CN115043957A - Polygonatum polysaccharide and preparation method and application thereof - Google Patents

Abstract

The invention provides a polygonatum polysaccharide and a pharmaceutical composition containing the polygonatum polysaccharide, and particularly discloses a preparation method and pharmacological activity of the polygonatum polysaccharide.

Description

Polygonatum polysaccharide and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to polygonatum polysaccharide and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is a degenerative Disease of the central nervous system that progresses with occult progression, with increasing prevalence in people over 65 years of age. The main clinical manifestations of AD patients are progressive cognitive function deterioration, including hypomnesis, slow thinking, abnormal behaviors and social disorders, etc., and finally the symptoms are that daily life can not be self-managed completely and a series of complications are caused, and the harm is very serious. The main pathological feature of AD is the presence of a large number of Senile Plaques (SPs) formed by abnormal aggregation of beta-amyloid (a β) outside the neurons of the hippocampal and cortical areas of the brain. Currently, first-line therapeutic drugs for AD are mainly acetylcholinesterase inhibitors (donepezil, galantamine, rivastigmine) and N-methyl-D-aspartate (NMDA) receptor blockers (memantine). From long-term clinical results, these drugs only alleviate symptoms in patients and do not slow or stop the progression of AD.

Traditional Chinese medicine rhizoma polygonati (Polygonatum polysaccharide) has the effects of tonifying kidney, benefiting essence, promoting intelligence, prolonging life, strengthening essence, strengthening marrow and the like, and the record of the Shenxian Cao Jing records that the rhizoma polygonati has the effects of strengthening the middle warmer, tonifying qi, regulating the five internal organs well, and keeping the hair white and blacker for many years. Sealwort is a large amount of medicinal materials which are mainly cultivated in Hunan Jiuwei at present and is cultivated and planted in large areas in Shaoyang, new areas and the like in Hunan. The polysaccharide (PSP) component rich in rhizoma Polygonati is the main active substance of rhizoma Polygonati. The department of pharmacy of Xiangya II Hospital, Zhongnan university, has long paid attention to the neuroprotective and anti-aging effects of PSP in combination with the geriatrics department. Studies on AD have shown that PSP can significantly inhibit A beta _1-42 The apoptosis of rat hippocampal tissue cells caused by injection improves the learning and memory ability of rats; in APP transgenic mouse experiments, PSP can obviously reduce A beta plaque deposition, increase the number of synapses in a mouse hippocampal CA1 region, reduce synaptic area and relieve synaptic degeneration degree, promote secretion of synaptic vesicles and protect synaptic structures and functions in a mouse hippocampal CA1 region. The research proves that the polygonatum polysaccharide has the function of improving learning and memory so as to achieve the effect of relieving AD symptoms. In view of the reports in the prior art that polygonatum polysaccharide can improve learning and memory and has a good effect of relieving the symptoms of the Alzheimer disease, monomer medicines with good effects are not developed for treating the disease. At the same time, there is a need in the art for more drugs that can be used to treat alzheimer's disease.

Disclosure of Invention

The invention mainly aims to overcome the defects of the prior art and provide a polygonatum polysaccharide and a preparation method and application thereof, and the following technical scheme is specifically adopted:

the invention provides a polygonatum polysaccharide in a first aspect, which is shown as a formula (I):

wherein n in the formula (I) is any one or more integers selected from 13-20; further, n in the formula (I) is selected from any one or more integers of 13, 14, 15, 16, 17, 18, 19 and 20;

the second aspect of the invention provides a pharmaceutical composition for preventing and/or treating alzheimer's disease, which comprises a compound as described in formula (I) and pharmaceutically acceptable auxiliary materials;

further, the compound shown in the formula (I) accounts for 1-95% of the weight of the pharmaceutical composition; furthermore, the compound shown in the formula (I) accounts for 1-10% of the weight of the pharmaceutical composition;

further, the pharmaceutically acceptable auxiliary materials are selected from diluents, lubricants, binders, disintegrants, stabilizers or solvents;

furthermore, the diluent is selected from any one or more of starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar or glucose;

further, the lubricant is selected from one or more of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate or poloxamer;

furthermore, the adhesive is selected from one or more of water, ethanol, starch slurry, syrup, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, sodium alginate and polyvinylpyrrolidone;

furthermore, the disintegrating agent is selected from any one or more of sodium bicarbonate, citric acid, tartaric acid or low-substituted hydroxypropyl cellulose;

further, the stabilizing agent is selected from any one or more of acacia gum, agar, alginic acid or cellulose ether;

still further, the solvent is selected from water or an equilibrated salt solution;

