CN111393534B

CN111393534B - Acorus gramineus sugar polymer and preparation method and application thereof

Info

Publication number: CN111393534B
Application number: CN202010110367.4A
Authority: CN
Inventors: 严春艳; 钟晶; 丘娴
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2020-02-21
Filing date: 2020-02-21
Publication date: 2022-04-22
Anticipated expiration: 2040-02-21
Also published as: CN111393534A

Abstract

The invention discloses a grassleaf sweelflag rhizome sugar polymer, which comprises AT1-3 and/or AT2-2, wherein AT1-3 is a sugar polymer consisting of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and xylose, and the structural formula is shown in a formula (I); AT2-2 is a sugar polymer composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, xylose and fucose, and has a structural formula shown in formula (II). AT1-3 and AT2-2 have the activity of preventing and treating neurodegenerative diseases, and are selected high-content and strong-activity grassleaf sweelflag rhizome glycopolymer parts. The invention also provides a preparation method of AT1-3 and AT 2-2.

Description

Acorus gramineus sugar polymer and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a grassleaf sweelflag rhizome sugar polymer and a preparation method and application thereof.

Background

Neurodegenerative diseases (Neurodegenerative diseases) are diseases that result from chronic progressive degenerative changes of the central nervous system and are characterized by apoptosis of neurons in the brain and spinal cord, which normally do not regenerate and gradually worsen over time. Common neurodegenerative diseases mainly include Alzheimer's Disease (AD), Parkinsonism (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease (HD), and the like. Statistically, nearly 5000 million people worldwide are affected by AD, and the number is expected to steadily increase in the next decade. The main manifestation of alzheimer's disease is acquired cognitive dysfunction syndrome, and numerous studies have shown that neuroinflammation is an important cause of neuronal degenerative loss in the pathogenesis of alzheimer's disease, while excessive activation of microglia is considered to be an important feature of neuroinflammation. Excessive activation of microglia can produce and secrete a large amount of neurotoxic factors (including chemokines and proinflammatory factors) to cause neuroinflammation, so that inhibition of neuroinflammation mediated by abnormal activation of microglia is an important strategy for the treatment of alzheimer's disease. At present, the related research on neurodegenerative diseases such as Alzheimer's disease and the like at home and abroad is still in an exploration stage, the types of medicines clinically used for treating the neurodegenerative diseases are few, the treatment effect only stays in a stage of delaying the deterioration of the disease, and certain toxic and side effects are realized. Therefore, the search for more effective therapeutic drugs is urgent, and in recent years, the use of natural active ingredients in traditional Chinese medicines for preventing and treating neurodegenerative diseases has become a hot point of research in the medical field.

Acorus tatarinowii (Acorus tatarinowii) belongs to Araceae and Acorus perennial herb, has odor of rhizome, is usually used as a medicine, can regulate qi, activate blood, dispel wind, remove dampness, has the functions of resolving dampness, stimulating appetite, inducing resuscitation, eliminating phlegm, inducing resuscitation and benefiting intelligence, and is used for treating epilepsy, phlegm syncope, fever coma, amnesia, qi block and deafness, chest distress, dysphoria, stomachache, abdominal pain, anemofrigid-damp arthralgia, superficial infection and pyogenic infections, traumatic injury and other symptoms. The main components of the grass-leaved sweetflag rhizome comprise volatile oil, alkaloids, carbohydrate polymers, flavonoids and the like; the research on volatile oil and alkaloids is reported more, and the research on crude sugar polymers and refined sugar polymers thereof and the research on preventing and treating neurodegenerative diseases of crude sugar polymers and refined sugar polymers thereof are not reported.

Disclosure of Invention

In a first aspect, the present invention provides an acorus-leaved sweets polymer.

The second aspect of the present invention is to provide the use of the above-mentioned grassleaved sweetflag sugar polymer in preparing a medicament for preventing and/or treating neurodegenerative diseases and complications thereof.

The third object of the present invention is to provide a method for preparing the above-mentioned polymers of acorus-leaved sweets.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, a grassleaved sweetflag sugar polymer is provided, which comprises AT1-3 and/or AT2-2, wherein AT1-3 is a sugar polymer composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and xylose, and the structural formula is shown in formula (I):

wherein a, b, c, d, e and f are in the range of 1-1000;

AT2-2 is a glycomer consisting of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, xylose and fucose, and has a structural formula shown in formula (II):

wherein a, b and c are in the range of 1-1000.

Both AT1-3 and AT2-2 contain mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and xylose, and are acidic polysaccharides.

In a second aspect of the present invention, there is provided a use of the grassleaved sweetflag sugar polymer according to the first aspect of the present invention in the preparation of a medicament for preventing and/or treating neurodegenerative diseases and complications thereof.

