CN114478816A

CN114478816A - White jade snail polysaccharide and preparation method and application thereof

Info

Publication number: CN114478816A
Application number: CN202210129044.9A
Authority: CN
Inventors: 方建平; 秦浩
Original assignee: Shanghai Xingtang Biotechnology Co ltd
Current assignee: Shanghai Xingtang Biotechnology Co ltd
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2022-05-13

Abstract

The invention provides white jade snail polysaccharide and a preparation method and application thereof. The white jade snail polysaccharide WJPS is obtained by extracting, separating and purifying the white jade snail by using a simple and effective polysaccharide extraction process and method. Pharmacological experiment results show that the polysaccharide WJPS can inhibit the aggregation of Abeta and inhibit Abeta at the level of human neuroblastoma cells₄₂Aggregation-induced cytotoxicity. Therefore, the compound has potential effect of treating the Alzheimer disease, and is expected to be developed into a medicine aiming at the Alzheimer disease.

Description

White jade snail polysaccharide and preparation method and application thereof

Technical Field

The invention relates to animal-derived polysaccharide, in particular to polysaccharide WJPS extracted from White Jade Snail (Achatina fulica), an extraction method thereof and application thereof in preparing a medicine for preventing and/or treating Alzheimer disease or a health-care product for assisting in improving memory function.

Background

Alzheimer's Disease (AD), also known as Alzheimer's disease, is a chronic progressive neurodegenerative disease, mainly manifested by progressive memory decline, cognitive dysfunction and loss of self-care ability (Int J Geriatr Psychiatry,2012,27(4): 364-.

With the continuous development of science and technology, people are continuously and deeply exploring the mechanism of AD occurrence, and researches show that a large amount of pathological changes such as senile plaques, neuron loss and the like can be detected in the brain of an AD onset patient. These pathological changes are also a major causative factor in amyloid cerebrovascular disease (International J. Anesthesia and Resuscitation, 2012,33(2): 99-102; Neuroc:. RTM.;)hem Res,2012,37(9): 1829-. Among them, abnormal expression and deposition of β -amyloid (a β) in the brain are central links in the initiation of AD (Nature Reviews Neuroscience,2007,8(7),499- & 509). Beta 0-amyloid has a relative molecular weight of 4.12kD, which is the product of hydrolysis of beta 1-amyloid precursor protein by endogenous proteases. A great deal of research shows that the pathological changes of AD mainly show that a great deal of amyloid precursor protein is secreted and aggregated on a cell membrane, the protein is converted into beta-amyloid protein in a great amount after being hydrolyzed by endogenous protease, A beta mainly exists in a monomeric alpha-helix conformation under the physiological state, and in AD patients, the microenvironment in brain is changed due to factors such as AD-related gene mutation, metal ion concentration increase and pH change, so that the A beta conformation is changed, namely, the A beta is converted into beta-folding and is easy to aggregate into fiber shape, and the soluble A beta oligomers have stronger toxicity than the A beta fibrils (Proc. Natl.Acad. Sci.2008,105(37), 14052-14057; Nature,2002 (6680), 535-539; J Alzheimer's Dis,2011,26(Suppl 3): 321; Pharmacoepidioid Drug (2013, 22(4):345-358), oligomers and aggregated fibrils that can interfere with Ca²⁺Homeostasis, induction of oxidative stress, mitochondrial dysfunction, inflammatory response, induction of Tau protein hyperphosphorylation, and neuronal loss grade-linked neurotoxicity, and ultimately dementia (Neurochemical Research,2007,33(3), 526-. In the occurrence and development of AD, A beta is A beta₄₀And Abeta₄₂The two forms, gamma secretase, occur at different cleavage sites at the C-terminus of APP, producing different a β C-terminal GGVV and GGVVIA. From time course and range Abeta₄₂All performance ratios of Abeta₄₀Stronger toxicity. Therefore, based on the neurotoxicity of a β, the search for drugs that reduce a β formation, inhibit a β aggregation, and accelerate a β degradation using a β as an action target is favored by various pharmaceutical companies, and is a research hotspot in the industry for treating AD. The A beta monoclonal antibody Aducangu of Biogen (Bohai Jian) obtains FDA rapid evaluation channel qualification in 2019, and obtains FDA approval in 2021 to be conditionally marketed (Can Geriaatr J,2021,24(4):373-378.), and the inhibition or elimination of A beta aggregation is an effective way for developing anti-Alzheimer disease.

