CN115975065A - High-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 and polygonatum oligosaccharide PSO, and methods and applications thereof - Google Patents
High-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 and polygonatum oligosaccharide PSO, and methods and applications thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a preparation method and pharmacological activity of high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 and polygonatum oligosaccharide PSO, and proves that the polysaccharide and the oligosaccharide can obviously delay CL4176 nematode paralysis caused by Abeta, have potential effect of treating Alzheimer's disease, and can be developed into a saccharide medicament for treating Alzheimer's disease.
Description
Technical Field
The invention relates to the field of biological medicine treatment, in particular to high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 and polygonatum oligosaccharide PSO, and a method and application thereof.
Background
Alzheimer's Disease (AD) is a progressive neurodegenerative disease with major clinical manifestations of memory impairment, bradycardia, impairment of visuospatial skills, dysfunction of executive functioning, and alterations in personality and behavior. There are currently approximately 5000 million AD patients worldwide, and with the aging problem of the world population exacerbated, it is estimated that the number of AD patients will triple, reaching 1.5 million, in 2050. Existing studies have shown that the main pathological features of AD patients are β -amyloid aggregation into senile plaques, intracellular Tau protein abnormalities and neuronal death. The deposition and abnormal expression of beta-amyloid in brain are currently considered as core links for triggering Alzheimer's disease, the beta-amyloid is taken as an action target, and the generation of the beta-amyloid is reduced from the source by interfering APP metabolism, so that the method is a research hotspot for treating Alzheimer's disease at present. The A beta monoclonal antibody medicament Aducanumab has been approved by FDA, and the inhibition or elimination of A beta aggregation is shown to be an effective way for developing anti-Alzheimer disease to a certain extent.
Rhizoma Polygonati (Polygonatum sibiricum Red.) is the rhizome of perennial herb of Polygonatum of Liliaceae, and has sweet taste, mild property, wide distribution, and various varieties, and has effects of nourishing kidney, moistening lung, invigorating spleen, and invigorating qi. Clinically, sealwort has the effects of resisting aging and oxidation, improving immunity, regulating hematopoiesis, resisting tumors, reducing blood sugar and the like. The rhizoma Polygonati contains polysaccharides, steroid saponin, anthraquinone, etc. Polygonatum Polysaccharides (PSP) are reported to have the effects of resisting tumors, resisting oxidation, improving learning and memory and the like as main active ingredients, but the effect of the Polygonatum Polysaccharides and Polygonatum oligosaccharides on treating Alzheimer's disease is not clear yet.
Although polysaccharides have been reported to have various biological activities, the original polysaccharides generally have a large molecular weight, are not favorable for crossing cell membranes into the body or binding to biologically active sites for exerting biological activities, and have low bioavailability. The invention provides a preparation method of polygonatum polysaccharide and polygonatum oligosaccharide with low molecular weight, and experiments prove that the biological activity of the polygonatum polysaccharide and polygonatum oligosaccharide is superior to that of the original polysaccharide. At present, no patent of the invention discloses a preparation method of low molecular weight polygonatum polysaccharide and polygonatum oligosaccharide and a patent of invention for treating AD. The extraction process of crude polysaccharide from rhizoma Polygonati is only described in patent CN 113501890B.
Disclosure of Invention
The invention provides a simple and effective extraction process and a method for polygonatum polysaccharide and oligosaccharide with high purity and low molecular weight, which take dried polygonatum as a raw material to obtain polygonatum polysaccharide PSP-1-1 and polygonatum oligosaccharide PSO, pharmacological experiments show that the polysaccharide and the oligosaccharide can remarkably delay CL4176 nematode paralysis caused by Abeta, have potential effect of treating Alzheimer disease, and can be developed into saccharide medicaments for treating Alzheimer disease.