In a third aspect of the present invention, there is provided a method for preparing the grassleaved sweetflag sugar polymer according to the first aspect of the present invention, comprising the steps of:

s1, soaking: taking dried rhizome of acorus calamus, cutting into sections, and soaking to obtain a soaking solution A;

s2, water extraction: heating and extracting rhizome of rhizoma Acori Graminei and soaking solution, and filtering to obtain extractive solution B and residue C;

s3, grading alcohol precipitation: concentrating the extracting solution B under reduced pressure, adding ethanol to make the volume concentration of the ethanol be a%, standing, and collecting precipitate and supernatant to obtain crude sugar polymer AT1 and supernatant D; concentrating the supernatant D again, adding ethanol to make the volume concentration of the ethanol be b%, standing, and collecting precipitate and supernatant to obtain crude sugar polymer AT2 and supernatant E; concentrating the supernatant E again, adding ethanol to make the volume concentration of the ethanol be c%, standing, collecting the precipitate to obtain a crude sugar polymer AT3, wherein a is more than or equal to 10 and less than b and less than c and less than 100;

s4, alkali extraction: soaking the residue C obtained in the step S2 in a NaOH solution, standing, collecting supernatant to obtain supernatant F, adding an HCl solution for neutralization, centrifuging, collecting supernatant, concentrating to obtain supernatant G, adding ethanol to make the volume concentration of the ethanol be 30-90%, standing, collecting precipitate to obtain an alkali-extracted crude sugar polymer ATB;

s5, purification: purifying the crude sugar polymers AT1, AT2, AT3 and ATB respectively to obtain purified crude sugar polymers AT1, AT2, AT3 and ATB of rhizoma Acori Graminei;

s6, ion exchange column chromatography: respectively carrying out ion exchange column chromatography on the purified crude sugar polymers AT1 and AT2 obtained in the step S5, carrying out gradient elution by using a NaCl solution with the concentration of 0-2M, tracking an elution curve by using a phenol-sulfuric acid method, respectively collecting sugar parts according to the elution curve, then concentrating, dialyzing, freeze-drying, respectively dissolving by using water, and respectively obtaining a supernatant H and a supernatant I after centrifugal separation;

s7, molecular sieve gel column chromatography: subjecting the supernatant H and the supernatant I in the step S6 to molecular sieve gel column chromatography, eluting with water, tracking elution curve by phenol-sulfuric acid method, collecting sugar fraction according to the elution curve, concentrating, and freeze drying to obtain the Acorus tatarinowii sugar polymers AT1-3 and AT2-2 of claim 1.

According to the method of the third aspect of the present invention, in step S3, a is 10. ltoreq. a < 60, b is 60. ltoreq. b < 80, and c is 80. ltoreq. c < 100.

According to the method of the third aspect of the present invention, the time of the step S3 of precipitating with ethanol and then standing is 10-28 hours.

According to the method of the third aspect of the present invention, the specific operations of purifying in step S5 are: removing proteins from the crude sugar polymers AT1, AT2, AT3 and ATB by a Sevag method, dialyzing and freeze-drying the protein-removed crude sugar polymers by a dialysis bag, wherein the cut-off molecular weight of the dialysis bag is 1000 Da.

According to the method of the third aspect of the present invention, the ion exchange column in step S6 is ion exchange cellulose or ion exchange gel, and the molecular weight cut-off of the dialysis bag is 100 Da.

According to the method of the third aspect of the present invention, in step S7, Sephadex G or Sephacryls series molecular sieve chromatographic column is used for molecular sieve gel chromatography.

According to the method of the third aspect of the present invention, the water extraction in step S2 specifically comprises: extracting the rhizome of the rhizoma acori graminei with hot water of 60-100 ℃ in an amount which is 5-15 times the volume of the rhizome of the rhizoma acori graminei for 1-10 hours.

According to the method of the third aspect of the present invention, the concentration in steps S3, S4, S6 and S7 is 40-70 ℃ concentration under reduced pressure.

The invention has the beneficial effects that:

1. the invention provides specific structures of the acorus gramineus sugar polymers AT1-3 and AT2-2, and carries out structural identification on the acorus gramineus sugar polymers, thereby defining the physicochemical properties of the acorus gramineus sugar polymers and providing a structural basis for researching the pharmacological activity mechanism of the acorus gramineus sugar polymers.

2. The acorus gramineus polysaccharide polymers AT1-3 and AT2-2 provided by the invention have the activity of preventing and treating neurodegenerative diseases, and are the screened acorus gramineus polysaccharide polymer parts with high content and strong activity.

3. The invention also provides an extraction method of the grassleaf sweelflag rhizome glycopolymer AT1-3 and AT2-2, the invention purifies the grassleaf sweelflag rhizome glycopolymer by using ion exchange column chromatography and molecular sieve gel column chromatography, prepares two grassleaf sweelflag rhizome refined glycopolymers for the first time, and carries out systematic analysis on the physicochemical properties, molecular weight, monosaccharide composition and the like of the two refined glycopolymers to successfully obtain the characteristic structures of the two refined glycopolymers. The invention provides a preparation method of crude sugar polymer and refined sugar polymer in grassleaf sweelflag rhizome, and the activity research of grassleaf sweelflag rhizome polymer in the aspect of resisting neurodegenerative diseases and complications thereof, and provides a basis for the application of grassleaf sweelflag rhizome sugar polymer in the fields of medicines, health products, functional foods and the like.

4. Compared with the traditional water boiling method for extracting the sugar polymer, the method for extracting the grassleaf sweelflag rhizome sugar polymer AT1-3 and AT2-2 combines a water extraction and alcohol precipitation method and an alkali extraction method, and carries out graded alcohol precipitation on the ethanol concentration from low to high to preliminarily separate the grassleaf sweelflag rhizome sugar polymer, and simultaneously, the high-concentration ethanol can separate the sugar polymer with large polarity and good water solubility from the sugar polymer with small polarity and poor water solubility, so that the extracted sugar polymer composition contains more various sugar polymer components. The preparation process is simple, convenient to operate and capable of realizing large-scale production.