further, the anti-inflammatory pharmaceutical composition is an oral preparation or an injection;

further, the oral preparation is an oral solid preparation or an oral liquid preparation;

further, the oral solid preparation is selected from common tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dripping pills or powder;

further, the oral liquid preparation is oral liquid or emulsion;

furthermore, the injection is selected from small water injection, transfusion or freeze-dried powder injection;

the third aspect of the invention provides a use of a compound as described in formula (I) or a pharmaceutical composition comprising the compound as described in formula (I) in the preparation of a medicament for preventing and/or treating neurodegenerative diseases;

further, the medicament for preventing and/or treating neurodegenerative diseases is a medicament for preventing and/or treating Alzheimer's disease;

further, the agent for preventing and/or treating alzheimer's disease has an effect of improving cognitive dysfunction; further, the improvement of cognitive dysfunction includes improvement of learning and memory functions and reduction of beta-amyloid (a β) deposition in brain;

further, the medicament for preventing and/or treating alzheimer's disease has the effect of restoring the balance of intestinal flora; further, the restoration of the intestinal flora balance includes the effects of inhibiting the growth of intestinal proinflammatory bacteria Helicobacter typhloinus and Helicobacter masturbinus and promoting the growth of probiotic akkermanophilin (akkermansia);

the fourth aspect of the present invention provides a process for the preparation of a compound of formula (I), comprising the steps of:

s1 water extraction and alcohol precipitation: adding water into dry polygonatum sibiricum medicinal material powder, heating and extracting, concentrating under reduced pressure, drying and crushing to obtain dry powder, adding water for dispersing after degreasing, then adding 70-95% v/v ethanol until the ethanol concentration in a sample solution reaches 60-80%, standing overnight, filtering, and repeating the alcohol precipitation and filtering operation for 1-2 times to obtain polygonatum sibiricum polysaccharide extract;

s2 deproteinization: taking a polygonatum polysaccharide extract, and mixing the polygonatum polysaccharide extract: adding deionized water according to the mass ratio of 1: 10-1: 50 to form a suspension, adding a Sevage reagent with the mass of 1/4-1/2 of the suspension, violently shaking for 20-30 min, standing for layering, slowly removing a lower organic solvent and middle layer denatured protein, retaining a supernatant, repeating the above operations for 6-10 times, combining the supernatants, concentrating to obtain a polygonatum polysaccharide concentrate, dialyzing for more than 12 hours, and freeze-drying the dialysate to obtain crude polygonatum polysaccharide;

and S3 cellulose column separation: dissolving crude polygonatum sibiricum polysaccharide in deionized water with the mass of 1/200-1/100, centrifuging after complete dissolution, filtering supernatant, separating by adopting a cellulose column, sequentially carrying out sectional elution by adopting NaCl aqueous solutions with the concentrations of 0.05, 0.1, 0.2, 0.3 and 0.5mol/L, tracking and detecting the light absorption value of eluent by adopting a phenol-sulfuric acid method, merging and collecting main peaks, and respectively carrying out dialysis desalting, reduced pressure concentration and freeze drying to obtain 7 fractions Fr.1-Fr.7;

s4 gel column separation: dissolving an elution component Fr.1 with the highest polysaccharide content by using deionized water with the mass of 1/100-1/50, centrifuging after complete dissolution, taking supernate, filtering, separating by using a gel column, taking 0.05-0.2 mol/L NaCl aqueous solution as an eluent, tracking the absorbance value of the eluent by using a phenol-sulfuric acid method, combining and collecting main peaks, concentrating and drying to obtain the polygonatum polysaccharide.

Advantageous effects

The invention further separates and purifies the polysaccharide at the effective part of the polygonatum sibiricum to obtain the homogeneous polygonatum sibiricum polysaccharide, and the structure identification is carried out on the polysaccharide (formula (I)). Pharmacological activity tests of the polysaccharide prove that the polysaccharide has the functions of remarkably improving learning and memory functions, reducing beta-amyloid deposition in brain, and recovering the steady state of host intestinal flora, particularly inhibiting the growth of intestinal proinflammatory bacteria Helicobacter typhimurium and Helicobacter mastorubium, and promoting the growth of probiotics Akkermansia mucina. Can be used for treating Alzheimer disease, has better activity than polysaccharide mixture, and can be used as effective lead compound for developing clinical medicine.

Drawings

FIG. 1: isolation and characterization spectra of formula (I). FIG. 1A is a diagram showing a DEAE-52 cellulose column separation; FIG. 1B is a diagram showing a gel column separation; FIG. 1C: an infrared spectrum of formula (I); FIG. 1D: ultraviolet spectrum of formula (I).