To achieve the purpose, the invention provides the following technical scheme:
in a first aspect of the invention, a preparation method of high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 is provided, which comprises the following steps:
s1, polysaccharide extraction: pulverizing dried rhizoma Polygonati, hot-soaking and extracting with pure water, filtering, concentrating, and precipitating with ethanol to obtain rhizoma Polygonati polysaccharide crude product;
s2, polysaccharide deproteinization and depigmentation: treating the crude product of the polygonatum polysaccharide by using a Sevage reagent and active carbon to obtain polygonatum polysaccharide PSP;
s3, polysaccharide cellulose column separation: dissolving rhizoma Polygonati polysaccharide in deionized water, centrifuging after completely dissolving, collecting supernatant, filtering, separating with DE-52 cellulose column, and sequentially eluting with pure water and sodium chloride solution of different concentrations; tracking and detecting the light absorption value of the eluent by using a phenol-sulfuric acid method, merging and collecting pure water elution components, desalting, concentrating, and freeze-drying to obtain PSP-1;
s4, polysaccharide gel chromatographic column separation: dissolving PSP-1 in pure water, filtering after complete dissolution, separating by Sephadex G-200, tracking and detecting the light absorption value of the eluent by a phenol-sulfuric acid method, merging and collecting main peaks, concentrating and drying to obtain PSP-1-1.
In the present invention, the Sevage reagent consists of chloroform and n-butanol (5:1).
Preferably, the weight of the pure water in the step S1 is 10-30 times of that of the rhizoma polygonati.
Preferably, the temperature of hot-dip extraction in step S1 is 70-100 deg.C, and the time is 1-4h.
Preferably, the hot water leaching times in the step S1 are 1 to 3 times.
Preferably, the alcohol precipitation treatment in step S1 is: adding anhydrous ethanol dropwise into the concentrated extract until the ethanol content is 80%; and standing the obtained solution at 4 ℃ overnight, performing suction filtration to obtain a filter cake, and then performing freeze drying to obtain a crude product of the polygonatum polysaccharide.
Preferably, in the step S2, the crude polygonatum polysaccharide product is dissolved in pure water, 1/5 Sevage reagent is added, after violent shaking for 20-40min, the mixture is kept stand to remove the precipitate, and the process is repeated for 8-20 times; adding 10% (w/v) of active carbon, stirring, filtering, precipitating the filtrate with ethanol, and freeze-drying to obtain the PSP.
Preferably, in step S3, the polygonatum polysaccharide PSP is taken, dissolved in 10-30 times of pure water, centrifuged after complete dissolution, the supernatant is taken for filtration, separated by a DE52 cellulose column, eluted by pure water, 0.03, 0.05, 0.1, 0.3 and 0.5M NaCl solutions in sequence, the pure water elution components are collected and combined, desalted by an ultrafiltration tube, and freeze-dried to obtain the polygonatum polysaccharide PSP-1.
Preferably, in the step S4, the polygonatum polysaccharide PSP-1 is taken, dissolved in 2 to 10 times of pure water, filtered, separated by a gel chromatography column Sephadex G-200, eluted by pure water, and subjected to light absorption value tracking detection of an eluent by a phenol-sulfuric acid method, and then the main peaks are combined and collected, concentrated and dried to obtain the PSP-1-1.
In the second aspect of the invention, the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 is provided and prepared by the preparation method of the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1.
Preferably, the polysaccharide PSP-1-1 of Polygonatum sibiricum has a total sugar content of 99.2% and an average molecular weight of 2.6kDa.
Preferably, the preparation method of the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 comprises the following steps:
s1, polysaccharide extraction: pulverizing dried rhizoma Polygonati, hot-soaking and extracting with pure water, filtering, concentrating, and precipitating with ethanol to obtain rhizoma Polygonati polysaccharide crude product;
s2, polysaccharide deproteinization and depigmentation: treating the crude product of the polygonatum polysaccharide by using a Sevage reagent and active carbon to obtain polygonatum polysaccharide PSP;
s3, polysaccharide cellulose column separation: dissolving rhizoma Polygonati polysaccharide in deionized water, centrifuging after completely dissolving, collecting supernatant, filtering, separating with DE-52 cellulose column, and sequentially eluting with pure water and sodium chloride solution of different concentrations; tracking and detecting the light absorption value of the eluent by using a phenol-sulfuric acid method, merging and collecting pure water elution components, desalting, concentrating, and freeze-drying to obtain PSP-1;
s4, polysaccharide gel chromatographic column separation: dissolving PSP-1 in pure water, filtering after complete dissolution, separating by Sephadex G-200, tracking and detecting the light absorption value of the eluent by a phenol-sulfuric acid method, merging and collecting main peaks, concentrating and drying to obtain PSP-1-1.