5. The method separates and purifies the crude sugar polymers of the grassleaf sweelflag rhizome by column chromatography, has obvious effect, and prepares two grassleaf sweelflag rhizome sugar polymers AT1-3 and AT2-2 for the first time.

Drawings

FIG. 1: morris water maze experiment of Acorus gramineus sugar polymer. FIG. A: a locus diagram of each group of mice in a positioning navigation experiment; and B: trajectory plots of each group of mice in a space exploration experiment; FIG. C, D: the latency and the total route of each group of mice in the positioning navigation experiment; FIG. E, F: the number of cross-over times and the second quadrant residence time of each group of mice in the space exploration experiment.^#P<0.05、^##P<0.01、^###P<0.001vs NC，^*P<0.05、^**P<0.01vs SCO, n.s: there was no significant difference.

FIG. 2: dark avoidance experiments with Acorus gramineus glycomer. FIG. A, B: latency and number of entries into dark boxes for each group of mice.^#P<0.05vs NC，^*P<0.05、^**P<0.01、^***P<0.001vs SCO, n.s: there was no significant difference.

FIG. 3: takeda jumping experiment of Acorus gramineus sugar polymer. FIG. A, B: latency and number of errors for each group of mice.^###P<0.001vs NC，^**P<0.01、^***P<0.001vs SCO, n.s: there was no significant difference.

FIG. 4: effect of tatarinow sweetflag rhizome glycomer on NO levels in AD mice. FIG. A, B: effects of AT1, AT2, ATB on NO levels in brain tissue and serum of AD mice.^#P<0.05、^###P<0.001vs NC，^*P<0.05、^**P<0.01、^***P<0.001vs SCO, n.s: there was no significant difference.

FIG. 5: effect of Acorus gramineus glycopolymer on proinflammatory cytokine levels in AD mice. Effect of AT1, AT2 and ATB on the release of IL-6 (FIG. A, B), IL-1 β (FIG. C, D), PGE-2 (FIG. E, F) and TNF- α (FIG. G, H) from the brain tissue and serum of AD mice.^#P<0.05、^##P<0.01、^###P<0.001vs NC，^*P<0.05、^**P<0.01、^***P<0.001vs SCO, n.s: there was no significant difference.

FIG. 6: infrared spectrum of AT 1-3.

FIG. 7: of AT1-3¹³C NMR spectrum.

FIG. 8: of AT1-3¹H NMR spectrum.

FIG. 9: of AT1-3¹H-¹H COSY map.

FIG. 10: HSQC spectrum of AT 1-3.

FIG. 11: HMBC mapping of AT 1-3.

FIG. 12: the anti-neuritic activity of the Acorus gramineus glycomer AT 1-3. FIG. A: effect of AT1-3 on BV2 cell viability; effect of AT1-3 on LPS-induced NO (panel B), IL-6 (panel C) and TNF-. alpha.release in BV2 cells (panel D). The results are expressed as mean + -SEM,^###P<0.001vs Control，^***P<0.001vsLPS, n.s: there was no significant difference.

FIG. 13: infrared spectrum of AT 2-2.

FIG. 14: of AT2-2¹³C NMR spectrum.

FIG. 15: of AT2-2¹H NMR spectrum.

FIG. 16: of AT2-2¹H-¹H COSY map.

FIG. 17: HSQC spectrum of AT 2-2.

FIG. 18: HMBC mapping of AT 2-2.

Detailed Description

The technical solutions of the present invention are further described below with reference to specific examples, but the present invention is not limited to these specific embodiments. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.

Example 1 extraction method of Acorus gramineus polysaccharide Polymer

1) Selecting materials: selecting dried rhizome of rhizoma acori graminei, cutting into small segments, and soaking in water for 6-12 h;

2) water extraction: extracting the rhizome of the grassleaf sweelflag rhizome soaked in the step 1) with hot water (80 ℃) with the volume being 10 times of that of the rhizome for 4 times, wherein each time lasts for 3 hours, collecting an extracting solution, and airing dregs of a decoction;

3) grading and alcohol precipitating: concentrating the extracting solution obtained in the step 2) AT 60 ℃ under reduced pressure, adding ethanol for alcohol precipitation until the volume concentration of the ethanol is 50%, standing AT room temperature for 24h, separating into a supernatant I and a precipitate I, and collecting the precipitate I to obtain a crude sugar polymer AT 1; concentrating the supernatant fluid I AT 60 ℃ under reduced pressure again, adding ethanol for alcohol precipitation until the volume concentration of the ethanol is 70%, standing AT room temperature for 24h, separating into a supernatant fluid II and a precipitate II, and collecting the precipitate II to obtain a crude sugar polymer AT 2; concentrating the supernatant II again, adding ethanol for alcohol precipitation until the volume concentration of the ethanol is 90%, standing AT room temperature for 24h to obtain a precipitate III, and collecting the precipitate III to obtain a crude sugar polymer AT 3;

4) alkali extraction: soaking the medicine residues aired in the step 2) in a 0.3M NaOH solution with the volume 15 times that of the medicine residues, and standing at room temperature for 2 hours to obtain an extracting solution. Extracting twice according to the method, combining the extracting solutions of the two times, neutralizing the extracting solution with 0.3M HCl solution until the pH value is 7, centrifuging (3500rpm, 10min), taking supernatant, concentrating at 60 ℃ under reduced pressure, adding ethanol until the volume concentration of the ethanol is 75%, standing at room temperature for 24h, and collecting precipitate to obtain crude sugar polymer ATB;

