CN114539439B

CN114539439B - Atractylodes macrocephala polysaccharide AMP1-1, and extraction method and application thereof

Info

Publication number: CN114539439B
Application number: CN202210339495.5A
Authority: CN
Inventors: 魏力军; 王蕊; 李桥; 闫秋鑫; 王金鹏; 侯晓玉; 姜宇; 于子昱
Original assignee: Harbin Institute of Technology
Current assignee: Harbin Institute of Technology
Priority date: 2022-04-01
Filing date: 2022-04-01
Publication date: 2023-03-17
Anticipated expiration: 2042-04-01
Also published as: CN114539439A

Abstract

The invention belongs to the technical field of polysaccharide extraction and weight loss bone loss protection, and particularly discloses an atractylodes macrocephala polysaccharide AMP1-1, and an extraction method and application thereof. The invention discloses a novel-structure atractylodes macrocephala polysaccharide AMP1-1, which consists of alpha-D-glucose and beta-D-fructose, has a molecular weight of 1253-1615 Da, has a main chain consisting of alpha-D-Glcp- (1 → and → 1) -beta-D-Fruf-2 → glycosidic bonds, is a linear-structure polysaccharide, and has a structural formula of alpha-D-Glcp-1 → (2-beta-D-Fruf-1) n (n = 6-8). The atractylis ovata polysaccharide AMP1-1 disclosed by the invention has strong capability of resisting weightlessness bone loss, and can remarkably or extremely remarkably stimulate the activity of osteoblasts and inhibit the activity of osteoclasts.

Description

Atractylodes macrocephala polysaccharide AMP1-1, and extraction method and application thereof

Technical Field

The invention relates to the technical field of polysaccharide extraction and weight loss bone loss protection, and particularly relates to atractylodes macrocephalaon AMP1-1, and an extraction method and application thereof.

Background

The weightlessness bone loss is a disease causing bone loss due to long-term exposure to a weightlessness state, which not only causes metabolic disorder of an organism, but also causes special conditions such as poor recovery and the like after astronauts return to the ground, thus seriously affecting the execution of space missions of astronauts and the body health of the astronauts.

Current protection against weight loss bone loss is mainly focused on exercise training and chemo-drug treatment. However, the effectiveness of the countertraining measures for preventing weight-loss bone loss is not significant. Although effective in alleviating the symptoms of bone loss, chemotherapy is not only highly toxic but also has a number of side effects. Therefore, the search for a bone loss protective agent with high protective activity, small toxic and side effect and safe use is an irreparable matter in aerospace medicine and is also one of important tasks.

The atractylodes macrocephala koidz is dried rhizome of perennial herb atractylodes macrocephala koidz, mainly produced in Jiangxi, hubei, hunan, zhejiang and other places, has various active ingredients and pharmacological actions as a traditional Chinese medicinal plant, and is widely applied to clinical treatment. The main active ingredients in the atractylodes macrocephala koidz are volatile ingredients, polysaccharides, lactones, flavonoids, glycosides and the like, and researches on the chemical ingredients of the atractylodes macrocephala koidz are mostly focused on the lactones, the volatile ingredients and the polysaccharides. The atractylodes can act on various aspects of organisms, has main effects on a gastrointestinal tract system, an immune system and a urinary system, and has various pharmacological effects of resisting tumors, repairing gastric mucosa, resisting inflammation and easing pain, protecting liver, improving memory, regulating lipid metabolism, reducing blood sugar, resisting blood platelet, resisting bacteria, regulating immunity, regulating water metabolism and the like.

The polysaccharide of atractylodes macrocephala has been in the favor of researchers as a main active substance and has various biological activities. Wherein, the influence on the immune system is particularly prominent, the atractylodes macrocephalaon polysaccharide can obviously increase 'T cells', increase the TH/Ts ratio, improve the distribution disorder state of T cell subsets, obviously increase the IL-2 level and increase the expression of IL-2R on the surface of T lymphocytes. In addition, it has significant effects on antioxidation and anti-aging. Numerous studies have found that the polysaccharide of Atractylodes macrocephala koidz can inhibit MDA content and promote the activity of cellular superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Besides, the Atractylodis rhizoma polysaccharide also has effects of lowering blood sugar, resisting tumor and acting on myocardial cells. However, the activity of the atractylenovata polysaccharide on resisting bone loss is not reported, and the structure and the biological activity of the atractylenovata polysaccharide AMP1-1 are discovered for the first time.