Preferably, the weight of the pure water in the step S1 is 10-30 times of that of the rhizoma polygonati.
Preferably, the temperature of hot-dip extraction in step S1 is 70-100 deg.C, and the time is 1-4h.
Preferably, the number of hot water leaching in step S1 is 1 to 3.
Preferably, the alcohol precipitation treatment in step S1 is: adding anhydrous ethanol dropwise into the concentrated extract until the ethanol content is 80%; and standing the obtained solution at 4 ℃ overnight, performing suction filtration to obtain a filter cake, and then performing freeze drying to obtain a crude product of the polygonatum polysaccharide.
Preferably, in the step S2, the crude polygonatum polysaccharide product is dissolved in pure water, 1/5 of Sevage reagent is added, after violent shaking for 20-40min, standing is carried out to remove the precipitate, and the steps are repeated for 8-20 times; adding 10% (w/v) active carbon, stirring, vacuum filtering, precipitating the filtrate with ethanol, and freeze drying to obtain the PSP.
Preferably, in the step S3, the polygonatum polysaccharide PSP is taken, dissolved in 10-30 times of pure water, centrifuged after complete dissolution, the supernatant is taken and filtered, separated by a DE52 cellulose column, eluted by pure water, 0.03, 0.05, 0.1, 0.3 and 0.5M NaCl solution in sequence, the pure water elution components are collected and combined, desalted by an ultrafiltration tube, and freeze-dried to obtain the polygonatum polysaccharide PSP-1.
Preferably, in the step S4, the polygonatum polysaccharide PSP-1 is taken, dissolved in 2-10 times of pure water, filtered, separated by gel chromatography column Sephadex G-200, eluted by pure water, and subjected to phenol-sulfuric acid method tracking detection of light absorption value of the eluate, and the main peak is collected, concentrated and dried to obtain the PSP-1-1.
The third aspect of the invention provides a preparation method of polygonatum sibiricum oligosaccharide PSO, which comprises the steps of hydrolyzing polygonatum sibiricum polysaccharide PSP-1-1 obtained by the preparation method by using a 0.2M sulfuric acid solution, neutralizing by using barium hydroxide, centrifuging to obtain a supernatant, precipitating by using 80% of alcohol, and freeze-drying to obtain the polygonatum sibiricum oligosaccharide PSO.
The fourth aspect of the invention provides a polygonatum sibiricum oligosaccharide PSO prepared by the preparation method of the polygonatum sibiricum oligosaccharide PSO.
In a fifth aspect of the invention, the invention provides the application of the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 and the polygonatum oligosaccharide PSO in the preparation of medicines for treating and/or preventing neurodegenerative diseases.
In a sixth aspect of the invention, the invention provides the use of the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 and the polygonatum oligosaccharide PSO in the preparation of medicines for treating and/or preventing Alzheimer's disease.
Compared with the prior art, the invention has the beneficial effects and remarkable progresses that: the invention further separates and purifies the polysaccharide at the effective part of the polygonatum sibiricum to obtain the polygonatum sibiricum polysaccharide with high purity and low molecular weight, and the polygonatum sibiricum oligosaccharide is obtained by hydrolysis. The polysaccharide and the oligosaccharide are subjected to pharmacological activity detection, and the polysaccharide and the oligosaccharide are proved to have the effects of remarkably delaying nematode paralysis caused by Abeta, have the activity superior to that of a polysaccharide mixture, have the potential effect of treating the Alzheimer's disease, and can be developed into saccharide medicaments for treating the Alzheimer's disease.
Drawings
To more clearly illustrate the technical solution of the present invention, the drawings required for the embodiment of the present invention will be briefly described below.
It should be apparent that the drawings in the following description are only drawings of some embodiments of the invention, and that other drawings can be obtained by those skilled in the art without inventive exercise, and the other drawings also belong to the drawings required by the embodiments of the invention.