5) and (3) purification: removing proteins from the crude sugar polymers by Sevag method, dialyzing the protein-removed crude sugar polymers with dialysis bag (molecular weight cutoff of 1000Da), and lyophilizing to obtain purified crude sugar polymers of rhizoma Acori Graminei AT1, AT2, AT3, and ATB;

example 2 extraction and separation method of Acorus gramineus solander sugar polymer AT1-3

3) grading and alcohol precipitating: concentrating the extractive solution obtained in step 2) AT 60 deg.C under reduced pressure, adding ethanol to make the volume concentration of ethanol 50%, standing AT room temperature for 24 hr, separating into supernatant I and precipitate I, and collecting precipitate to obtain crude sugar polymer AT 1;

4) and (3) purification: removing protein from the crude sugar polymer AT1 by Sevag method, dialyzing the protein-removed crude sugar polymer with dialysis bag (molecular weight cutoff is 1000Da), and lyophilizing to obtain purified crude sugar polymer AT1 of rhizoma Acori Graminei;

5) ion exchange column chromatography: 200mg of AT1 was dissolved in 10mL of deionized water, and the solution was separated by using DEAE-Cellulose 52 column, and 5 peaks appeared in eluents of different salt concentrations, wherein the third peak was 0.1M NaCl elution fraction (elution was followed by phenol-sulfuric acid method, and sugar fractions were collected from the elution fraction). Concentrating the eluate at 60 deg.C under reduced pressure, dialyzing with dialysis bag (molecular weight cut-off of 100Da), and freeze drying to obtain peak trisaccharide polymer.

6) Molecular sieve gel column chromatography: taking 20mg of the peak trisaccharide polymerization sample obtained in the step 6), dissolving with water, separating with a Sephadex G75 column, using a phenol-sulfuric acid method to track an elution curve to generate a single symmetric peak, collecting a main peak, concentrating under reduced pressure AT 60 ℃, and freeze-drying to obtain the acorus gramineus sugar polymer AT 1-3.

And (3) purity detection: the Acorus gramineus polysaccharide AT1-3 is prepared into 2% concentration (W/V) water solution and detected by HPGPC method. The results showed that AT1-3 has a single symmetrical peak, indicating that AT1-3 is a homogeneous polymer of acorus gramineus sugars.

Further structural analysis of AT1-3 extracted and isolated in the above example is described below.

Structural analysis of acorus gramineus polysaccharide AT1-3

(1) Monosaccharide composition analysis

AT1-3 monosaccharide composition was mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and xylose as determined by PMP-pre-column derivatization-high performance liquid chromatography.

(2) Infrared spectroscopy detection

The infrared spectrum (shown in figure 6) of AT1-3 shows that AT1-3 contains the characteristic absorption peak of sugar.

(3) Methylation analysis

After the AT1-3 sample is methylated, hydrolyzed, reduced and acetylated, the GC-MS analysis shows that the AT1-3 contains L-Araf- (1 →, D-Xylp- (1 →, → 3) -L-Araf- (1 →, → 5) -L-Araf- (1 →, → 3,4) -L-Rhap- (1 →, → 3,4) -D-Xylp- (1 →, → 2) -L-Araf- (1 →, → 4) -D-Glcp- (1 →, → 3) -D-Galp- (1 →, → 6) -D-Galp- (1 →, → 2,3,6) -D-Glcp- (1 → 4,6) -D-Manp- (1 → 3,6) -D-Galp- (1 → and → 3,4,6) -D-Galp- (1 → a sugar residue.

(4) NMR analysis of sugar polymers

A sample of the homogeneous sugar polymer AT1-3 was placed in a nuclear magnetic tube and examined with D₂The results of the measurement of the spectrum after dissolving O are shown in FIGS. 7 to 11.

The assignment of each carbon and hydrogen is known from the nuclear magnetic spectra of FIGS. 7 to 11, and is shown in Table 1 below.

TABLE 1 AT1-3 NMR analysis results

The results of the above-mentioned complete acid hydrolysis, methylation analysis, infrared spectrum detection and nuclear magnetic resonance analysis showed that AT1-3 is a carbohydrate polymer composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and xylose, and it was found from GC-MS analysis and nuclear magnetic resonance analysis that it contains α -L-Araf- (1 →, α -D-Xylp- (1 →, → 3) - α -L-Araf- (1 →, → 5) - α -L-Araf- (1 →, → 3,4) - α -L-Rhap- (1 →, → 3,4) - α -D-Xylp- (1 →, → 2) - α -L-Araf- (1 →, → 4) - β -D-GlcpA- (1 →, and → 3,4) - α -D-Xylp- (1 →, and → 2) - α -L-Araf- (1 → 4) - β -D-GlcpA- (1 →, and →, The sequence of linkages between the different sugar residues in → 3) - β -D-Galp- (1 → 3) - β -D-GalpA- (1 → 6) - β -D-Galp- (1 →, → 2,3,6) - β -D-Glcp- (1 →, → 4,6) - β -D-Galp- (1 →, → 3,6) - β -D-Galp- (1 → and → 3,4,6) - β -D-Galp- (1 → sugar residues, as a result of two-dimensional nuclear magnetic HMBC spectrogram analysis, the structure of AT1-3 from the above analysis is shown below:

wherein a, b, c, d, e, f and g are in the range of 1-1000.