Therefore, how to provide the atractylodes macrocephalaon AMP1-1 which can be applied to the protective agent for weight-loss bone loss, enhance the treatment effect on the weight-loss bone loss and avoid the toxic and side effects of chemical drugs is a difficult problem to be solved in the field. In addition, the identification and disclosure of the novel structure of the polysaccharide AMP1-1 of Atractylodes macrocephala is also an important part for solving the problem.

Disclosure of Invention

In view of the above, the invention provides the atractylodes macrocephala polysaccharide AMP1-1 and the extraction method and application thereof, and provides a new choice for the weight-loss bone loss protective agent.

In order to achieve the purpose, the invention adopts the following technical scheme:

an Atractylodes macrocephala polysaccharide AMP1-1, the structure of the Atractylodes macrocephala polysaccharide AMP1-1 is as follows:

wherein the molecular weight of the atractylodes macrocephalaon AMP1-1 is 1253-1615 Da.

The invention also aims to provide an extraction method of the atractylodes macrocephala polysaccharide AMP1-1, which comprises the following steps:

step 1, extracting rhizome powder of bighead atractylodes rhizome with water, and concentrating an extracting solution to obtain a concentrated solution;

step 2, adding ethanol into the concentrated solution to obtain a crude extract of the atractylodes macrocephala polysaccharide;

step 3, separating, purifying and eluting the crude extract of the atractylodes macrocephala polysaccharide by using DEAE-52 ion exchange chromatography, and collecting eluent;

and 4, separating and purifying the eluent obtained in the step 3 by using Sephadex G-100 molecular exclusion chromatography to obtain the bighead atractylodes rhizome polysaccharide AMP1-1.

Preferably, the volume ratio of the rhizome powder of the white atractylodes rhizome to the water in the step 1 is 1:10 to 30.

Preferably, the temperature of the extraction in the step 1 is 70-90 ℃; the extraction time is 3-8 h.

Preferably, the extraction in step 1 further comprises ultrasonic-assisted extraction, wherein the power of the ultrasonic-assisted extraction is 300-800W, the time of the ultrasonic-assisted extraction is 20-40 min, and the number of times of the ultrasonic-assisted extraction is 2-4.

Preferably, the ethanol in the step 2 is absolute ethanol, and the volume ratio of the concentrated solution to the absolute ethanol is 1.

Preferably, the elution conditions in step 3 are: loading the column with DEAE-52, wherein the diameter-length ratio is 1-30, the eluent is distilled water, and the flow rate is 1-1.5 mL/min.

Preferably, the elution conditions in step 4 are as follows: packing the column by Sephadex G-100, wherein the diameter-length ratio is 1-30, the eluent is distilled water, and the flow rate is 1-1.5 mL/min.

The invention also aims to provide application of the atractylenovata polysaccharide AMP1-1 in preparation of a weight-loss bone loss protective agent.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:

the obtained atractylodes macrocephala polysaccharide AMP1-1 has good weight loss bone loss protection activity, and the activity can directly promote osteogenesis and inhibit bone resorption under the microgravity effect, so that the protection effect on bones is achieved. Wherein AMP1-1 promotes osteogenesis by promoting and inhibiting ALP and TRAP activities in bone tissue, respectively. In addition, the promotion of osteogenesis is achieved by up-regulating the expression of differentiation genes of osteoblasts (Runx 2, OSX) and down-regulating differentiation genes of osteoclasts (NFATc 1, cathepsin), respectively.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the distribution of the weight-average molecular weight of the polysaccharide AMP1-1 obtained in example 1;

FIG. 2 is an IR spectrum of the polysaccharide AMP1-1 from example 1;

FIG. 3 shows how the Atractylodes macrocephala Koidz polysaccharide AMP1-1 obtained in example 1 ¹ H NMR and ¹³ a C NMR spectrum;

FIG. 4 is a two-dimensional nuclear magnetic spectrum of the Atractylodes macrocephala polysaccharide AMP1-1 obtained in example 1;

FIG. 5 is a graph showing the effect of polysaccharide AMP1-1 obtained in example 1 on the microstructure of rat femur under weightlessness effect, wherein Control represents the ground Control group; HLS stands for tail suspension treatment group; HLS +20 represents the group treated with 20mg/kg/dAMP1-1 while the tail was suspended; HLS +40 represents the group treated with 40mg/kg/dAMP1-1 while the tail was suspended; HLS +60 represents the group treated with 60mg/kg/dAMP1-1 while the tail was suspended; drawing notes: comparison with the ground control group: * P <0.05, P <0.01, P <0.001; compared with the simulated weight loss effect group, # P <0.05, # P <0.01, # P <0.001.