FIG. 1 is a flow chart of the extraction and separation of Polygonatum sibiricum polysaccharide PSP-1-1 according to an embodiment of the present invention;
FIG. 2 is a diagram showing the separation of polysaccharides from Polygonatum sibiricum according to an embodiment of the present invention, wherein FIG. 2A is a diagram showing the separation of a DE-52 cellulose column; FIG. 2B is a diagram of Sephadex G-200G-2 gel column separation;
FIG. 3 is an HPGPC purity identification chart of Polygonatum sibiricum polysaccharide PSP-1-1 according to one embodiment of the present invention;
FIG. 4 is a flow chart of the extraction of Polygonatum sibiricum oligosaccharide PSO according to an embodiment of the present invention;
FIG. 5 is a graph of prolonged CL4176 nematode paralysis of Polygonatum polysaccharide PSP-1-1 according to one embodiment of the present invention;
FIG. 6 is a picture of prolonged CL4176 nematode paralysis of polygonatum oligosaccharide PSO according to one embodiment of the present invention.
Detailed Description
In order to make the purpose, technical solution, beneficial effect and significant progress of the embodiments of the present invention clearer and more complete, the technical solution of the embodiments of the present invention will be described in detail below with reference to the embodiments of the present invention.
It is to be understood that all of the described embodiments are only some, and not all, of the present invention; all other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
It is to be understood that:
the specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
It should be further noted that the following embodiments may be combined with each other, and the same or similar concepts or processes may not be described in detail in some embodiments.
The technical means of the present invention will be described in detail with reference to specific examples.
Example 1 extraction, separation and purification of Polygonatum polysaccharide
The extraction and separation flow chart of Polygonatum sibiricum polysaccharide PSP-1-1 is shown in figure 1.
1.1, polysaccharide extraction: pulverizing dried rhizoma Polygonati, adding 20 times of water, and extracting at 90 deg.C for 2 times each for 2 hr. Filtering, mixing, concentrating under reduced pressure, adding anhydrous ethanol until the ethanol concentration in the sample solution reaches 80%, stirring, standing at 4 deg.C overnight, filtering, precipitating for 2 times by the same method, mixing the precipitates, concentrating under reduced pressure, and freeze drying to obtain crude product of rhizoma Polygonati polysaccharide.
1.2, deproteinization and depigmentation: taking 10g crude product of polygonatum polysaccharide, adding 400mL pure water for dissolving, adding 1/5 volume of Sevage reagent (chloroform: n-butanol = 5:1), violently shaking for 25min, standing for layering, slowly discarding lower layer organic solvent and middle layer denatured protein, keeping supernatant, and repeating for 8 times. Adding 10% (w/v) of active carbon, stirring, filtering, precipitating the filtrate with ethanol, and freeze-drying to obtain the PSP.
Comparing the crude product of polygonatum polysaccharide obtained in step 1.1 with the PSP of polygonatum polysaccharide obtained in step 1.2, the results are shown in table 1.
TABLE 1 comparison of Polygonatum sibiricum polysaccharide PSP and crude Polygonatum sibiricum polysaccharide
| Yield (%) | Protein content (%) | Sugar content (%) | |
| Crude polysaccharide of rhizoma Polygonati | 11.5 | 47.8 | 37.0 |
| Rhizoma Polygonati polysaccharide | 47.0 | 3.2 | 95.3 |
1.3 separation and purification of polysaccharides
1) DE-52 cellulose pretreatment
The dry powder of cellulose 100gDE-52 is fully expanded in deionized water and washed to remove impurities. Soaking with 0.5M NaOH for half an hour, washing with pure water to neutral, soaking with 0.5M NaCl solution for half an hour, and washing with pure water for 5 times; then soaked for half an hour by 0.5M NaOH and washed to be neutral by pure water.
2) Column mounting
Before column filling, deionized water is used for emptying air at the bottom of the glass column, the pretreated DEAE-52 cellulose is gently stirred and uniformly mixed, a glass rod is used for drainage, the mixture is poured into the chromatographic column as soon as possible, and suspension is left along the inner wall of the chromatographic column to prevent bubbles from being generated. After the column is filled, whether the column bed is regular or not and whether bubbles or interfaces exist are checked.
3) Column equilibration and elution
Pure water eluted 5 column volumes. 250mg of PSP was dissolved in 10mL of pure water, centrifuged at 4000rpm for 5min, and the supernatant was filtered to remove impurities and then was completely and uniformly applied. Sequentially eluting with pure water, 0.03, 0.05, 0.1, 0.3 and 0.5M NaCl solution in stages. The absorbance of the eluate at 490nm was followed by phenol-sulfuric acid method (FIG. 2A). Mixing and collecting main peaks to obtain PSP-1, PSP-2 and PSP-3, desalting with ultrafiltration tube, concentrating, and freeze drying. The assay results were followed for binding activity (Table 2) to obtain PSP-1.
