

[0029] p-GSK-3β protein expression Figure 5 As shown in B, compared with the blank group, the expression of p-GSK-3β protein in the injury group was significantly downregulated (p<0.05); compared with the injury group, the expression levels of p-GSK-3β protein in the 1 µg / mL and 10 µg / mL sample groups were significantly upregulated. 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 with the positive control group (p>0.05), indicating that the low-concentration Auricularia auricula low-molecular-weight polysaccharide has a certain effect of upregulating the expression of p-GSK-3β protein, thereby affecting the central nervous system and reducing the toxicity of Aβ.

[0030] like Figure 5 As shown in B, compared with the blank group, the expression of β-catenin protein in the injury group was significantly downregulated (p<0.05); compared with the injury group, the protein expression levels of the sample groups significantly upregulated the expression of β-catenin protein, but there was no significant difference between the two groups of samples (p>0.05), indicating that the two components could not regulate the upregulation of β-catenin protein expression levels in a concentration-dependent manner. However, it can be seen from the figure that the 1 µg / mL and 10 µg / mL groups were significantly different from the positive control group (p<0.05), indicating that the sample can upregulate β-catenin protein expression.

[0031] In summary, the present invention provides a low molecular weight polysaccharide with anti-Alzheimer's disease activity prepared from Auricularia auricula. The polysaccharide is prepared using the CGMCC No.15181 Alternaria sp. W-1 fungus fermented Auricularia auricula collected from the Weihai waters of my country. In vitro experiments showed that the low molecular weight polysaccharide prepared by it can effectively inhibit Aβ aggregation. Further studies have found that SLP has an inhibitory effect on Aβ. 1 - 42 The induced SH-SY5Y cell injury model has a preventive and protective effect, which can promote cell proliferation, reduce cell morphological damage, reduce LDH leakage rate, and regulate key proteins in the PI3K / Akt and Wnt / β-catenin signaling pathways associated with Alzheimer's disease (such as upregulating p-Akt, p-GSK-3β, and β-catenin protein expression). The results show that the low molecular weight polysaccharide of Auricularia auricula has significant neuroprotective effects and anti-Alzheimer's disease activity, and has great potential in the development of anti-Alzheimer's disease functional foods or drugs, providing a new direction and valuable material basis for the treatment of Alzheimer's disease.

Claims

1. A low molecular weight polysaccharide with anti-Alzheimer's disease activity prepared from Auricularia auricula, characterized in that: The low molecular weight polysaccharide is a polysaccharide with a deposit number of CGMCC No.15181. Alternaria sp .Prepared by fermenting sea fungus with fungi.

2. The low molecular weight polysaccharide according to claim 1, characterized in that The preparation method of the low molecular weight polysaccharide 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 the Auricularia auricula low molecular weight polysaccharide.

3. The low molecular weight polysaccharide according to claim 2, characterized in that The concentration of the ethanol solution in 1) is 85%.

4. The low molecular weight polysaccharide according to claim 2, characterized in that The fermentation in 2) is cultured at 37°C for 24 hours.

5. The low molecular weight polysaccharide according to claim 2, characterized in that The centrifugation in 2) is performed at 8000 r / min for 15 min.

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