A large number of researches show that the polysaccharide compound has a plurality of physiological functions in vivo, such as the functions of regulating immunity, resisting viruses, inhibiting tumor cell proliferation and the like. The role of polysaccharides in neurodegenerative disorders has also been reported. Such as lentinan, ganoderan and agaricus blazei polysaccharide, researches show that the agaricus blazei polysaccharide has the effects of reducing inflammatory reaction, delaying apoptosis and the like, and has the potential in the aspects of reducing neurodegenerative diseases, weakening nerve cell injury and the like. Algal polysaccharide and cistanche polysaccharide exist in plants mostly, and can inhibit nerve cell apoptosis by accelerating the scavenging speed of organisms on free radicals, so that the AD deterioration process can be delayed. Chitosan oligosaccharide and sea cucumber polysaccharide are mostly present in animal bodies, have better biocompatibility, can reduce the biological activity of beta-amyloid protein, and are beneficial to the differentiation and synaptic growth of neurons, thereby relieving the nerve injury of AD patients and delaying the disease progression (Stroke,2014,45(5): 1492-1494). These studies suggest that the use of polysaccharides for the treatment of AD is still in the early exploration phase and has broad application prospects.

Snails are terrestrial mollusks of shell class, and are of various types, about 25000 types worldwide and thousands types in China. The White Jade Snail (White Jade Snail) is a variant Snail of the fusca, is named as the White Jade Snail because the head and the abdomen of the Snail are White like Jade, the Snail is a special variety in China, the breeding history of the White Jade Snail in China has been over 20 years, the breeding scale is continuously expanded in recent years, and the annual output exceeds million tons (proceedings of spring state institute of faculty and profession, 2017,35(6): 6-11). The modern Chinese medicine believes that the snails are salty in taste, enter large intestine, lung, liver and kidney channels, and have the effects of clearing away heat and toxic materials, reducing phlegm and swelling, relieving asthma and managing hernia. In the course of traditional Chinese medicine treatment, snails are often decocted in water to obtain a Ru yao, and documents indicate that freshwater and marine shellfish polysaccharide have the functions of resisting tumors and viruses and strengthening collective immunity, so that the medicinal value of snails is presumed to be mainly reflected in polysaccharide components (medical guidance, 2012,31(1): 14-16.). The African snail polysaccharide, acranan sulfate, extracted from African giant snails is a relatively more studied snail polysaccharide, has the structural formula of → 4) -alpha-L-IdoA 2S- (1 → 4) -alpha-D-GlcNAc- (1 →, the molecular weight of 29,000Da, and has been found to have various biological activities such as anticoagulation, antiangiogenesis and injury repair (J Biol chem.,1996,271 (20): 11750-5; Carbohydr Res. (2015, 413: 41-50).

Disclosure of Invention

The inventor uses a simple and effective separation and purification method to extract a polysaccharide WJPS from white jade snails, and researches the effect of the polysaccharide WJPS on inhibiting the aggregation of Abeta. The inventor finds that the white jade snail polysaccharide WJPS can obviously inhibit Abeta in vitro₄₂And inhibit a β at the level of human neuroblastoma cells₄₂Aggregation-induced cytotoxicity, and has potential effects of treating Alzheimer disease and assisting in improving memory function.

Therefore, it is an object of the present invention to provide a polysaccharide WJPS.

