阅读说明：本技术 一种多糖及其制备方法和应用 (Polysaccharide and preparation method and application thereof ) 是由 丁侃 曾晖 厉飘飘 于 2020-05-13 设计创作，主要内容包括：本发明提供一种多糖及其制备方法和应用。本发明通过药理实验表明上述多糖可以剂量依赖性地抑制稳定转染APP和BACE1的CHO/APPBACE1细胞中和稳定转染APP Switish突变的HEK293-APP<Sup>sw</Sup>细胞中Aβ<Sub>42</Sub>的生成,并且所述多糖能明显抑制Aβ<Sub>42</Sub>的聚集；同时具有潜在的治疗阿尔兹海默症的作用,能开发成治疗阿尔兹海默症的糖类药物。(The invention provides a polysaccharide and a preparation method and application thereof. Pharmacological experiments show that the polysaccharide can inhibit the neutralization of CHO/APPABCE 1 cells stably transfected with APP and BACE1 and HEK293-APP with APP Switish mutation stably transfected sw A β in cells 42 And the polysaccharide can obviously inhibit A β 42 (ii) aggregation of (ii); meanwhile, the compound has the potential effect of treating the Alzheimer's disease and can be developed into a carbohydrate medicament for treating the Alzheimer's disease.)

1. A polysaccharide RP02-1 having the structure:

wherein a and b each independently represent the number of 1, 5-arabinose (1,5-Ara), and a + b is 21; n is an integer of 1 to 33.

2. The polysaccharide RP02-1 according to claim 1, wherein the polysaccharide RP02-1 has a weight-average molecular weight in the range of 11-239 kDa.

3. The polysaccharide RP02-1 according to claim 1 or 2, wherein the monosaccharide composition of the polysaccharide RP02-1 comprises arabinose, galactose, rhamnose and galacturonic acid, wherein the molar ratio of the monosaccharides is 63.5:19.8:8.3: 8.4.

4. A method for extracting polysaccharide RP02-1 from cortex et radix Polygalae comprises the following steps:

(1) polysaccharide extraction: soaking dried cortex et radix Polygalae in ethanol water solution (preferably 95% ethanol water solution) for defatting, removing ethanol, and drying; extracting with boiling water until no obvious reaction is detected by a sulfuric acid-phenol method; centrifuging, concentrating the filtrate, dialyzing, concentrating again, adding 1-20 times (preferably 5 times) ethanol water solution (preferably 95% ethanol water solution) of the volume of the concentrated solution, and centrifuging to obtain precipitate; washing the obtained precipitate with anhydrous ethanol and acetone alternately, and drying to obtain crude cortex et radix Polygalae polysaccharide;

(2) polysaccharide purification: dissolving crude Polygala tenuifolia polysaccharide in 10-15 times of water, centrifuging, purifying supernatant by stages, sequentially adding H₂Eluting with O, 0.05M NaCl, 0.1M NaCl and 0.2M NaCl, collecting the eluted component of 0.2M NaCl, concentrating, dialyzing, and drying to obtain primarily purified polysaccharide RP 02; dissolving the obtained polysaccharide RP02 in 0.2M NaCl, centrifuging, separating the supernatant, eluting with 0.2M NaCl, collecting the eluate, concentrating, dialyzing, and drying to obtain polysaccharide RP 02-1.

5. The method of claim 4, wherein:

the dialysis operation in the step (1) is to put the concentrated filtrate into a dialysis bag and dialyze the filtrate for 2 to 3 days by flowing tap water, preferably, the dialysis bag with the molecular cut-off of 3,500Da is used for dialysis; and/or

In the step (1), the absolute ethyl alcohol and the acetone are alternately washed and then are dried in vacuum, and the operating temperature of the vacuum drying is 40-60 ℃.

6. The method of claim 4 or 5, wherein:

in the step (2), the elution speed is 3-6mL/15 min; and/or

And (3) in the step (2), the centrifugation time is 10min, and the rotating speed is 4000 r/min.