Structural analysis of Acorus gramineus polysaccharide AT2-2

(1) Monosaccharide composition analysis

AT2-2 monosaccharide composition is mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, xylose and fucose, as determined by PMP-pre-column derivatization-high performance liquid chromatography.

(2) Infrared spectroscopy detection

The infrared spectrum (shown in FIG. 13) of AT2-2 shows that AT2-2 contains the characteristic absorption peak of sugar.

(3) Methylation analysis

After the AT2-2 sample is methylated, hydrolyzed, reduced and acetylated, the GC-MS analysis shows that AT2-2 contains L-Araf- (1 →, D-Xylp- (1 →, L-Rhap- (1 →, → 3) -L-Araf- (1 →, → 5) -L-Araf- (1 →, → 4) -D-Xylp- (1 →, → 3) -L-Rhap- (1 →, D-Glcp- (1 →, → 3,5) -L-Araf- (1 →, → 3,4) -D-Xylp- (1 →, → 2) -L-Fucp- (1 →, → 4) -D-Galp- (1 → 3,6) -D-Manp- (1 → and → 2,3,5) -L-Araf- (1 → a sugar residue.

(4) NMR analysis of sugar polymers

A sample of the homogeneous sugar polymer AT2-2 was placed in a nuclear magnetic tube and examined with D₂The results of the measurement of the spectrum after dissolving O are shown in FIGS. 14 to 18.

The assignment of each carbon and hydrogen is known from the nuclear magnetic spectra of FIGS. 14 to 18, and is shown in Table 2 below.

TABLE 2 AT2-2 NMR analysis results

As a result of the above-mentioned complete acid hydrolysis, methylation analysis, infrared spectrum detection and nuclear magnetic resonance analysis, AT2-2 is a carbohydrate polymer composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, xylose and fucose, and it was found from GC-MS analysis and nuclear magnetic resonance analysis that it contains L-Araf- (1 →, D-Xylp- (1 →, L-Rhap- (1 →, → 3) -L-Araf- (1 →, → 5) -L-Araf- (1 →, → 4) -D-Xylp- (1 →, → 3) -L-Rhap- (1 →, D-Glcp- (1 →, → 3,5) -L-Araf- (1 →,3, 4) -D-Xylp- (1 →, D-Xylp- (1 →, and, → 2) -L-Fucp- (1 → 4) -D-Galp- (1 → 3) -D-Galp- (1 →, → 3,6) -D-Manp- (1 → and → 2,3,5) -L-Araf- (1 → sugar residues, the order of linkage between the different sugar residues is derived from two-dimensional nuclear magnetic HMBC spectroscopy analysis, from which the structure of AT2-2 is:

wherein a, b and c are in the range of 1-1000.

The uniform carbohydrate polymer can be an active ingredient of the acorus gramineus for preventing and treating the neurodegenerative diseases, so the structural identification of the uniform carbohydrate polymer provides a powerful basis for the subsequent research on the mechanism of the acorus gramineus for preventing and treating the neurodegenerative diseases.

Example 3 study of anti-Alzheimer's disease Activity of Acorus gramineus crude sugar polymers AT1, AT2, ATB

1. Experimental methods

1.1 Experimental design and grouping:

SPF grade 6-week-old male KM mice, 120, were randomly divided into 6 groups of 20 mice each. The following components are respectively administered: normal group NC: physiological saline; model group SCO: physiological saline; positive control group HA: huperzine A (2 mg/kg); AT1 group: acorus gramineus glycomer AT1(500 mg/kg); AT2 group: acorus gramineus glycomer AT2(500 mg/kg); ATB group: acorus gramineus sugar polymer ATB (500 mg/kg). After 30min of administration by intragastric administration every day, normal group was injected with normal saline, and the other groups were injected with scopolamine (1mg/kg, injection volume of 0.1mL/10g) for molding. Weighing every two days, adjusting the dosage according to the weight change, and continuously performing intragastric administration for 14 days.

1.2 behavioural testing

The behavioral tests were performed 30min after the mice of each group were modeled.

1.2.1 Morris Water maze experiment

After 14 days of continuous administration, i.e., day 15, the Morris water maze test, including the localized voyage test and the space exploration test, was started and continuously tested for 6 days. In a positioning navigation experiment, the escape platform is fixed in the II-th quadrant and is hidden underwater for 1 cm. The test is started by placing the mouse in water facing to the pool wall, training the mouse 4 times every day, 60s each time, continuously training for 4 days, and performing the test of the positioning navigation experiment on the 5 th day. If the mouse climbs the escape platform within 60s, the time the animal reached the platform (i.e., the escape latency) and total distance traveled are recorded. If the mouse did not find a platform within 60s, it was directed to the platform and escape latency was noted as 60 s. All mice were allowed to remain on the platform for 20s, whether or not the platform was found. After the positioning navigation experiment is completed, the escape platform is removed, a space exploration experiment is carried out on the 6 th day, the mouse is placed in the pool for 60s, and the time of activity in each quadrant and the times of passing through the original platform position are recorded.

1.2.2 dark avoidance experiments

On day 21. Before training, the mouse head is put into an open box with the opening facing away from the hole, and the mouse is adapted to the environment for 3 min. Then, the copper grid of the dark box is continuously electrified with 0.3mA current, the mouse is shocked as soon as entering the dark box, and the normal reaction is that the mouse returns to the bright box. Training for the first time, observing for 5min, and recording the time when the mouse enters a dark box for the first time as the score obtained by memory. And (3) carrying out memory test on the mice after 24h, recording the time when the mice enter a dark box for the first time, namely the dark-avoiding latency, and recording the times of the mice entering the dark box within 5min, wherein the latency of the mice which do not enter the dark box within 5min is calculated according to 300 s.