Another object of the present invention is to provide a method for preparing the above polysaccharide WJPS. Compared with the common extraction method of African snails (J Biol chem.,1996,271 (20):11750-5), the method is simpler and more convenient to operate, simple in process, higher in yield and easy to realize industrial production.

It is still another object of the present invention to provide a pharmaceutical composition comprising an effective amount of the above polysaccharide WJPS as an effective ingredient, and preferably further comprising pharmaceutically acceptable carriers and excipients.

It is still another object of the present invention to provide an edible composition comprising an effective amount of the above polysaccharide WJPS as an effective ingredient, preferably further comprising a food acceptable carrier and an adjuvant.

The invention also aims to provide application of the polysaccharide WJPS in preparing a medicament for preventing and/or treating neurodegenerative diseases and application in preventing and/or treating neurodegenerative diseases.

The invention further aims to provide application of the polysaccharide WJPS in preparing health-care products for assisting in improving memory functions.

To achieve the above object, in one aspect, the present invention provides a polysaccharide WJPS having a disaccharide repeating unit structure shown below:

→4)-α-L-IdoA2S-(1→4)-α-D-GlcNAc-(1→

the weight average molecular weight of the polysaccharide WJPS is about 409KDa, and the molecular weight range is about 350-450 KDa.

In another aspect, the invention provides a method for preparing polysaccharide WJPS, comprising the steps of separating, extracting and purifying the polysaccharide WJPS from white jade snail.

In one embodiment, the method of the present invention for preparing the polysaccharide WJPS comprises the steps of:

s1, degreasing the hulled white jade snail, drying, crushing and then carrying out enzymolysis by papain;

s2, stopping the enzyme reaction, centrifuging, collecting supernatant, and dialyzing the supernatant for 48-72 hours;

s3, adding absolute ethyl alcohol with the volume 2-3 times that of the dialyzed solution, standing to enable polysaccharide to precipitate, centrifugally collecting the precipitate, and drying to obtain crude polysaccharide;

s4, dissolving the crude polysaccharide with water, adding into a balanced DEAE-cellulose column for chromatography, eluting the column gradually with sodium chloride solutions with concentration gradients of 0.1M, 0.2M, 0.3M, 0.5M, 0.8M, 1.5M and 3M as eluents, dialyzing the polysaccharide eluent eluted with the sodium chloride solution of 0.8M with distilled water, and freeze-drying to obtain the polysaccharide WJPS.

Preferably, in the method S1 of the present invention, the degreasing step may be to soak the hulled white jade snail in 95% ethanol water solution for 16-20 hours, and then to soak the hulled white jade snail in acetone for 16-20 hours twice.

Preferably, in the method S1 of the present invention, the drying may be drying for 24 hours at 60 ℃ by a forced air dryer or airing for one week in a fume hood at room temperature.

Preferably, in the method S1, the enzymolysis may be performed by adding the defatted, dried and pulverized white jade snail into a papain digestion buffer solution, and then adding papain to perform enzymolysis, wherein the papain digestion buffer solution has a pH of 5.5 and a formula of 50mM sodium acetate, 5mM EDTA,5mM L-cysteine, and/or wherein the volume usage amount of the papain digestion buffer solution is about 15mL/g of the dried and pulverized white jade snail tissue dry weight, and/or wherein the weight ratio of the papain to the dried and pulverized white jade snail tissue is 15-25 mg enzyme/g tissue, preferably 20mg enzyme/g tissue. More preferably, the papain can be added once or more, for example, the white jade snail which is degreased, dried and crushed is added into the papain digestion buffer solution, after the mixture is stirred evenly, the papain is added according to the proportion that 20mg of the papain is added into each g of the dried and crushed tissue, the mixture is subjected to enzymolysis for 24 to 48 hours by gentle shaking, the same amount of the papain is added, and the enzymolysis for 24 to 48 hours by gentle shaking is continued.