7. The method according to claim 4 or 5, wherein the drying in the step (2) is freeze drying, the refrigeration temperature of the freeze drying treatment is-50 ℃, the temperature is raised by 5 ℃ per hour, the final temperature is 40 ℃, and the drying is continued for 48 hours.

8. Use of the polysaccharide RP02-1 according to any of claims 1 to 3 for the preparation of a medicament or nutraceutical for the prevention and/or treatment of neurodegenerative diseases.

9. Use of the polysaccharide RP02-1 of any one of claims 1 to 3 in the preparation of a medicament for inhibiting A β₄₂The produced and aggregated medicament or health product.

10. Use of the polysaccharide RP02-1 according to any of claims 1 to 3 for the preparation of a medicament or health product for the prevention and/or treatment of Alzheimer's disease.

Technical Field

The invention relates to polysaccharide and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is a progressive neurodegenerative disease that is common in the elderly and is prone to behavioral and cognitive impairment. With the increasing global aging problem, the prevalence of AD is increasing year by year, and AD will become a non-negligible health and social problem throughout the world. The pathogenesis of AD is complex, according to the amyloid cascade hypothesis, amyloid protein (A beta) is one of the main pathological features of AD, amyloid precursor protein is cut by beta-secretase and gamma-secretase together to generate, and the A beta is aggregated and deposited to form senile plaques to further damage the nervous system. Therefore, the search for drugs capable of preventing and curing Alzheimer's disease by targeting A β is the focus of the current research (see, for example, Mattson M P. Pathways tolwards and away from Alzheimer's disease [ J ] Nature,2004,430(7000): 631-639.).

Polygala tenuifolia is a dried root of Polygala tenuifolia Willd belonging to Polygala of Polygalaceae, has neuroprotective effects such as tranquilization, sleep promotion, anti-aging, and anti-dementia according to the record of Chinese dictionary, and has been used for over two thousand years in China. Polysaccharides have been reported to have antitumor, antioxidant and other effects as one of the main components of polygala tenuifolia, but polygala tenuifolia polysaccharides have not been reported to have an effect of treating alzheimer's disease.

Disclosure of Invention

It is an object of the present invention to provide a polysaccharide. The invention extracts and separates a pectin polysaccharide RP02-1 from dried roots of polygala tenuifolia, pharmacological experiments show that the prepared polysaccharide RP02-1 can obviously reduce and can dose-dependently inhibit CHO/APPACE 1 cells stably transfecting APP and BACE1 and HEK293-APP stably transfecting APP Switish mutant^swA β in cells₄₂RP02-1 has no obvious cytotoxicity and can obviously inhibit A β₄₂(ii) aggregation of (ii); has the function of treating the Alzheimer disease, and can be developed into a saccharide candidate drug for treating the Alzheimer disease.

Another object of the present invention is to provide a process for producing the above polysaccharide.

It is a further object of the present invention to provide the use of the above polysaccharide.

Thus, according to one aspect, the present invention provides a polysaccharide RP02-1 having the structure:

wherein a and b each independently represent the number of 1, 5-arabinose (1,5-Ara), and a + b is 21; n is an integer from 1 to 33, for example from 16 to 17.

In the present invention, the weight-average molecular weight of the polysaccharide RP02-1 is preferably in the range of 11 to 239 kDa.

In the present invention, the monosaccharide composition of the polysaccharide RP02-1 preferably comprises arabinose, galactose, rhamnose and galacturonic acid, wherein the molar ratio of the monosaccharides is 63.5:19.8:8.3: 8.4.

According to another aspect, the present invention provides a method for extracting polysaccharide RP02-1 from polygala tenuifolia, comprising the steps of:

(1) polysaccharide extraction: soaking dried cortex et radix Polygalae in ethanol water solution (preferably 95% ethanol water solution) for defatting (for example, two to three weeks), defatting, removing ethanol, and drying (for example, air drying); extracting with boiling water (e.g., in an amount of 1 kg/4L) (e.g., 4-6h each time) until no significant reaction is detected by sulfuric acid-phenol method (e.g., 4-6 total extractions); centrifuging, concentrating the filtrate, dialyzing, concentrating again, adding 1-20 times (preferably 5 times) ethanol water solution (preferably 95% ethanol water solution) of the volume of the concentrated solution, and centrifuging to obtain precipitate; washing the obtained precipitate with anhydrous ethanol and acetone alternately (for 3 times), and drying to obtain crude polysaccharide of cortex et radix Polygalae;