FIG. 2: molecular weight determination of formula (I). FIG. 2A: an HPGPC chromatogram of formula (I); FIG. 2B: the molecular weight results of formula (I) are shown in the figure.

FIG. 3: formula (I) ¹ HNMR atlas

FIG. 4: of formula (I) ¹³ C NMR spectrum

FIG. 5: HSQC spectra of formula (I)

FIG. 6: COSY map of formula (I)

FIG. 7 is a schematic view of: HMBC map of formula (I)

FIG. 8: NOESY map of formula (I)

FIG. 9: pharmacological experiment chart of formula (I) for improving spatial learning and memory functions of 5xFAD mice

FIG. 10: formula (I) reduces intracerebral A beta deposition of 5xFAD

FIG. 11: assay of intestinal flora 16S rRNA in 5xFAD mice before and after administration of formula (I)

FIG. 12: the activity of formula (I) is compared with that of crude polysaccharide of rhizoma Polygonati

Detailed description of the preferred embodiments

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1 extraction, isolation and purification of polysaccharides

1.1 extraction of polysaccharides

Adding 10 times of water into dried rhizoma Polygonati powder, and extracting with boiling water for 2 times, each time for 2 hr. Filtering, merging, decompressing and concentrating to obtain dry powder. Sequentially Soxhlet extracting with acetone and methanol for 12h, and defatting. And (4) pouring out the residual powder, and drying for 24 hours at 50 ℃ to obtain the defatted rhizoma polygonati dry powder. Taking dried defatted rhizoma Polygonati powder, adding 4 times of 95% ethanol until the ethanol concentration in the sample solution reaches 80%, stirring thoroughly, refrigerating at 4 deg.C overnight, filtering, precipitating for 2 times according to the same method, mixing the precipitates, and concentrating under reduced pressure to obtain rhizoma Polygonati polysaccharide extract.

1.2 deproteinization

Taking 20g of polygonatum polysaccharide extract, adding 400ml of deionized water for dissolving, transferring the extract into a separating funnel, adding a Sevage reagent (chloroform: n-butyl alcohol is 4:1) with the volume of 1/3, violently shaking for 20-30 min, shaking a large amount of floccules, standing for layering, slowly discarding a lower-layer organic solvent and middle-layer denatured protein, keeping a supernatant, adding the Sevage reagent for repeated extraction for 7 times, combining the supernatants, and concentrating under reduced pressure to obtain the polygonatum polysaccharide concentrated solution. Dialyzing rhizoma Polygonati polysaccharide concentrated solution in dialysis bag (molecular weight cutoff is 3500Da) in deionized water for 24h, and freeze drying dialysate to obtain rhizoma Polygonati crude polysaccharide (PSP).

1.3 isolation and purification of polysaccharides

1.3.1 DEAE-52 cellulose pretreatment

80g of DEAE-52 cellulose dry powder is taken to be fully expanded in deionized water and washed to remove impurities. Soaking for half an hour with 0.5M NaOH, removing alkali liquor in a Buchner funnel, washing with deionized water to neutrality, soaking for half an hour with 0.5M HCl, removing acid liquor in the Buchner funnel, and washing with deionized water to neutrality; after repeating twice, washing with deionized water until the solution is neutral.

1.3.2 column packing

Before column filling, the air at the bottom of the glass column is emptied by deionized water, and the pretreated DEAE-52 cellulose is gently stirred and uniformly mixed to ensure that the DEAE-52 cellulose: deionized water (v/v) ═ 3:1, and the mixture was poured into the column as once as possible by using a glass rod to drain, and the suspension was left along the inner wall of the column to prevent the generation of bubbles. After the column is filled, whether the column bed is regular or not and whether bubbles or interfaces exist are checked.

1.3.3 column equilibration and elution

To ensure that the functional groups of the cellulosic material are in equilibrium with the starting eluent, 5 column volumes were eluted with 0.05M NaCl as the starting eluent. 600mg of PSP was dissolved in 4mL of deionized water, centrifuged at 1000rpm for 5min, and the supernatant was further purified by filtration (0.75 μm) to remove impurities and then applied to a uniform sample. The eluent is sequentially eluted by stages with the concentrations of 0.05, 0.1, 0.2, 0.3 and 0.5M NaCl. The absorbance of the eluate at 490nm was followed by phenol-sulfuric acid method (FIG. 1A). The main peaks are combined and collected to obtain 7 components Fr.1-Fr.7. Dialyzing to remove salt, concentrating, and freeze drying.