TABLE 2 determination of yield and activity of each component of rhizoma Polygonati polysaccharide after DE-52 separation
| Yield (%) | PT 50 Percentage of elongation (%) | |
| PSP | - | 42.5 |
| PSP-1 | 26.0 | 40.6 |
| PSP-2 | 34.6 | 28.6 |
| PSP-3 | 21.3 | 11.1 |
4) Sephadex G-200 gel pretreatment
Dry dextran gel powder is soaked in distilled water at room temperature for at least 24 hours, and is continuously stirred to ensure that the gel swells until the gel swells fully and the volume of the gel does not change.
5) Sephadex G-200 gel packing column
And placing the swelled gel into the column at one time according to the column packing requirement, keeping the column packing in a wet state, and avoiding generating bubbles or faults in the column.
6) Equilibration and elution for Sephadex G-200
5 column volumes of pure water are used for balancing the gel column, PSP-1 is taken, pure water is added, centrifugation is carried out after complete dissolution, supernatant is taken, filtration is carried out, then uniform sample loading is carried out, pure water is taken as eluent, and the phenol-sulfuric acid method is used for tracking the light absorption value of the eluent at 490nm (figure 2B). Combining and collecting main peaks, concentrating under reduced pressure, vacuum drying to obtain polysaccharide PSP-1-1, and weighing.
Example 2 identification of Polygonatum polysaccharide PSP-1-1
The polysaccharide PSP-1-1 of the polygonatum obtained in the example 1 is subjected to sugar content determination, molecular weight determination and purity identification:
the total sugar content of the polygonatum polysaccharide PSP-1-1 is determined by adopting a phenol-sulfuric acid method, a glucose solution is used for preparing a standard solution, and the total sugar content of the polygonatum polysaccharide PSP-1-1 is determined to be 99.2%.
Preparing standard curve with standard Dextran (Dextran) of different molecular weight by using high performance gel permeation chromatography system TSKgel G3000PWxl (7.8 × 300mm, tosoh of Japan); setting the column temperature at 40 deg.C, using 0.7% Na2SO4 solution as mobile phase, flow rate at 0.8mL/min, and concentration of rhizoma Polygonati polysaccharide PSP-1-1 at 5mg/mL, and determining molecular weight of rhizoma Polygonati polysaccharide PSP-1-1.
As shown in figure 3, the determined PSP-1-1HPGPC of the polygonatum polysaccharide is a single symmetrical peak, the relative molecular mass is 2.657kDa, the polydispersity index is 1.3878, and the polygonatum polysaccharide PSP-1-1 is a homogeneous polysaccharide.
Example 3 preparation of Polygonatum sibiricum oligosaccharide PSO
The extraction flow chart of the polygonatum sibiricum oligosaccharide PSO is shown in figure 4.
The polygonatum polysaccharide PSP-1-1 obtained in example 1 is added into 30 times (w/v) of 0.2M sulfuric acid solution, heated for 1h at 80 ℃, and immediately added into 0.2M Ba (OH) 2 solution for neutralization after cooling. Centrifuging to remove precipitate, adding ethanol until ethanol content is 80%, refrigerating at 4 deg.C overnight, removing ethanol from supernatant, and freeze drying.
Example 4 protective Effect of Polygonatum polysaccharide PSP-1-1 and Polygonatum oligosaccharide PSO on CL4176 nematode paralysis caused by Abeta
CL4176 nematode paralysis experiments were carried out on the polygonatum polysaccharides PSP-1-1 obtained in example 1 and the polygonatum oligosaccharides PSO obtained in example 3. The CL4176 nematode has human A beta gene transferred into muscle cell of body wall specifically, and the gene is expressed under temperature induction, i.e., after culturing at 16 deg.c to L3 stage, raising the culture temperature to 23 deg.c can induce the A beta protein to express greatly, and the expressed A beta will accumulate in muscle cell of the CL4176 nematode to make the nematode show the symptom of dyskinesia, which is called paralysis phenotype. The paralysis experiment step comprises:
4.1, calculating the density of the synchronized CL4176 nematodes, placing a certain volume of synchronized nematode liquid into a 0.6mL sterile centrifuge tube to ensure that the number of nematodes in the obtained solution is not less than 30, then adding 5 muL of OP50 bacterial liquid and 10 muL of drugs into each tube, supplementing the system to 100 muL by using an S-complete solution, lightly blowing and beating by using a micropipettor, uniformly mixing, transferring to an NGM culture medium prepared by a freshly prepared 35mm culture dish, and placing in a 16 ℃ constant temperature incubator for culturing for 36 hours.