(2) polysaccharide purification: dissolving crude Polygala tenuifolia Willd polysaccharide in 10-15 times of water, centrifuging, purifying supernatant (e.g. by DEAE anion exchange column) by fractionation, sequentially using H₂Eluting with O, 0.05M NaCl, 0.1M NaCl and 0.2M NaCl, collecting the eluate of 0.2M NaCl, concentrating, dialyzing, and drying (such as freeze drying) to obtain primarily purified polysaccharide RP 02; the resulting polysaccharide RP02 is dissolved in 0.2M NaCl, centrifuged, the supernatant separated (e.g., by Sephacryl HR S-300 column), eluted with 0.2M NaCl, the eluted fraction is collected, concentrated, dialyzed, and dried (e.g., lyophilized) to give polysaccharide RP 02-1.

In the above method of the present invention, preferably, the dialysis operation in step (1) is to put the concentrated filtrate into a dialysis bag and dialyze the filtrate with flowing tap water for 2 to 3 days.

In the above method of the present invention, preferably, the drying in step (1) is performed by vacuum drying, and the operating temperature of the vacuum drying is 40-60 ℃.

In the method of the present invention, dialysis can be carried out, for example, with a dialysis bag having a molecular cut-off of 3,500 Da.

In the above method of the present invention, preferably, in the step (2), the elution rate is 3 to 6mL/15 min.

In the above method of the present invention, preferably, the centrifugation time in the step (2) is 10min, and the rotation speed is 4000 r/min.

In the above method of the present invention, preferably, the refrigeration temperature of the lyophilization treatment in the step (2) is-50 ℃, and then the temperature is raised by 5 ℃ per hour, and the final temperature is 40 ℃, and the drying is continued for 48 hours.

According to a further aspect, the invention provides specific application of polysaccharide RP02-1, which comprises application of polysaccharide RP02-1 in preparation of medicines or health products for preventing and/or treating neurodegenerative diseases, and application of polysaccharide RP02-1 in preparation of medicines or health products for inhibiting A β₄₂The resultant and aggregated medicament or health product of (a); and the application of polysaccharide RP02-1 in preparing medicaments or health products for preventing and/or treating Alzheimer's disease.

Pharmacological experiments show that the polysaccharide can inhibit the neutralization of CHO/APPABCE 1 cells stably transfected with APP and BACE1 and the Swedish mutant HEK293-APP stably transfected with APP through dose dependence^swA β in cells₄₂And the polysaccharide can obviously inhibit A β₄₂(ii) aggregation of (ii); meanwhile, the compound has the potential effect of treating the Alzheimer's disease and can be developed into a carbohydrate medicament for treating the Alzheimer's disease.

Drawings

FIG. 1A is a characterization of polysaccharide RP02-1¹³A C NMR spectrum;

FIG. 1B is a DEPT135 map of polysaccharide RP 02-1;

FIG. 2 is a characteristic HMBC profile of polysaccharide RP 02-102I;

FIG. 3 is a characteristic HMBC profile of polysaccharide RP 02-1;

FIGS. 4A and 4B are graphs showing inhibition of HEK293-APP by polysaccharide RP02-1, respectively^swA β in CHO/APPACE 1 cells₄₂Histograms of secretion volume;

FIGS. 5A and 5B are polysaccharide RP02-1 vs HEK293-APP, respectively^swHistograms of cell and CHO/APPACE 1 cell viability effects;

FIG. 6 shows inhibition of A β by polysaccharide RP02-1₄₂A line graph of the collection of (a).

Detailed Description

In order that the objects and advantages of the invention will be more clearly understood, the following description is given in conjunction with the accompanying examples. It is to be understood that the following text is merely illustrative of one or more specific embodiments of the invention and does not strictly limit the scope of the invention as specifically claimed.