1.2.3 diving platform experiment

On day 24. The method comprises the steps of firstly placing a mouse in a box chamber to adapt for 5min, then placing the mouse on an insulated circular table, electrifying and stimulating the mouse, setting the voltage to be 36V alternating current, recording the time of the mouse jumping off from an insulating table for the first time in each chamber, wherein the time is a latency period, the electrifying time is set to be 5min, and recording the total times of jumping off from the insulating table for the mouse within 5min, wherein the times is error times. After 24h, the test was repeated and the latency and the number of errors in 5min were recorded, the latency for mice that did not jump from the bench within 5min was calculated as 300 s.

1.2.4 Cross elevated experiment

At day 27. The elevated intersecting maze consists of an open arm (50cm × 10cm), a closed arm (50cm × 10cm) and a central area (10cm × 10 cm). At the beginning of the experiment, mice were placed in the maze in the central grid of the closed arm and activity was recorded for 5 min.

1.2.5 open field experiments

At day 28. The open field analysis system is used for observing and researching neuropsychiatric changes of experimental animals and various behaviors after the experimental animals enter an open environment, for example, the animals fear a new open environment and mainly move in a peripheral area, the animals move less in a central area, but the exploration characteristics of the animals promote the animals to generate motivation for moving in the central area, and the anxiety psychology generated by the motivation can also be observed. The bottom surface of the open field is divided into a plurality of grids, the grids are called as peripheral grids along the wall, and the rest are central grids. The activity of the mouse starting running from the center of the bottom surface within 3min was recorded. The groups of mice were compared for differences in spontaneous performance in a new and different environment.

1.3 obtaining and preserving materials

1) Weighing and anaesthetizing;

2) the femoral artery of the mouse was bled and the brain was removed rapidly. The blood samples were gently shaken, centrifuged at a high speed refrigerated centrifuge (3000rpm, 10min, 4 ℃), serum was separated and stored at-80 ℃ for analysis. Washing brain tissue with PBS buffer solution, and drying with filter paper; brains for histopathological observation and immunohistochemical detection were fixed with 10% neutral formaldehyde solution; the remaining samples were stored at-80 ℃ for analysis.

1.4 Biochemical index detection

Brain tissue homogenate samples and serum samples were prepared, tested for NO content according to the Nitric Oxide (NO) kit instructions, and assayed for IL-6, IL-1 β, TNF- α, and PGE-2 content by enzyme-linked immunosorbent assay (ELISA).

2. Results of the experiment

2.1 results of behavioral experiments

The Morris water maze experiment result is shown in figure 1, compared with the NC group, the AD model mouse shows longer escape latency (figure 1C, P <0.05), and the learning and memory ability is reduced, which indicates that the AD model is successfully constructed. Compared with SCO group, the escape latency of the crude sugar polymers AT1, AT2 and AB administration group of the grassleaf sweelflag rhizome and the positive medicine HA group is obviously shortened (P <0.05 or P <0.01), which shows that the learning and memory ability of AD mice is obviously improved; the administration groups of the crude sugar polymers AT1, AT2 and AB of the grassleaf sweelflag rhizome have no significant difference from the positive medicine group and no significant difference from the NC group, which shows that the improvement effects of AT1, AT2 and AB on SCO-induced AD mouse dysmnesia are equivalent to HA and can be improved to normal level. The results of the localization voyage experiments are shown in fig. 1A, D, and the total distance of AT1, AT2 and ATB group is not significantly different from that of HA group, and is not significantly different from that of NC group, which also indicates that AT1, AT2 and AB have the same improvement effect on SCO-induced AD mouse memory impairment as HA, and can improve to normal level. The space exploration experiment result is shown in fig. 1B, E, F, the activity track of the SCO group is obviously deviated from the target quadrant, and the NC group, the HA group, the AT1 group, the AT2 group and the ATB group all have obvious activity tracks in the target quadrant; the times of the AT1 group crossing the original platform position is not significantly different from that of the HA group, the activity time in the target quadrant is not significantly different from that of the HA group, and is also not significantly different from that of the NC group, which indicates that the treatment effect of the AT1 is equivalent to that of the HA, and the treatment effect can be recovered to a normal level. In conclusion, in the water maze experiment, the AT1 group HAs the most obvious effect, and the effect of improving the dysmnesia is equivalent to that of HA; the AT2 and ATB groups also have good therapeutic effect.

As shown in FIG. 2, compared with the NC group, the AD model mouse shows a shorter dark-avoiding latent period (P <0.05), and the number of times of entering the dark room is obviously increased (P <0.05), thus proving that the AD model is successfully constructed. Compared with SCO group, the AT1, AT2 administration group and positive medicine HA group have significant difference in dark-avoiding latency and dark-avoiding frequency (P <0.05, P <0.01 or P <0.001), which shows that AT1, AT2 and positive medicine HA can effectively prolong the dark-avoiding latency of AD mice, reduce the frequency of AD mice entering dark box and have no significant difference (P >0.05) compared with HA group, thus the improvement effect of AT1 and AT2 on AD mouse memory disorder is equivalent to that of HA.