4.2 and 36 hours later, observing the living state of the nematodes in the culture dish under a microscope, observing that the nematodes in the culture dish all grow to the L3 stage, and increasing the culture temperature to 23 ℃ for continuous culture for 24 hours.
4.3, after culturing for 24 hours at 23 ℃, beginning to record the paralyzed condition of the nematodes in the culture dish, and observing and recording the number of the paralyzed nematodes every 1 hour (the judgment standard of nematode paralysis is that platinum wires are used for lightly touching the head and the tail of the nematodes, and if the nematodes cannot move and only the head slightly shakes or a transparent round sterile circle appears in the head area of the nematodes, the nematodes are considered to be paralyzed nematodes).
And 4.4, recording the number of nematode paralysis in the culture dish every hour until all nematodes in the culture dish are paralyzed, repeating the experiment at least three times, and drawing a paralysis survival curve and a nematode half paralysis time histogram (nematode half paralysis time histogram) by using the experiment result obtained by using Graphpad Prism 9 statistical analysis.
The results are shown in fig. 5 and fig. 6, the polygonatum polysaccharide PSP-1-1 and the polygonatum oligosaccharide PSO can obviously delay the paralysis time of CL4176 nematodes caused by A beta. Wherein p is less than 0.05; p < 0.01; p < 0.001; indicating the degree of significant difference compared to the control group.
In conclusion, the embodiments show that the scheme is adopted to further separate and purify the polysaccharide at the effective part of the polygonatum sibiricum so as to obtain the polygonatum sibiricum polysaccharide with high purity and low molecular weight, and the polygonatum sibiricum oligosaccharide is obtained through hydrolysis. The pharmacological activity detection of the polysaccharide and the oligosaccharide proves that the polysaccharide and the oligosaccharide both have the function of obviously delaying nematode paralysis caused by Abeta, and the activity of the polysaccharide and the oligosaccharide is superior to that of a polysaccharide mixture. The results show that the polygonatum polysaccharide PSP-1-1 and the polygonatum oligosaccharide PSO are expected to become saccharide candidate drugs for treating the Alzheimer disease.
During the description of the above description:
the description of the terms "this embodiment," "an embodiment of the invention," "as shown in … …," "further improved technical solution," etc., means that a particular feature, structure, material, or characteristic described in this embodiment or example is included in at least one embodiment or example of the invention; in this specification, the schematic representations of the terms used above are not necessarily for the same embodiment or example, and the particular features, structures, materials, or characteristics described, etc., may be combined or brought together in any suitable manner in any one or more embodiments or examples; furthermore, those of ordinary skill in the art may combine or combine features of different embodiments or examples and features of different embodiments or examples described in this specification without undue conflict.
Finally, it should be noted that:
the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same;
although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that various changes and modifications may be made, and equivalents may be substituted for elements thereof without departing from the scope of the embodiments of the present invention.
Claims (14)
1. A method for preparing high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 is characterized by comprising the following steps:
s1, polysaccharide extraction: pulverizing dried rhizoma Polygonati, hot-soaking and extracting with pure water, filtering, concentrating, and precipitating with ethanol to obtain rhizoma Polygonati polysaccharide crude product;
s2, polysaccharide deproteinization and depigmentation: treating the crude product of the polygonatum polysaccharide by using a Sevage reagent and active carbon to obtain polygonatum polysaccharide PSP;
s3, polysaccharide cellulose column separation: dissolving rhizoma Polygonati polysaccharide in deionized water, centrifuging after completely dissolving, collecting supernatant, filtering, separating with DE-52 cellulose column, and sequentially eluting with pure water and sodium chloride solution of different concentrations; tracking and detecting the light absorption value of the eluent by using a phenol-sulfuric acid method, merging and collecting pure water elution components, desalting, concentrating, and freeze-drying to obtain PSP-1;
s4, polysaccharide gel chromatographic column separation: dissolving PSP-1 in pure water, filtering after complete dissolution, separating by Sephadex G-200, tracking and detecting the light absorption value of the eluent by a phenol-sulfuric acid method, merging and collecting main peaks, concentrating and drying to obtain PSP-1-1.