1.3.4 Sephacryl S-200 gel pretreatment

Soaking Sephacryl S-200 gel in deionized water for half an hour, repeatedly soaking and washing with deionized water after the filler is fully swelled, vacuum-filtering with Buchner funnel to remove alcohol smell, and suspending gel particles with deionized water.

1.3.5 Sephacryl S-200 gel column

Removing air bubbles in the Sephacryl S-200 suspension, and discharging air in the bottom of the chromatographic column by using deionized water before column filling so that the ratio of the Sephacryl S-200: deionized water (v/v) ═ 3:1, gently stirring and mixing, and pouring the medium into the chromatographic column by using a glass rod for drainage as once as possible.

1.3.6 equilibration and elution of Sephacryl S-200

The deionized water used for the column was replaced with 5 column volumes of 0.1M NaCl, the elution fraction fr.1 with the highest polysaccharide content was taken and dissolved in 1/50 mass of deionized water, after complete dissolution, centrifugation was carried out, the supernatant was filtered and homogenized, and the absorbance of the eluate at 490nm was followed by the phenol-sulfuric acid method using 0.1M NaCl as the eluent (fig. 1B). Combining and collecting main peaks, concentrating under reduced pressure, vacuum drying to obtain polysaccharide formula (I), and weighing.

Example 2 structural characterization of polysaccharides of formula (I)

2.1 determination of the UV, IR and molecular weight of the polysaccharide formula (I)

2.2.1 UV analysis of formula (I)

Dissolving 1mg of polysaccharide formula (I) in 2ml of water, and diluting to a concentration of 0.5 mg/ml; and (3) scanning in a wavelength region of 200-800 nm of an ultraviolet visible spectrum by taking distilled water as a blank control. The results are shown in FIG. 1C, formula (I) has no absorption peak at 260nm, indicating the absence of nucleic acid components; the curve was substantially smooth at 280nm, indicating that formula (I) is protein free. UV overall indicates that formula (I) is a purified polysaccharide.

2.2.2 IR analysis of formula (I)

KBr was ground thoroughly in an agate mortar and tabletted as a blank control. 5mg of a powder of the formula (I) and KBr are mixed in a ratio of 1: putting the mixture into an agate mortar at a ratio of 100, uniformly grinding and tabletting. Scanning and analyzing by using a Fourier infrared spectrometer, wherein the scanning range is 4000-400 ^-1 Resolution of the instrument 2cm ^-1 And scanning for 16 times to obtain an infrared spectrogram. As shown in FIG. 1D, at 3371.73cm ^-1 A wider characteristic peak is an intermolecular O-H stretching vibration peak; at 2927.39cm ^-1 The absorption peak is C-H stretching vibration; 1374.32cm ^-1 Is the C-H bending vibration peak; 1703.20cm ^-1 、 1647.61cm ^-1 The absorption peak should be the stretching vibration of carboxyl (-COO-); 1200cm ^-1 ～1000cm ^-1 The characteristic peak is the angle-variable vibration of C-O-C and C-O-H of the pyran ring; 861.81cm ^-1 Is a characteristic peak of the beta-glycosidic bond of the saccharide. IR generally indicates that formula (I) is a polysaccharide structure.

2.2 molecular weight analysis of formula (I)

2.2.1 reagents and instruments

2.2.1.1 reagents

The main reagents used are reflected in table 1 below.

TABLE 1

2.2.2 Main Instrument

The main instruments used are reflected in table 2 below.

TABLE 2

2.2.3 sample information

The samples were numbered as shown in table 3 below.

TABLE 3

2.2.4 Experimental methods

2.2.4.1 molecular weight calibration Curve establishment

Dextran standards ( molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000 and 670000 series analysis standards) with different molecular weights are weighed, 0.05M NaCl solution is respectively added to prepare 5mg/ml dextran standard solution, 0.22 mu M of microfiltration membrane is used for filtration for later use, HPGPC method is adopted, high performance gel permeation chromatography series columns are used for detection, Waters Empower software is used for analyzing results, logarithm value of relative molecular mass Mp of the standard is used as ordinate, retention time of corresponding chromatographic peak is used as abscissa for linear regression, and a calibration curve is obtained.

2.2.4.2 preparation of test sample solutions

Weighing purified polysaccharide sample, adding 0.05M NaCl solution into the sample to prepare 5mg/ml sample solution, filtering the supernatant with 0.22 μ M microporous filter membrane, and transferring the sample into 2ml sample bottle for use.