The result of the platform jump experiment is shown in fig. 3, compared with the NC group, the escape latency of the AD model mouse is obviously shortened (P <0.05), the error frequency is obviously increased (P <0.05), and the AD model is proved to be successfully constructed. Compared with SCO group, the incubation period of Acorus gramineus glycopolymer AT1, AT2 and ATB administration group and positive drug HA group is obviously prolonged, and the error frequency is obviously reduced (P <0.01 or P < 0.001). The incubation period and error frequency of the AT1, AT2 and ATB administration groups are not significantly different from those of the HA group (P >0.05), and the improvement effect of AT1, AT2 and ATB on AD mouse dysmnesia is equivalent to that of HA.

The results of open field experiments and cross elevated experiments show that the grassleaf sweelflag rhizome crude sugar polymers AT1, AT2 and ATB can obviously improve the anxiety and depression psychology of AD model mice, regulate the mental changes of the AD model mice and have good effects of preventing and treating nervous system diseases and complications thereof.

2.2 Biochemical index detection results

The effect of crude sugar polymers of acorus gramineus on NO levels in brain tissues of AD model mice is shown in fig. 4A, compared with the NC group, NO levels in brain tissues of SCO group mice are significantly increased (P <0.05), compared with the SCO group, acorus gramineus sugar polymers AT1 and AT2 group, NO levels in brain tissues of positive drug HA group mice are significantly reduced (P <0.05 or P <0.01), NO level of AT1 and AT2 group is significantly different from that of HA group (P >0.05), and NO level of AT1 and AT2 group is significantly different from that of NC group (P >0.05), which indicates that the inhibition effect of AT1 and AT2 on NO release in brain tissues of AD mice is equivalent to that of HA; AT1 and AT2 can down-regulate NO in brain tissues of AD mice to normal level, namely, the anti-inflammatory effect can be restored to normal level.

The effect of the grassleaf sweelflag rhizome glycopolymer on the serum NO level of AD model mice is shown in fig. 4B, compared with the NC group, the serum NO level of SCO group mice is significantly increased (P <0.001), compared with the SCO group, the grassleaf sweelflag rhizome glycopolymer AT1, AT2, ATB group, positive drug HA group mouse serum NO level is significantly reduced (P <0.001), compared with the HA group, the NO level of AT1, AT2, ATB group HAs NO significant difference (P >0.05), and compared with the NC group HAs NO significant difference (P >0.05), which indicates that AT1, AT2, ATB have the same inhibitory effect on the serum NO release of AD mouse NO as HA; AT1, AT2 and ATB can down-regulate NO in the serum of AD mice to normal level, namely the anti-inflammatory effect can be restored to normal level.

The influence of the acorus gramineus glycopolymer on the proinflammatory cytokine level in the AD model mouse is shown in figure 5, and compared with the NC group, the levels of the proinflammatory cytokines IL-6, IL-1 beta, PGE-2 and TNF-alpha in the brain tissue and serum of the AD model mouse are obviously increased, which indicates that the inflammation level in the AD model mouse is increased. Compared with the SCO group, the AT1 administration group remarkably inhibits the IL-6, IL-1 beta, PGE-2 and TNF-alpha levels in the brain tissues and serum of the AD model mice, and HAs no remarkable difference with the HA group, which shows that the inhibition effect of AT1 on proinflammatory cytokines in AD model mice is equivalent to HA, namely the anti-inflammatory effect is equivalent, and partial indexes are restored to the normal group level. Compared with SCO group, the AT2 administration group significantly reduces IL-6 and TNF-alpha level in the brain tissue and serum of AD mice to NC group level, and HAs no significant difference with HA group effect, which shows that the effect of AT2 on IL-6 and TNF-alpha in the serum of AD mice is equivalent to HA and can be recovered to normal level. The levels of IL-1 beta, PRG-2 and TNF-alpha in the brain tissues of AD mice and the levels of IL-6 and TNF-alpha in serum of AD mice are obviously reduced compared with the SCO group and have no obvious difference compared with the HA group, wherein the levels of PGE-2 and TNF-alpha in the brain tissues and the levels of IL-6 and TNF-alpha in serum have no obvious difference compared with the NC group, which indicates that the inhibiting effect of ATB on the inflammatory factors is equivalent to that of positive medicine HA and the inflammatory factors can be restored to the normal level.

Combining the results, the AT1 administration group HAs the most obvious effect, the inhibition effect on the release of proinflammatory cytokines is equivalent to that of positive medicine HA, namely, the anti-neuritis effect is equivalent, and the anti-neuritis effect can be restored to the normal level; AT2 and ATB administration groups also have good anti-neuritic effect.

In the crude sugar polymer of the grassleaf sweelflag rhizome extracted by the invention, AT1, AT2 and ATB can effectively prevent and treat neurodegenerative diseases, wherein the AT1 effect is more obvious. Wherein the seminal glycomer AT1-3 separated and purified from AT1 may be effective component for exerting the above activity, and also has neuritis resisting and neurodegenerative disease treating effects.