2. The method for preparing the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 according to claim 1, wherein the weight of the pure water in the step S1 is 10-30 times of that of the polygonatum.
3. The method for preparing the high-purity low-molecular weight polygonatum polysaccharides PSP-1-1 according to claim 1, wherein the temperature of the hot-dip extraction in the step S1 is 70-100 ℃ for 1-4 hours.
4. The method for preparing the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 according to claim 1, wherein the hot water leaching in the step S1 is performed for 1 to 3 times.
5. The method for preparing the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 according to claim 1, wherein the alcohol precipitation treatment in the step S1 is as follows: adding anhydrous ethanol dropwise into the concentrated extract until the ethanol content is 80%; and standing the obtained solution at 4 ℃ overnight, performing suction filtration to obtain a filter cake, and then performing freeze drying to obtain a crude product of the polygonatum polysaccharide.
6. The method for preparing the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 according to claim 1, wherein in the step S2, the crude polygonatum polysaccharide is dissolved in pure water, 1/5 Sevage reagent is added, after violent shaking for 20-40min, the mixture is kept stand to remove the precipitate, and the steps are repeated for 8-20 times; adding 10% (w/v) active carbon, stirring, vacuum filtering, precipitating the filtrate with ethanol, and freeze drying to obtain the PSP.
7. The method for preparing the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 according to claim 1, wherein in the step S3, the polygonatum polysaccharide PSP is taken, 10-30 times of pure water is added for dissolution, centrifugation is carried out after complete dissolution, the supernatant is taken for filtration, separation is carried out by a DE52 cellulose column, elution is sequentially carried out by pure water, 0.03, 0.05, 0.1, 0.3 and 0.5M NaCl solution, the elution components of the pure water are collected and combined, the salt is removed by an ultrafiltration tube, and freeze drying is carried out, so as to obtain the polygonatum polysaccharide PSP-1.
8. The method for preparing the high-purity low-molecular-weight polygonatum polysaccharide PSP-1-1 according to claim 1, wherein in the step S4, the polygonatum polysaccharide PSP-1 is taken, dissolved by adding 2-10 times of pure water, filtered, separated by a gel chromatography column Sephadex G-200, eluted by pure water, subjected to tracking detection of the light absorption value of the eluate by a phenol-sulfuric acid method, combined to collect the main peak, concentrated and dried to obtain the PSP-1-1.
9. A high purity low molecular weight polygonatum polysaccharide PSP-1-1, characterized in that it is prepared by the method of any one of claims 1-8.
10. The high purity low molecular weight polygonatum polysaccharide PSP-1-1 according to claim 9, wherein the total sugar content is 99.2% and the average molecular weight is 2.6kDa.
11. A preparation method of polygonatum sibiricum oligosaccharide PSO is characterized in that polygonatum sibiricum polysaccharide PSP-1-1 obtained by the preparation method of any one of claims 1 to 8 is hydrolyzed by using 0.2M sulfuric acid solution, neutralized by barium hydroxide, centrifuged to obtain supernatant, precipitated by 80% alcohol, and freeze-dried to obtain polygonatum sibiricum oligosaccharide PSO.
12. A polygonatum sibiricum oligosaccharide PSO, characterized in that the polygonatum sibiricum oligosaccharide PSO is prepared by the method of claim 11.
13. Use of the high purity low molecular weight polygonatum polysaccharides PSP-1-1 of claim 9 or 10 and the polygonatum oligosaccharides PSO of claim 12 for the preparation of a medicament for the treatment and/or prevention of neurodegenerative diseases.
14. Use of the high purity low molecular weight polygonatum polysaccharides PSP-1-1 of claim 9 or 10 and the polygonatum oligosaccharides PSO of claim 12 for the preparation of a medicament for the treatment and/or prevention of alzheimer's disease.
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