FIG. 6 is a graph showing the effect of the polysaccharide AMP1-1 obtained in example 1 on the biomechanical properties of rat femur under the effect of weight loss, wherein Control represents a ground Control group; HLS stands for tail suspension treatment group; HLS-AMP represents the group treated with 60mg/kg/dAMP1-1 while the tail was suspended; drawing notes: comparison with the ground control group: * P <0.05,. P <0.01; compared with the simulated weightlessness effect group, # P <0.05, # P <0.01.

FIG. 7 is a graph showing the effect of the polysaccharide AMP1-1 obtained in example 1 on ALP and TRAP activities of rat femurs under the effect of weight loss, wherein Control represents a ground Control group; HLS stands for tail suspension treatment group; HLS-AMP represents the group treated with 60mg/kg/dAMP1-1 while the tail is suspended; and (4) illustration: comparison with the ground control group: * P <0.05,. P <0.01; compared with the simulated weightlessness effect group, # P <0.05, # P <0.01.

FIG. 8 is a graph showing the effect of the polysaccharide AMP1-1 of Atractylodis rhizoma obtained in example 1 on osteoblast differentiation and maturation genes, wherein SMG +30ng/mL represents the treatment with 30ng/mLAMP1-1 under the effect of weight loss; SMG +60ng/mL represents that 60ng/mLAMP1-1 is added under the weight loss effect; SMG +90ng/mL represents that 90ng/mLAMP1-1 is added under the weight loss effect; and (4) illustration: comparison with the ground control group: * P <0.05,. P <0.01; compared with the simulated weightlessness effect group, # P <0.05, # P <0.01.

FIG. 9 is a graph showing the effect of the polysaccharide AMP1-1 from example 1 on RAW246.7 cell line, wherein SMG +30ng/mL represents the treatment with 30ng/mLAMP1-1 under weightlessness; SMG +60ng/mL represents that 60ng/mLAMP1-1 is added under the weight loss effect; SMG +90ng/mL represents that 90ng/mLAMP1-1 is added under the weight loss effect; drawing notes: comparison with the ground control group: * P <0.05,. P <0.01; compared with the simulated weightlessness effect group, # P <0.05, # P <0.01.

Detailed Description

The invention provides an Atractylodes macrocephala polysaccharide AMP1-1, wherein the Atractylodes macrocephala polysaccharide AMP1-1 consists of alpha-D-glucose and beta-D-fructose, has a molecular weight of 1253-1615 Da, has a main chain consisting of alpha-D-Glcp- (1 → and → 1) -beta-D-Fruf-2 → glycosidic bonds, and is a polysaccharide with a linear structure. The structural formula of the atractylodes macrocephalaon AMP1-1 is as follows: α -D-Glcp-1 → (2- β -D-Fruf-1) n (n =6 to 8),

the invention also provides an extraction method of the atractylodes macrocephala polysaccharide AMP1-1, which comprises the following steps:

step 3, separating and purifying the crude extract of the atractylodes macrocephala polysaccharide by using DEAE-52 ion exchange chromatography, and collecting eluent;

In the invention, the separation and purification treatment by Sephadex G-100 molecular exclusion chromatography also comprises the detection of relative molecular weight by GPC.

In the present invention, the volume ratio of the rhizome powder of atractylodes macrocephala in step 1 to water is 1:10 to 30, preferably 1:15 to 25, and further preferably 1:20.

in the present invention, distilled water is preferably used for extraction.

In the invention, the temperature of the extraction in the step 1 is 70-90 ℃, preferably 80 ℃; the extraction time is 3-8 h, preferably 4h.

In the invention, the extraction in step 1 further comprises ultrasonic-assisted extraction, wherein the power of the ultrasonic-assisted extraction is 300-800W, preferably 400-600W, and more preferably 500W; the ultrasonic-assisted extraction time is 20-40 min, preferably 20-30 min, and more preferably 25min; the number of ultrasonic-assisted extractions is 2 to 4, preferably 3.

In the present invention, the end point of the concentration is 1/5 of the original volume.