2.2.4.3 chromatographic method

By HPGPC, a difference detector was prepared by using a high performance liquid chromatograph, and polymer matrix water-soluble SEC (GFC) columns OHpak SB-803HQ, Ohpak SB-804HQ and Ohpak SB-805HQ (8X 300mm) were connected in series to detect them, while a mobile phase was 0.05M NaCl solution at a flow rate of 0.6ml/min, a column temperature of 40 ℃ and a sample introduction amount of 30. mu.l.

2.2.4.4 results of the experiment

(1) Molecular weight calibration curve results

The calibration curve was obtained by performing a third order linear regression using Waters Empower software, taking the logarithm of the relative molecular mass Mp of the standard as ordinate and the retention time (T) of the corresponding chromatographic peak as abscissa, as follows:

the Log Mp-T correction curve equation is: log Mp ═ 0.0004T ³ +0.0500T ² -2.1475 T+36.9321，R ² ＝0.9997。

(2) Molecular weight assay of formula (I)

And (4) obtaining a calculation formula according to the standard product calibration curve so as to calculate the molecular weight range of the formula (I). The results are shown in FIG. 2, and PSP-1-2 in FIG. 2B is a representation of the compound of formula (I).

2.3 NMR measurement of the formula (I)

2.3.1 reagents and instruments

(1) Reagent: deuterated water (D) ₂ O), spectral grade, shanghai alading biochemical science and technology, ltd; 3- (trimethylsilyl) propionic acid-D4 sodium salt (TMSP), 98 atom% D, Sigma Co.

(2) The instrument comprises: 600MHz Bruker nuclear magnetic resonance spectrometer (Bruker AVANCE HD III 600MHz Spectromete), Bruker, Germany.

2.3.2 NMR test

The lyophilized sample was dissolved in 0.5ml of D ₂ O, TMSP was added as an internal standard. One-dimensional Nuclear Magnetic Resonance (NMR) measurements using a 600MHz Bruker NMR spectrometer ¹ H-NMR、 ¹³ C-NMR and two-dimensional Nuclear magnetism ¹ H- ¹ H COSY, TOCSY, HSQC, HMBC, NOESY. Calibration: HDO hydrogen δ H δ 4.70ppm, methyl carbon δ C of TMSP δ -1.80 ppm. .

In order to further obtain the structural characteristic information of the sample, the sample is subjected to one-dimensional nuclear magnetism ¹ H-NMR、 ¹³ C-NMR and two-dimensional Nuclear magnetism ¹ H- ¹ H COSY, HSQC, HMBC and NOESY, wherein the spectrograms of the H COSY, the HSQC, the HMBC and the NOESY are shown in figures 2-7, all H and C chemical shift information of each main sugar residue is obtained, and the connection sequence of each sugar residue is deduced.

Using one-dimensional nuclear magnetic resonance hydrogen spectrum ( ¹ H-NMR, see FIG. 3) further identifies the glycosidic bond configuration of the maltodextrin samples, and the hydrogen spectrum signals of the polysaccharide are mostly between delta 3.0 and 5.5ppm, usually between delta 4.5 and 5.5ppm, which are anomeric proton (H-1) resonance regions. Of maltodextrin samples ¹ On the H-NMR spectrum, a large number of proton resonance signals are concentrated in a delta 3.0-5.5ppm area, the signals are overlapped seriously, 3 main anomeric proton coupling signals are found in the anomeric area, and the anomeric proton signals are delta 5.31ppm, delta 5.25ppm and delta 4.87ppm respectively, which shows thatThe compound possibly contains 3 monosaccharide residues, the corresponding 3 monosaccharide residues are respectively marked as A, B, C according to the chemical shift signal intensity, other hydrogen signals are all concentrated in a delta 4.5-3.0 ppm region, the signal overlapping is serious, and the signal is difficult to be assigned.

TABLE 4 polysaccharide samples of the respective sugar residues ¹ H and ¹³ chemical shift assignment of C

"- -" indicates that no signal was found

By passing ¹³ The cross peaks in the anomeric region of the C NMR spectrum (FIG. 4) and the HSQC spectrum (FIG. 5) determined that the anomeric carbon signal at residue A, B, C was δ 98.59ppm, δ 99.98ppm, and δ 98.05ppm, respectively. After the off-head signal attribution was determined, binding was by COSY (FIG. 6), HMBC (FIG. 7) and NOESY (FIG. 8) ¹ H-NMR、 ¹³ C-NMR spectra were compared to chemical shift data of the relevant literature andsample (I)Of the main type of sugar residue ¹ H and ¹³ c chemical shift signal is assigned, and the resultSee Table 1. The structural analysis of each sugar residue is as follows:

to be provided withSugar residue AFor example, the 1D and 2D NMR analysis procedures for each sugar residue are illustrated: anomeric signals delta 5.31ppm (H-1) and delta 98.59ppm (C-1) indicate that sugar residue A is in the alpha configuration. According to ¹ H NMR confirmed the H-1 chemical shift of sugar residue A of delta 5.31ppm, the signals of H-2, H-3, H-4 and H-5 are clear by COSY spectrum cross peaks, the H-2, H-3, H-4 and H-5 chemical shifts of sugar residue A are respectively assigned to delta 3.53ppm, delta 03.86ppm, delta 13.56ppm and delta 3.74ppm, and H-6 can be assigned to delta 3.74ppm by HSQC related spectrum. After assigning the chemical shifts of the hydrogens on the sugar rings, the chemical shifts of the carbons on the sugar rings can be assigned by HSQC correlation spectra as δ 98.59ppm, δ 71.33ppm, δ 73.27ppm, δ 76.62ppm, δ 71.19ppm, and δ 60.36ppm, respectively, see Table 1. Wherein the chemical potential of C-1 and C-4 is shifted to low field, which indicates that the residue is substituted at the C-1 and C-4 positions of the sugar ring, and the sugar residue A is inferred to be → 4) -delta 2-D-Glcp- (1 →) according to the report of combined literature.

According to a similar method, the hydrogen and carbon signals of other main residues are deduced, the chemical shift assignments of the main monosaccharide residues H and C in the polysaccharide sample are shown in Table 1, and the combination literature reports that B is inferred to be → 4,6) -alpha-D-Glcp- (1 →andC is inferred to be alpha-D-Glcp- (1 →).

The sequence of the interconnections between the individual sugar residues can be further deduced by correlating the coupling signals of anomeric hydrogens to carbons on the individual sugar residues on the HMBC remote correlation spectrum, or the coupling signals of anomeric carbons to hydrogens on the individual sugar residues. The HMBC correlation spectrum of the sample is shown in FIG. 7, from which the coupling signals are found, for residue A, H-1 (. delta.5.31 ppm) with residue A C-4 (. delta. 76.62ppm) having a coupling signal (AH-1/AC-4), for residue A, H-1 (. delta.5.31 ppm) with residue B, C-4 (. delta. 76.58ppm) having a coupling signal (AH-1/B C-4), for residue A, H-4 (. delta.3.56 ppm) with residue A, C-1 (. delta.98.59 ppm) having a coupling signal (A H-4/A C-1). The order of attachment of the residues of the polysaccharide samples was further verified by NOESY spectra (FIG. 7) in which there were cross peaks (A H-1/A H-4) for H-1 of residue A (Δ 5.31ppm) and H-4 of residue A (Δ 3.56ppm), cross peaks (B H-1/AH-4) for H-1 of residue B (Δ 5.25ppm) and H-4 of residue A (Δ 3.56ppm), and cross peaks (C H-1/B H-6) for H-1 of residue C (Δ 4.87ppm) and H-6 of residue B (Δ 3.84 ppm). This indicates that the sample has a sugar backbone formed by residue A linked to residues A and B via a1 → 4 glycosidic bond, and that residue B is linked to residue C via position 6 on the side chain.

Based on the one-dimensional and two-dimensional nuclear magnetic information analysis, the deduced primary structure of the sample is a glucan with → 4) -alpha-D-Glcp- (1 → as the main chain, and alpha-D-Glcp- (1 → link 6 site, nuclear magnetic H spectrum integral shows that the ratio of → 4,6) -alpha-D-Glcp- (1 → and → 4) -alpha-D-Glcp- (1 → is about 1: 6, and the de-H in the sugar unit linking process is combined with the molecular weight information (FIG. 2B) ₂ The number of groups O and n is estimated to be in the range of 13 to 20. The structure of (I) was determined as follows:

wherein n in the formula (I) is one or more integers selected from 13-20.

EXAMPLE 3 pharmacological Activity test of formula (I) (PSP-1)

3.1 experiment for improving spatial learning and memory function of 5xFAD mice

3-month-old SPF breeding-grade Wild Type (WT) mice and AD transgenic model mice 5xFAD were divided into 2 groups of 9 mice each, and the weights were recorded. The experimental group was gavaged with 30mg/kg of formula (I) daily, the control group was gavaged with an equal volume of physiological saline daily, and the water maze behavioural test was performed after 3 months of continuous administration. Dosing and frequency of dosing were maintained during the behavioral testing period (fig. 9A). FIGS. 9B and 9C show that 5xFAD mice in the administered group had significantly shorter time to reach the escape platform than 5xFAD mice in the saline group after 5 days of training; fig. 9D and 9E show that the 5xFAD mice in the dosing group had significantly increased residence time in the platform quadrant and number of crossing over the original platform position compared to the 5xFAD mice in the saline group after the escape platform was removed; FIG. 9F is a graph of the water maze movement of the mouse; figure 9G shows that the swimming speed of the mice given the group did not change. Data are mean ± standard deviation (n ═ 9); denotes p < 0.05, p < 0.01 and p < 0.001, respectively, n.s. denotes no statistical significance. The above results show that the gavage administration of formula (I) can significantly improve the spatial learning and memory function of AD mice.