Preferably, in the method S2 of the present invention, the enzyme reaction is terminated by heating to about 80-100 ℃ for 20-40 minutes, the supernatant is collected by centrifugation at 4 ℃ and dialyzed for 48-72 hours. More preferably, the centrifugal rotating speed is 8000-12000 r/min, and the centrifugal time is 20-40 min. Still more preferably, the dialysis is dialysis against running water.

Preferably, in the method S3, the dialyzed solution is concentrated and then added with 2-3 times of volume of absolute ethanol.

In still another aspect, the present invention provides a pharmaceutical composition for preventing and/or treating neurodegenerative diseases such as alzheimer's disease, comprising an effective amount of the polysaccharide WJPS of the present invention as an effective ingredient, and preferably further comprising pharmaceutically acceptable carriers and excipients.

In still another aspect, the present invention provides an edible composition comprising an effective amount of the polysaccharide WJPS of the present invention as an effective ingredient, and preferably further comprising a food-acceptable carrier and adjuvant.

The polysaccharide according to the present invention may be administered orally or non-orally, and in the case of oral administration, the polysaccharide may be administered after being mixed with conventional health care products or pharmaceutical excipients including solvents, sweeteners, preservatives, flavors, fillers, diluents, binders, lubricants, flavors, pigments, and the like. For parenteral administration, conventional administration forms such as injection or suppository can be used. The preparation of the product can adopt the conventional preparation technology.

The pharmaceutical composition or the edible composition can be prepared into different dosage forms such as oral liquid, oral tablets, solid beverages and the like.

In the present invention, the pharmaceutical composition or the edible composition may further contain other pharmaceutical active ingredients or edible active ingredients.

In yet another aspect, the present invention provides the use of the polysaccharide WJPS of the present invention for the preparation of a medicament for the prevention and/or treatment of neurodegenerative diseases.

Preferably, the neurodegenerative disease is a neurodegenerative disease caused by abnormal deposition of amyloid beta in the brain, for example, alzheimer's disease and the like.

In still another aspect, the invention provides the use of the polysaccharide WJPS or the edible composition of the invention in the preparation of a health product for assisting in improving memory function.

The white jade snail polysaccharide is obtained by extracting, separating and purifying the white jade snail by using a simple and effective polysaccharide extraction process and method. Pharmacological experiment results show that WJPS can inhibit the aggregation of A beta and inhibit the A beta on the level of human neuroblastoma cells₄₂Aggregation-induced cytotoxicity. Therefore, the compound has the functions of potentially treating the Alzheimer's disease and assisting in improving the memory function, and is expected to be developed into a medicine aiming at the Alzheimer's disease or a health-care product assisting in improving the memory function.

The present invention has been described in detail hereinabove, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. Furthermore, there is no intention to be bound by any theory presented in the preceding prior art or the summary or the following examples.

Unless expressly stated otherwise, a numerical range throughout this specification includes any sub-range therein and any numerical value incremented by the smallest sub-unit within a given value. Unless expressly stated otherwise, numerical values throughout this specification represent approximate measures or limitations to the extent that such deviations from the given values, as well as embodiments having approximately the stated values and having the exact values stated, are included. Other than in the operating examples provided at the end of the detailed description, all numbers expressing quantities or conditions of parameters (e.g., quantities or conditions) used in the specification (including the appended claims) are to be understood as being modified in all instances by the term "about" whether or not "about" actually appears before the number. "about" means that the numerical value so stated is allowed to be somewhat imprecise (with some approach to exactness in that value; about or reasonably close to that value; approximately). As used herein, "about" refers to at least variations that can be produced by ordinary methods of measuring and using such parameters, provided that the imprecision provided by "about" is not otherwise understood in the art with this ordinary meaning. For example, "about" can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%.