Example 4 study of the anti-neuritic Activity of AT1-3

1. Experimental methods

1.1 cell viability assay:

BV2 microglia in logarithmic growth phase at 4X 10⁴The cells were seeded in 96-well plates (100. mu.L/well) at a density of/mL and cultured at 37 ℃ for 24 hours. To each well of the administration group, 100. mu.L of a solution containing different concentrations of AT1-3(2.5, 5, 10. mu.M) and LPS (1. mu.g/mL) was added and incubated AT 37 ℃ for 24 h. Control group was added with 100. mu.L of blank medium and model group was added with 100. mu.L of 1. mu.g/mL LPS. Adding 20 μ L MTT (5mg/mL) into each well, culturing for 4h, adding 150 μ L DMSO into each well, shaking for 10min, measuring OD value of each well at 490nm as detection wavelength, setting 4 multiple wells for each concentration, and adding medicine group, positive medicine group or model group with OD value of A₁The OD value of the control group was A₀The cell survival rate calculation formula is as follows: cell survival (%) ═ a₁/A₀×100％。

1.2 Effect of AT1-3 on NO, IL-6 and TNF-. alpha.Release from BV2 cells:

the inhibitory effect of AT1-3 on LPS-induced NO release in BV2 cells was assessed by the Griess method. BV2 cells in good condition and logarithmic growth phase were cultured at 4X 10⁴The cells were seeded in 96-well plates (100. mu.L/well) at a density of/mL and cultured at 37 ℃ for 24 hours. To each well of the administration group, 100. mu.L of a solution containing different concentrations of AT1-3(2.5, 5, 10. mu.M) and LPS (1. mu.g/mL) was added and incubated AT 37 ℃ for 24 h. Subsequently, 50 μ L of cell supernatant was transferred to a new well, and equal volumes of nitric oxide kit solution i and solution ii were added thereto, shaken well, and absorbance was measured at a wavelength of 540nm with a microplate reader, and 4 duplicate wells were set for each concentration, with sodium nitrite standard solution as a reference.

2. Results of the experiment

In vitro experimental results as shown in fig. 12A, the MTT method showed that LPS has no significant effect on the viability of BV2 cells, and further, the results showed that AT1-3 AT different concentrations had no significant effect on the viability of LPS-induced BV2 cells (P > 0.05). LPS treatment significantly increased NO, TNF- α and IL-6 production by BV2 cells (P <0.001), 5, 10 μ M AT1-3 significantly inhibited LPS-induced NO, IL-6 and TNF- α production (P <0.001), 2.5 μ M AT1-3 significantly inhibited LPS-induced NO and TNF- α production (P <0.001) compared to controls. Overall, AT1-3 has good anti-neuritic activity.

3 conclusion

In the core pathological mechanism of neurodegenerative diseases, neuroinflammation is an important cause of neuronal degeneration loss, microglial over-activation is considered as an important characteristic of neuroinflammation, and a large number of neurotoxic factors (including chemokines and proinflammatory factors) inhibiting the production and secretion of microglial over-activation are important strategies for the treatment of neurodegenerative diseases. The acorus gramineus saccharide polymer extracted by the method shows obvious anti-neuritis effect. Therefore, the activity research of the acorus gramineus sugar polymer in preventing and treating neurodegenerative diseases and complications thereof provides basis for the application of the acorus gramineus sugar polymer in the fields of medicines, health-care foods, functional foods and the like.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A rhizoma Acori Graminei saccharide polymer comprises AT1-3 and/or AT2-2, wherein AT1-3 is saccharide polymer composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and xylose, and its structural formula is shown in formula (I):

wherein a, b, c, d, e and f are in the range of 1-1000;

wherein a, b and c are in the range of 1-1000.

2. The use of the acorus-leaved sweetflag saccharide polymer according to claim 1 in the preparation of a medicament for the prevention and/or treatment of neurodegenerative diseases and their complications.

3. The method for preparing the acorus-leaved sweetflag sugar polymer according to claim 1 or 2, comprising the steps of:

s2, water extraction: heating and extracting rhizome of rhizoma Acori Graminei and the soaking solution A, and filtering to obtain extract B and residue C;

s3, grading alcohol precipitation: concentrating the extracting solution B under reduced pressure, adding ethanol to make the volume concentration of the ethanol be a%, standing, and collecting precipitate and supernatant to obtain crude sugar polymer AT1 and supernatant D; concentrating the supernatant D again, adding ethanol to make the volume concentration of the ethanol be b%, standing, and collecting precipitate and supernatant to obtain crude sugar polymer AT2 and supernatant E; concentrating the supernatant E again, adding ethanol to make the volume concentration of the ethanol be c%, standing, collecting the precipitate to obtain a crude sugar polymer AT3, wherein a is more than or equal to 10 and less than 60, b is more than or equal to 60 and less than 80, and c is more than or equal to 80 and less than 100;

4. The method according to claim 3, wherein the standing time after the alcohol precipitation in the step S3 is 10-28 h.

5. The method according to claim 3, wherein the specific operations of purifying in step S5 are: removing proteins from the crude sugar polymers AT1, AT2, AT3 and ATB by a Sevag method, dialyzing and freeze-drying the protein-removed crude sugar polymers by a dialysis bag, wherein the cut-off molecular weight of the dialysis bag is 1000 Da.

6. The method of claim 3, wherein the ion exchange column in step S6 is ion exchange cellulose or ion exchange gel, and the cut-off molecular weight of the dialysis bag is 100 Da.

7. The method of claim 3, wherein the molecular sieve gel chromatography in step S7 is performed using Sephadex G or Sephacryl S series molecular sieve chromatography column.

8. The method of claim 3, wherein the water extraction in step S2 comprises the following steps: extracting the rhizome of the rhizoma acori graminei with hot water of 60-100 ℃ in an amount which is 5-15 times the volume of the rhizome of the rhizoma acori graminei for 1-10 hours.

9. The method of claim 3, wherein the concentration in steps S3, S4, S6 and S7 is 40-70 ℃ concentration under reduced pressure.