3.25 xFAD brain tissue section Abeta plaque staining detection

The brain tissues of WT mice, 5xFAD mice physiological saline and 5xFAD mice PSP-1 mice were frozen and sliced to a thickness of 30 μm. The hippocampal and cortical regions of the brain were then stained separately using a Thioflavin S staining and immunohistochemical staining (using specific ab antibodies) method and analyzed for statistical differences. The results show that FIGS. 10A and 10B show that PSP-1 significantly reduces the deposition of Abeta plaques in the hippocampal region of the brain; FIGS. 10C and 10D show that PSP-1 significantly reduced deposition of Abeta plaques in the cerebral cortex. A representative picture scale is 100 μm. Data are mean ± standard deviation (n ═ 5); denotes p < 0.01. The above results indicate that PSP-1 can reduce the deposition of A beta in the brain tissue of AD mice.

3.35 xFAD mouse intestinal flora 16S rRNA detection

5xFAD mice were collected before dosing (3 months of age), and 5xFA mice after dosingD mice normal saline group (6 months old), 5 xad mice PSP-1 group (6 months old) feces, and intestinal flora were detected by 16S rRNA. FIGS. 11A and 11B illustrate that the lysosome group (6 months old) of 5xFAD mice with intestinal flora is significantly different from that of 5xFAD mice before administration (3 months old), indicating that 5xFAD mice with 6 months old are disturbed by intestinal flora, whereas the intestinal flora of 5xFAD mice after administration is restored; FIG. 11C shows the intestinal flora

No change in diversity indicates that PSP-1 did not change the species of the flora, but fig. 11D shows that the β -diversity of the intestinal flora clearly differs from the saline group after administration, indicating that the abundance of each flora changes and that PSP-1 remodels the host intestinal flora. FIGS. 11E, 11F and 11G illustrate that PSP-1 has inhibitory effects on the intestinal pro-inflammatory bacteria Helicobacter typhloinus and Helicobacter mastorubius, respectively, and growth promoting effects on the probiotic bacteria Akkermansia mucina, and PSP-1 has prebiotic functions. Wherein in fig. 11A, (a): 5xFAD mice pre-dose (3 months of age), (b): 5xFAD mouse saline group (6 months old), (c): 5xFAD mice PSP-1 group (6 months of age). Data are mean ± standard deviation (n ═ 6); and p < 0.05 and p < 0.01 and p < 0.001, n.s. indicates no statistical significance. The results show that oral administration of PSP-1 can remodel the intestinal flora of a host, and the intestinal flora is in a steady state.

3.4 comparison of the Activity of formula (I) with crude polysaccharide of Polygonatum sibiricum

3-month-old 5xFAD mice were divided into two groups of 9 mice each. The crude polysaccharide and PSP-1 of rhizoma Polygonati were administered by intragastric administration at a concentration of 30 mg/kg. The water maze behavioural experiment was performed 3 months after gavage. During the course of the behavioral experiments, oral gavage was maintained. FIG. 12 shows that the time for the 5xFAD mice given PSP-1 to find the escape platform on both day 4 and day 5 of training is shorter than that of the crude polysaccharide from Polygonati officinalis rhizoma, which indicates that PSP-1 has stronger activity in improving the spatial learning and memory function of AD mice than that of the crude polysaccharide from Polygonati rhizoma.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (8)

1. A polygonatum polysaccharide is characterized by being shown in a formula (I):

wherein n in the formula (I) is any one or more integers selected from 13-20; further, n in the formula (I) is selected from any one or more integers of 13, 14, 15, 16, 17, 18, 19 and 20.

2. A pharmaceutical composition for preventing and/or treating alzheimer's disease, comprising a compound of formula (I) according to claim 1 and pharmaceutically acceptable excipients.

3. The pharmaceutical composition for preventing and/or treating alzheimer's disease according to claim 2, wherein the compound of formula (I) accounts for 1-95% of the weight of the pharmaceutical composition; further, the compound shown in the formula (I) accounts for 1-10% of the weight of the pharmaceutical composition.