In the present invention, the ethanol in step 2 is absolute ethanol, and the volume ratio of the concentrated solution to the absolute ethanol is 1:3.

in the present invention, the elution conditions in step 3 are: loading the column with DEAE-52, wherein the diameter-length ratio is 1; the eluent is distilled water, and the flow rate is 1-1.5 mL/min, preferably 1.3mL/min.

In the present invention, the elution conditions in step 4 are: packing the column by Sephadex G-100, wherein the diameter-length ratio is 1-30, and preferably 1; the eluent is distilled water, and the flow rate is 1-1.5 mL/min, preferably 1.3mL/min.

Still another objective of the invention is to provide a preparation method of the atractylenovase AMP1-1 and an application thereof in prevention of weight loss bone loss.

The technical solutions in the embodiments of the present invention will be clearly and completely described below, 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

Rhizome powder of white Atractylodes rhizome (Atractylodes macrocepha) and distilled water were mixed at a ratio of 1:10 volume ratio in water bath at 80 deg.C for 3 hr (during which 500W ultrasonic wave assisted extraction is performed three times for 20 min), collecting supernatant, and concentrating to 1/5 of original volume. Adding anhydrous ethanol with the volume 2 times of the concentrated solution to form flocculent precipitate, collecting the precipitate, and drying to obtain crude extract of the rhizoma Atractylodis Macrocephalae polysaccharide. Separating and purifying the crude extract by using ion exchange chromatography, and loading the crude extract into a column by using DEAE-52, wherein the diameter-length ratio is 1. After collecting the eluent, separating and purifying the eluent by using a molecular exclusion chromatography, and loading the eluent into a Sephadex G-100 column with the diameter-length ratio of 1. Collecting eluate, and detecting relative molecular mass by GPC to obtain novel water soluble polysaccharide, atractylodis rhizoma polysaccharide AMP1-1.

Example 2

Mixing powder of rhizome of Atractylodes macrocephala Koidz with distilled water at a ratio of 1:20 in a water bath at 70 ℃ for 4 hours (during which the extraction is carried out twice with 30min of ultrasound assisted power of 800W), the supernatant is collected and concentrated to 1/5 of the original volume. Adding anhydrous ethanol with the volume 3 times of the concentrated solution to form flocculent precipitate, collecting the precipitate, and drying to obtain crude extract of the rhizoma Atractylodis Macrocephalae polysaccharide. Separating and purifying the crude extract by using ion exchange chromatography, and loading the crude extract into a column by using DEAE-52, wherein the diameter-length ratio is 1. After collecting the eluent, separating and purifying the eluent by using molecular exclusion chromatography, and packing the eluent into a Sephadex G-100 column with the diameter-length ratio of 1:10, wherein the eluent is distilled water and the flow rate is 1.2mL/min. Collecting eluate, and detecting relative molecular mass by GPC to obtain Atractylodis rhizoma polysaccharide AMP1-1.

Example 3

Mixing powder of rhizome of Atractylodes macrocephala Koidz with distilled water at a ratio of 1:30 volume ratio in 90 deg.C water bath for 5 hr (during which 300W power ultrasonic wave assisted extraction is adopted for 4 times, each time for 40 min), collecting supernatant, and concentrating to 1/5 of original volume. Adding 4 times volume of anhydrous ethanol to form flocculent precipitate, collecting precipitate, and drying to obtain crude extract of Atractylodis rhizoma polysaccharide. Separating and purifying the crude extract by using ion exchange chromatography, accurately weighing DEAE-52 by using an electronic balance, packing the DEAE-52 into a column, wherein the diameter-length ratio is 1. After collecting the eluent, separating and purifying the eluent by using a molecular exclusion chromatography, and loading the eluent into a Sephadex G-100 column with the diameter-length ratio of 1:30, wherein the eluent is distilled water and the flow rate is 1.5mL/min. Collecting eluate, and detecting relative molecular mass by GPC to obtain Atractylodis rhizoma polysaccharide AMP1-1.

Example 4 structural characterization of the Atractylodes macrocephala polysaccharide AMP1-1

The structure of the polysaccharide fragment AMP1-1 of Atractylodis rhizoma obtained in example 1 was analyzed by chemical methods (acid hydrolysis, methylation), spectroscopic techniques (IR, HPLC, GC-MS, NMR) and the like, and the following procedure was followed:

1. determination of molecular weight

5mg of the Atractylodes macrocephala polysaccharide AMP1-1 sample was weighed, and 1mL ddH was added ₂ Dissolving O, performing ultrasonic treatment for 5min, and performing GPC analysis. As shown in figure 1, the weight average molecular weight of the largehead atractylodes rhizome polysaccharide AMP1-1 is 1433Da, and the largehead atractylodes rhizome polysaccharide AMP1-1 obtained by the invention is high in purity through the conjecture of peak appearance.