Drawings

FIG. 1 is a flow chart of the extraction and separation of white jade snail polysaccharide WJPS according to one embodiment of the present invention;

FIG. 2 is a HPGPC purity identification chart of white jade snail polysaccharide WJPS according to one embodiment of the present invention;

FIG. 3 is a depiction of white jade snail polysaccharide WJPS according to one embodiment of the present invention¹H NMR spectrum;

FIG. 4 is a 1H,1H two-dimensional correlation spectrum (COSY) of white jade snail polysaccharide WJPS according to one embodiment of the present invention

FIG. 5 shows inhibition of Abeta of WJPS according to one embodiment of the present invention₄₂Results of aggregation activity assay. Wherein, FIG. 5A shows the results of thioflavine fluorescence detection; FIG. 5B shows A.beta.₄₂Atomic Force Microscope (AFM) test results after incubation alone for 7 days; FIG. 5C shows WJPS and Abeta₄₂After 7 days of co-incubation the results were examined by atomic force microscopy.

FIG. 6 shows inhibition of A β by white jade snail polysaccharide WJPS according to one embodiment of the present invention₄₂Aggregation-induced neuronal cytotoxicity results. Wherein, FIG. 6A shows that different concentrations of polysaccharide WJPS alone or together with Abeta₄₂Effect on survival of neuronal cells SH-SY5Y after co-incubation; FIG. 6B is a photograph of SH-SY5Y as a control cell taken by microscope in bright field; FIG. 6C shows A.beta.₄₂Effect of incubation alone on SH-SY5Y cell growth by microscopic observationThe field photographing result; FIG. 6D shows 100. mu.g/mL of polysaccharide WJPS and Abeta₄₂Effect of Co-incubation on SH-SY5Y cell growth the results of bright field photographs were observed microscopically.

Detailed Description

The invention will now be further illustrated by reference to the following examples, which are given by way of illustration only. It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention within the scope and spirit of the invention. It is to be understood that the invention is intended to cover such alternatives and modifications as may be included within the scope of the appended claims.

The instrument comprises the following steps:

high performance liquid chromatography (GPC-PDA-RI): US Waters 2695 connects diode array UV-visible light detectors (PDA, US Waters) and differential refraction detectors (RI, US Waters)

A chromatographic column: TSKgel G4000PWxl tandem G2500PWxl (TOSOH, Japan)

Vacuum freeze drier (Freezone 6, Labconco USA)

Nuclear magnetic resonance spectrometer: bruker AVANCEIII USA

Microplate reader (Molecular Device, M2, USA), inverted fluorescence microscope (Nikon, Ni-E, Japan)

Atomic force microscope (NanosceopeIII a, Veeco instruments Inc. USA)

Drugs and reagents:

the common reagents of white jade snail (China Beijing Changpi white jade snail breeding base), papain, sodium acetate, EDTA, L-cysteine, absolute ethyl alcohol and the like are all purchased from Chinese medicine group, Abeta₄₂Lyophilized powder (Shanghai Jier Biochemical company), Thioflavin ThT (Merck, USA), dialysis bag (3500MWCO, Spectrum lab, USA)

Human neuroblastoma cell SH-SY5Y (purchased from Chinese academy of sciences cell bank)

Cell counting Kit (CellCountying Kit-8, CCK-8) purchased from Homony chemistry, Japan

DEAE-sephorose fillers are available from Cytiva, USA

Example 1: preparation of WJPS polysaccharide

Fresh living white jade snails are shelled, soaked in 95% ethanol water solution overnight, soaked in acetone twice overnight for degreasing, and dried for 24 hours at 60 ℃ by a blast drier. 120g of dried white jade snail is taken and ground in a fruit grinder. Taking 2L of papain digestion buffer (pH is about 5.5, containing 50mM sodium acetate, 5mM EDTA,5mM L-cysteine), adding all the ground white jade snail powder into the buffer, stirring uniformly, adding 2.4 g of papain (Chinese medicine reagent), stirring uniformly, and placing at 55 ℃ for gentle shaking for 24 hours. Then 2.4 g papain was added and gently shaken at 55 ℃ for 24 hours. After the reaction is finished, the papain is boiled at 100 ℃ for 30 minutes to denature the papain. The solution was centrifuged at 9000g for 30 minutes at 4 ℃ and the precipitate was discarded, and the supernatant was collected. The supernatant was dialyzed against running water for 72 hours and concentrated to about 800 ml under rotary vacuum. Adding anhydrous ethanol with three times volume, standing overnight at 4 deg.C to precipitate polysaccharide precipitate. The overnight product was centrifuged at 9000g for 30min, the precipitate was collected, washed with 200 ml of pure ethanol and lyophilized overnight to give approximately 8 g of crude polysaccharide from white jade snail (6.67% yield).