4. The pharmaceutical composition for preventing and/or treating alzheimer's disease according to claim 2 or 3, wherein said pharmaceutically acceptable excipients are selected from any one or more of diluents, lubricants, binders, disintegrants, stabilizers or solvents;

preferably, the diluent is selected from any one or more of starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar or glucose;

preferably, the lubricant is selected from one or more of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate or poloxamer;

preferably, the adhesive is selected from one or more of water, ethanol, starch slurry, syrup, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, sodium alginate and polyvinylpyrrolidone;

preferably, the disintegrant is selected from any one or more of sodium bicarbonate, citric acid, tartaric acid or low-substituted hydroxypropyl cellulose;

preferably, the stabilizing agent is selected from any one or more of acacia gum, agar, alginic acid or cellulose ether;

preferably, the solvent is selected from water or an equilibrated salt solution;

preferably, the anti-inflammatory pharmaceutical composition is an oral preparation or an injection;

further preferably, the oral preparation is an oral solid preparation or an oral liquid preparation; still more preferably, the oral solid preparation is selected from common tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dripping pills or powder; or even more preferably, the oral liquid preparation is an oral liquid or an emulsion;

or further preferably, the injection is selected from small water injection, transfusion or freeze-dried powder injection.

5. Use of a compound of formula (I) according to claim 1 or a pharmaceutical composition comprising a compound of formula (I) for the preparation of a medicament for the prevention and/or treatment of a neurodegenerative disease; further, the medicament for preventing and/or treating the neurodegenerative disease is a medicament for preventing and/or treating Alzheimer disease.

6. The use according to claim 5, wherein the medicament for the prevention and/or treatment of Alzheimer's disease has an effect of improving cognitive dysfunction; further, the improvement of cognitive dysfunction includes improvement of learning and memory functions and reduction of beta-amyloid (a β) deposition in brain.

7. The use according to claim 5, wherein the medicament for the prevention and/or treatment of Alzheimer's disease has an effect of restoring the balance of intestinal flora; further, the restoration of the intestinal flora balance includes the effects of inhibiting the growth of the intestinal proinflammatory bacteria Helicobacter typhimurium and Helicobacter mastorinus and promoting the growth of the probiotic bacteria Akkermansia muciniphila (Akkermansia muciniphila).

8. A process for the preparation of a compound of formula (I) according to claim 1, comprising the steps of:

s1 water extraction and alcohol precipitation: adding water into dry polygonatum sibiricum medicinal material powder, heating and extracting, concentrating under reduced pressure, drying and crushing to obtain dry powder, adding water for dispersing after degreasing, then adding 70-95% v/v ethanol until the ethanol concentration in a sample solution reaches 60-80%, standing overnight, filtering, and repeating the alcohol precipitation and filtering operation for 1-2 times to obtain polygonatum sibiricum polysaccharide extract;

s2 deproteinization: taking a polygonatum polysaccharide extract, and mixing the polygonatum polysaccharide extract: adding deionized water according to the mass ratio of 1: 10-1: 50 to form a suspension, adding a Sevage reagent with the mass of 1/4-1/2 of the suspension, violently shaking for 20-30 min, standing for layering, slowly removing a lower organic solvent and middle layer denatured protein, retaining a supernatant, repeating the above operations for 6-10 times, combining the supernatants, concentrating to obtain a polygonatum polysaccharide concentrate, dialyzing for more than 12 hours, and freeze-drying the dialysate to obtain crude polygonatum polysaccharide;

and S3 cellulose column separation: dissolving crude polygonatum polysaccharide in deionized water with the mass of 1/200-1/100, centrifuging after complete dissolution, filtering supernatant, separating by adopting a cellulose column, sequentially carrying out sectional elution by adopting NaCl aqueous solutions with the concentrations of 0.05, 0.1, 0.2, 0.3 and 0.5mol/L, tracking and detecting the light absorption value of eluent by adopting a phenol-sulfuric acid method, merging and collecting main peaks, and respectively carrying out dialysis desalting, reduced pressure concentration and freeze drying to obtain 7 fractions Fr.1-Fr.7;

s4 gel column separation: dissolving an elution component Fr.1 with the highest polysaccharide content by using deionized water with the mass of 1/100-1/50, centrifuging after complete dissolution, taking supernate, filtering, separating by using a gel column, taking 0.05-0.2 mol/L NaCl aqueous solution as an eluent, tracking the absorbance value of the eluent by using a phenol-sulfuric acid method, combining and collecting main peaks, concentrating and drying to obtain the polygonatum polysaccharide.

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