2. Infrared spectrum analysis of Atractylodes macrocephala polysaccharide AMP1-1

Weighing 5mg of atractylenovata polysaccharide AMP1-1 sample, mixing with dried KBr powder, tabletting, and placing in an infrared spectrometer at 4000cm ^-1 ～400cm ^-1 An in-range scan.

As shown in FIG. 2, at 3423cm ^-1 The strong absorption peak at (A) indicates that AMP1-1 polysaccharide has hydroxyl groups (O-H), 925cm ^-1 The signals at (A) indicate that AMP1-1 contains a furan ring and 1141.10cm ^-1 ，598cm ^-1 And 1030.2cm ^-1 The peak at (a) is also due to the presence of the pyranose ring. Further according to 872cm ^-1 And 819.73cm ^-1 Characteristic absorption peaks at (a), indicating that AMP1-1 contains predominantly the alpha configuration.

3. Nuclear magnetic resonance analysis of Atractylodes macrocephala polysaccharide AMP1-1

A50 mg sample of the atractylenovase AMP1-1 was weighed out, dissolved in 0.5mL of heavy water and freeze-dried. And then dissolving the freeze-dried powder in 0.5mL of heavy water to continue freeze drying, and repeating the processes to fully exchange active hydrogen. The sample was then dissolved in 0.5mL of heavy water and subjected to NMR measurement at 600MHz at 25 ℃ at room temperature ¹ H NMR spectrum, ¹³ C NMR spectra, DEPT135 one-dimensional spectra and two-dimensional spectra.

In the hydrogen spectrum and in the carbon spectrum (see FIG. 3)First, the anomeric proton resonance region was analyzed, as shown in FIG. 3 for AMP1-1 ¹ The H-NMR spectrum shows that the product has a weaker signal peak at delta 5.33, and analysis of the combination result shows that delta 5.32 belongs to anomeric hydrogen of-alpha-D-Glcp-1 → the product has the property of reducing the hydrogen content of the head. Such as ¹³ C-NMR showed that there were two signal peaks in the anomeric carbon signal region, δ 104.59 and δ 93.83, respectively, so that the signal peak at δ 104.59 belongs to the main anomeric carbon signal of AMP1-1. The analysis revealed that the signal peak at δ 104.59 was a fructose residue, and the signal peak at δ 93.83 was a glucose residue. In addition, the peak of the anomeric signal of AMP1-1 was significantly shifted downward, indicating that AMP1-1 contains a beta-type glycosidic linkage.

It was found from a two-dimensional nuclear magnetic spectrum (see FIG. 4) that there was a cross peak in H1a,1b (. Beta. -D-Fruf-2, 1) -C2 (. Beta. -D-Fruf-2, 1), indicating that the fructose residues were linked in a chain by a linkage of 2,1 glycosidic bonds. H3 (. Beta. -D-Fruf-2, 1) -C2 (. Beta. -D-Fruf-2, 1), H3 (. Beta. -D-Fruf-2, 1) -C4 (. Beta. -D-Fruf-2, 1) were also detected. And H4 (. Beta. -D-Fruf-2, 1) -C6 (. Beta. -D-Fruf-2, 1) was observed; h4 (beta-D-Fruf-2, 1) -C3 (beta-D-Fruf-2, 1); h4 Three crossing peaks of (. Beta. -D-Fruf-2, 1) -C5 (. Beta. -D-Fruf-2, 1). In addition, the anomeric proton resonance region of AMP1-1 has a strong cross peak H1 (. Alpha. -D-Glcp-1) -C2 (. Beta. -D-Fruf-2, 1), indicating that there is a peak of. Alpha. -D-Glcp-1 → 2-. Beta. -D-Fruf-1 →. Based on Table 1, it is found that four sets of chemical shifts at δ 5.35/3.46, δ 3.46/3.70, δ 3.70/3.39, and δ 3.39/3.78 represent the correlations of H1-H2, H2-H3, H3-H4, and H4-H5 at glucose residues, respectively, and further that three sets of chemical shifts at δ 4.18/4.01, δ 4.01/3.78, and δ 3.78/3.68 represent the correlations of H3-H4, H4-H5, and H5-H6 at fructose residues, respectively.