Dissolving the crude polysaccharide with a proper amount of distilled water, centrifuging, carrying out primary separation on supernate by using DEAE-sephorose column chromatography, gradually eluting the chromatographic column by using eluent (50mM sodium acetate, pH4.0) containing NaCl (0.1, 0.2, 0.3, 0.5, 0.8, 1.5 and 3M) with different concentrations at the speed of 1 ml/min, eluting 3-5 column volumes per concentration until the sugar content can not be detected by a phenol sulfate method, wherein the eluent eluted and collected by 0.8M NaCl is dialyzed completely (three times by 100 times of volume ratio) by using a 3500M CaCl dialysis bag, and then carrying out vacuum freeze drying to obtain the white jade snail polysaccharide WJPS.

Experimental example 1: structural analysis of polysaccharides

Analysis by high performance gel column chromatography (HPGPC) showed that the weight average molecular weight Mw of the polysaccharide WJPS was 409 kDa. The purity measurement is shown in FIG. 2.¹In H-NMR and COSY two-dimensional spectra (FIG. 3, FIG. 4), the signals were assigned according to literature reports (Eur.J. biochem.,2004,271,845-54; J.biol.chem.,1996,271,11750-5), and the results show that the phases of IdoA H1(5.16ppm) and H2(4.28ppm) located at a lower field can be seen in the COSY spectraOff signal, indicating the presence of a sulfuric acid substituent at position 2, whereas H1(5.06ppm) for GlcNAc correlates with H2(4.00ppm), and a chemical shift at low field for H2 indicates substitution of the amino group with an acetyl group. Of the polysaccharide¹The H NMR signal is very similar to that reported in the literature, and it can be concluded that the polysaccharide has a repeating disaccharide unit structure → 4) - α -L-IdoA2S- (1 → 4) - α -D-GlcNAc- (1 → this unit structure. Indicating that the structure of WJPS is

→4)-α-L-IdoA2S-(1→4)-α-D-GlcNAc-(1→

Pharmacological test example

Pharmacological experiments example 1: inhibition of A beta₄₂Aggregation assay

1) Thioflavin fluorescence assay (Thioflavin T, ThT)

Mixing A beta₄₂The lyophilized powder (Shanghai Jier Biochemical company) was dissolved in Hexafluoroisopropanol (HFIP) to prepare a 5mg/mL solution, and after dissolution by sonication and vortexing, it was left at room temperature for 24 hours. Then subpackaging and vacuum freeze-drying to obtain Abeta₄₂Monomers were used in ThT experiments.

Aβ₄₂The monomers were dissolved in anhydrous DMSO (concentration 1.62mM) to prepare a mother liquor. mu.L of this solution was dissolved in 20. mu.L of phosphate buffer (pH about 7.5, 50mM sodium phosphate, 100mM NaCl, 0.02% NaN)₃PBS), or 2 μ L of the solution was added with 10 μ L of WJPS polysaccharide solutions of different concentrations (0.5mg/mL,1.0mg/mL,5.0mg/mL), 8 μ L of PBS buffer was added, incubated at 37 ℃ for 20 minutes, 80 μ L of thioflavin ThT solution (1.25 μ M thioflavin in 50mM glycine-NaOH, ph8.5) was added, incubated at 37 ℃ and detected by a microplate reader every 30min at a detection wavelength of Ex 440/10nm and Em 470/10 nm.