TABLE 1 preparation of polysaccharide AMP1-1 from Atractylodes macrocephala ¹³ Chemical shifts in C NMR spectra

4. GC-MS spectrum analysis of Atractylodes macrocephala polysaccharide AMP1-1

AMP1-1 sample (2.5 mg) was weighed out and placed in a glass reaction flask for methylation reaction under conditions of magnetically stirring in a water bath at 30 ℃ for 60min, followed by termination of the reaction.

The methylated polysaccharide is acetylated, hydrolyzed by adding 1mL of 2mol/L trifluoroacetic acid (TFA) for 90min, then reduced by 60mg of sodium borohydride for 8h, and then acetylated for 1h at 100 ℃ by adding 1mL of acetic anhydride, and cooled.

The acetylated product was washed with 3mL CH ₂ Cl ₂ After dissolution, transfer to a separatory funnel where CH ₂ Cl ₂ The layer was dried over anhydrous sodium sulfate, and the volume was set to 10mL, and the mixture was placed in a liquid phase vial. The sample of the acetylated product was determined by Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer.

GC-MS conditions: RXI-5SILMS chromatographic column 30m × 0.25mm × 0.25um; the temperature programming conditions are as follows: the initial temperature is 120 ℃, and the temperature is increased to 250 ℃/min at the speed of 3 ℃/min; keeping for 5min; the temperature of the sample inlet is 250 ℃, the temperature of the detector is 250 ℃/min, the carrier gas is helium, and the flow rate is 1mL/min.

From table 2, AMP1-1 has Fruf- (2 →, glcp- (1 → and → 1) -Fruf- (2 → three different glycosidic linkages and its molar ratio is 3.32.

TABLE 2 methylated GC-MS data for the Atractylodes macrocephala polysaccharide AMP1-1

EXAMPLE 5 study of weight loss bone loss prevention Activity of Atractylodes macrocephala polysaccharide AMP1-1

Effect of Atractylodes macrocephala polysaccharide AMP1-1 on osteoblasts and RAW246.7 cell line differentiation and maturation genes

A simulated weightlessness effect (SMG) is established using a bi-directional multi-sample gyrator (2D-RWVS). Primary osteoblasts and RAW246.7 cell lines at 5X 10, respectively ⁵ Perml and 1X 10 ⁵ The cell suspension at a concentration of/mL was inoculated in a 6-well plate already covered with blood slices, and cultured for 12 hours and then subjected to 72 hours of culture for a simulated weight loss effect. After the treatment of simulating the weightlessness effect, collecting cells and extracting mRNA of the cells.

The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit, followed by loading according to the procedures of the real-time quantitative PCR kit instructions, and detection of the mRNA surface using the ABI 7500 SystemAnd (4) obtaining the amount. Finally pass through 2 ^-ΔΔct The method of (3) calculates the expression levels of Runx2 and OSX mRNA in primary osteoblasts and NFATc1 and Cathepsin KmRNA in a RAW246.7 cell line.

1.2 Effect of Atractylodes Macrocephala polysaccharide AMP1-1 on the weight loss Effect treated femoral Structure

A rat tail suspension model is used for establishing a weightlessness bone loss model. 30 healthy male Sprague-Dawley (SD) rats of age 12 weeks were purchased and weighed 200 + -20 g. After 1 week of acclimation, the rats were randomly divided into 3 groups of 10 animals each. The rats in the control group were free to move without any treatment. The tails were suspended in the other two groups for free feeding for 4 weeks during which time the rats were gavaged with AMP1-1 (60 mg/kg/d) while the control group was given an equivalent amount of water. After 4 weeks, the rats were euthanized and their femurs were examined using a micct.

1.3 Effect of Atractylodes Macrocephala polysaccharide AMP1-1 on the mechanical Properties of femurs treated with weightlessness Effect

After 4 weeks from tail-lifting of the rats, all rats were euthanized and femurs were collected for biomechanical testing. The rat femur was placed on the universal tester console so that the distance between the two brackets on which the femur was placed was 16mm. Then, the pressure regulator was moved downward at a constant speed of 0.4mm/min until the femur broke.

1.4 femoral ALP and TRAP Activity treated with the Effect of Atractylodes macrocephala polysaccharide AMP1-1 on weight loss

Femurs in animal models were manipulated and tested and analyzed as required by the instructions of the ALP and TRAP activity test kits.

2. Statistics and analysis

All data are expressed as mean ± SD, the significance of the difference is examined by t-test, and compared to the ground control group, the significance is expressed as # with P <0.05, the extreme significance is expressed as # # with P <0.01; p <0.05, very significantly P <0.01, compared to the simulated weightlessness effect group.

3. Results

As shown in FIG. 5, the microstructure of rat femur is obviously damaged under the weight loss effect, but AMP1-1 has obvious protective effect on the microstructure damage of femur induced by the weight loss effect. In addition, the reduction of Tb.N, tb.Th, BV/TV, BS/TV and BMD induced by the weight loss effect is also protected by AMP1-1, and the indexes can be obviously promoted after the AMP1-1 treatment. Wherein, the AMP1-1 also has a protective effect on Tb.Sp, and can reduce the increase of Tb.Sp caused by the weight loss effect.

As shown in FIG. 6, the elastic modulus (MPa), elastic load (N), maximum strain (%) and flexural rigidity (N.mm) of rat femur ^-1 ) Are all significantly reduced by the effect of weight loss. AMP1-1 in the AMP1-1 gastric lavage treatment group has obvious inhibition effect on reduction of biological performance caused by weight loss effect.

As can be seen from FIG. 7, the weight loss effect can significantly reduce the activity of ALP, a marker enzyme for osteogenesis, and the reduction of ALP activity caused by the weight loss environment after AMP1-1 treatment is obviously inhibited. In addition, the weight loss effect can also cause the TRAP activity to be increased, and the occurrence of bone resorption is promoted. However, AMP1-1 also provides significant protection against this phenomenon, which inhibits TRAP activity under the microgravity effect.

As shown in FIG. 8, the expression levels of mRNA of Runx2 and OSX in osteoblasts extracted from the skull were both affected by the effect of simulated weightlessness, resulting in a decrease in the expression levels. However, the expression of Runx2 and OSX was significantly up-regulated in osteoblasts treated with the atractylenide AMP1-1 compared to the group mimicking the effect of weightlessness. And its promoting effect is positively correlated with the dose of AMP1-1.

As can be seen from fig. 9, the mRNA expression levels of NFATc1 and Cathepsin K in osteoclasts simulated by the RAW246.7 cell line were significantly increased by the effect of the simulated weightlessness effect, whereas the increase in the mRNA expression levels of NFATc1 and Cathepsin K induced by the weightlessness effect was significantly suppressed by the simulated weightlessness treatment with the addition of AMP1-1, and the suppression effects thereof were increased in a dose-dependent manner.

In the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The method for extracting the atractylodes macrocephala polysaccharide AMP1-1 is characterized by comprising the following steps:

step 4, separating and purifying the eluent obtained in the step 3 by using Sephadex G-100 molecular exclusion chromatography to obtain bighead atractylodes rhizome polysaccharide AMP1-1;

the temperature of the extraction in the step 1 is 70 to 90 ℃; the extraction time is 3 to 8 hours;

the extraction in the step 1 further comprises ultrasonic auxiliary extraction, wherein the power of the ultrasonic auxiliary extraction is 300-800W, the time of the ultrasonic auxiliary extraction is 20-40 min, and the number of times of the ultrasonic auxiliary extraction is 2-4;

the elution conditions in step 3 are as follows: packing the mixture into a column by using DEAE-52, wherein the length ratio of the diameter to the length is 1 to 10 to 30, the eluent is distilled water, and the flow rate of the eluent is 1 to 1.5 mL/min;

the eluent in the step 4 is distilled water;

in the step 2, the ethanol is absolute ethanol, and the volume ratio of the concentrated solution to the absolute ethanol is 1 to 2-4.

2. The method for extracting the atractylenovata polysaccharide AMP1-1, as claimed in claim 1, wherein the volume ratio of the rhizome powder of atractylenovata in step 1 to water is 1:10 to 30.

3. The method for extracting the atractylenovata polysaccharide AMP1-1, as claimed in claim 1, wherein the elution conditions in step 4 are: packing the mixture into a Sephadex G-100 column with the diameter-length ratio of 1 to 30 and the flow rate of the eluent of 1 to 1.5mL/min.

4. Use of the atractylodes macrocephala polysaccharide AMP1-1 extracted by the extraction method of any one of claims 1 to 3 in preparation of a bone loss prevention agent.