As can be seen in FIG. 5A, the polysaccharide WJPS was able to significantly inhibit A β dose-dependently₄₂To (3) is performed.

2) Atomic Force Microscope (AFM) detection experiment

To further verify that polysaccharide WJPS inhibits A beta₄₂As a result of aggregation, the polysaccharide WJPS was observed for A.beta.using an atomic force microscope₄₂Influence of aggregation morphology. 1 μ L of A β at a concentration of 1.68mM₄₂10 μ L of WJPS polysaccharide solution at 5mg/mL was dissolved in 89 μ L of double distilled water and incubated at 37 ℃ for 7 days. 1 μ L of A β at a concentration of 1.68mM₄₂Dissolved in 99. mu.L of Millipore water and incubated at 37 ℃ for 7 days as a control. A5. mu.L sample solution was dropped onto a clean mica plate and carefully dried by blowing and tested in tapping mode with an atomic force microscope (NanosceopeIII a, Veeco instruments).

As can be seen in fig. 5B, a β 42 showed a large number of filamentous fiber aggregates after 7 days of incubation alone, whereas the reduction of filamentous fiber aggregates was significantly reduced after 7 days of co-incubation with the polysaccharide WJPS (fig. 5C). The result is identical with the result of the thioflavin fluorescence detection, and the result proves that the polysaccharide has the function of inhibiting A beta₄₂Activity of aggregation.

Pharmacological test example 2: white jade snail polysaccharide WJPS inhibits Abeta₄₂Aggregation-induced neuronal cytotoxicity assay

1) Cell culture

Human neuroblastoma SH-SY5Y (purchased from cell bank of Chinese academy of sciences) was cultured in 5% CO with MEM and Ham's F12 medium (containing 10% fetal calf serum) at a volume ratio of 1:1₂Culturing at 37 deg.C in incubator, and changing the culture solution 1 time for 2-3 days. After the cells were overgrown, they were digested with 0.25% trypsin (Invitrogen), and then seeded into 96-well cell culture plates at 35000 cells/well in a volume of 100. mu.L per well. Culturing at 37 deg.C for 16h to make the cells adhere to the wall.

Polysaccharide WJPS solutions (0,5,50, 500. mu.g/mL) at different concentrations were mixed with A.beta.₄₂(200. mu.M) or an equal volume of DMSO, after incubation in a 37 ℃ water bath for 4 days, the supernatant of the cultured overnight cells was aspirated, and 100. mu.L of the above-mentioned solution diluted with medium and incubated for 4 days was added to each well, so that the final concentration of AB42 was 40. mu.M and the final concentration of polysaccharide WJPS was (0, 1. mu.g/mL, 10. mu.g/mL, 100. mu.g/mL). Culturing at 37 deg.C for 48 hr, and determining cell survival rate by CCK-8.

2) Cell counting Kit (CellCountying Kit-8, CCK-8) assay

Add 10. mu.L of CCK-8 solution to each well. Incubation was continued for 4h, the wavelength was selected at 450nm, the absorbance of each well was measured on an enzyme linked immunosorbent assay (Molecular Device, M2, USA), the results were recorded, and the cell viability was calculated.

As shown in FIG. 6A, l) A β₄₂After the single incubation for 4 days and the addition of SH-SY5Y cells, the cell survival rate is reduced by about 40 percent compared with the normal group, which shows that the A beta₄₂The oligomer is cytotoxic to SH-SY5Y cells. 2) When A beta₄₂After the cells are incubated for 4 days with the polysaccharide WJPS with different concentrations, the survival rate of the cells is recovered in a dose-dependent manner after SH-SY5Y cells are added, and the cells have statistical significance, while the polysaccharide WJPS with different concentrations has no obvious influence on the cell production.

At the same time, from the morphology of the cells observed under the microscope, A.beta.was compared with the control (FIG. 6B)₄₂Incubation alone (FIG. 6C) resulted in cell damage, while the polysaccharide WJPS was 100. mu.g/mL with A.beta.₄₂Incubation with water significantly reduced the damage to the cells (FIG. 6D).

These results indicate that the polysaccharide WJPS can protect against neuronal cell SH-SY5Y damage by inhibiting A β 42 aggregation. The addition of the polysaccharide WJPS alone to SH-SY5Y cells had no significant effect on the survival of the cells.

Claims

1. A polysaccharide WJPS having a disaccharide repeating unit structure shown below:

→4)-α-L-IdoA2S-(1→4)-α-D-GlcNAc-(1→

the weight average molecular weight of the polysaccharide WJPS is 409KDa, and the molecular weight range is 350-450 KDa.

2. A method for preparing the polysaccharide WJPS of claim 1, which comprises the steps of separating, extracting and purifying the polysaccharide WJPS from the white jade snail.

3. The method of claim 1, comprising the steps of:

4. The method of claim 3, wherein the first and second light sources are selected from the group consisting of,

wherein in S1, the degreasing step is to soak the hulled white jade snail in 95 percent ethanol water solution for 16 to 20 hours, and then to soak the snail in acetone for 16 to 20 hours twice; and/or

In S1, the drying is carried out by drying for 24 hours at 60 ℃ by using a blast dryer or airing for a week in a fume hood at room temperature; and/or

In S1, the enzymolysis is to add degreased, dried and crushed white jade snail into papain digestion buffer solution, and then add papain for enzymolysis, wherein the pH of the papain digestion buffer solution is 5.5, and the formula is 50mM sodium acetate, 5mM EDTA,5mM L-cysteine, and/or, wherein the volume of the papain digestion buffer solution is about 15mL/g of dried and crushed white jade snail tissue dry weight, and/or, wherein the weight ratio of the papain to the dried and crushed white jade snail tissue is 15-25 mg enzyme/g tissue, preferably 20mg enzyme/g tissue, preferably, the papain can be added once or multiple times, for example, the degreased, dried and crushed white jade snail is added into the papain digestion buffer solution, and after uniform stirring, the papain is added according to the proportion of 20mg papain per g of dried and crushed tissue And (3) carrying out enzymolysis for 24-48 hours by gentle shaking, then adding the same amount of papain, and continuing to carry out enzymolysis for 24-48 hours by gentle shaking.

5. The method of claim 3, wherein the first and second light sources are selected from the group consisting of,

wherein, in S2, enzyme reaction is stopped by heating to 80-100 ℃ for 20-40 minutes, supernatant is collected by centrifugation at 4 ℃, and the supernatant is dialyzed for 48-72 hours; preferably, the centrifugal rotating speed is 8000-12000 r/min, and the centrifugal time is 20-40 min; still more preferably, the dialysis is dialysis against running water; and/or

In S3, the dialyzed solution is concentrated and then added with 2-3 times of volume of absolute ethyl alcohol.

6. A pharmaceutical composition comprising an effective amount of the polysaccharide WJPS of claim 1 as an effective ingredient, preferably further comprising pharmaceutically acceptable carriers and excipients.

7. An edible composition comprising an effective amount of the polysaccharide WJPS of claim 1 as an effective ingredient, preferably further comprising a food acceptable carrier and adjuvants.

8. The pharmaceutical composition of claim 6, further comprising an additional pharmaceutically active ingredient.

9. Use of the polysaccharide WJPS according to claim 1 or the pharmaceutical composition according to claim 6 for the preparation of a medicament for the prevention and/or treatment of neurodegenerative diseases (in particular neurodegenerative diseases caused by the abnormal deposition of amyloid beta in the brain, e.g. Alzheimer's disease).

10. Use of the polysaccharide WJPS according to claim 1 or the edible composition according to claim 7 in the preparation of a nutraceutical product to assist in improving memory function.