Oligo-oligosaccharide of mangiferin and preparation method and application thereof

Abstract

The application discloses an allicol oligosaccharide as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Alkaline phosphatase staining and alizarin red staining tests prove that the allicol oligosaccharide AMO-1 can significantly improve the activity level of alkaline phosphatase in MC3T3 cells and osteogenic mineralization nodules, and can significantly up-regulate the expression levels of osteogenic differentiation related proteins RUNX2, OSX, OCN and COL1; animal experiments prove that AMO-1 can obviously relieve bone loss of DEX-induced osteoporosis mice, and can be used for preparing medicines for preventing and treating osteoporosis. The oligosaccharide provided by the application has high purity, does not contain impurities such as proteins and nucleic acids, has small toxic and side effects, has a wide application prospect in preparation of low-toxicity and high-efficiency anti-osteoporosis medicines, and is favorable for further development and utilization of allicol resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a kind of Avicennia marina oligosaccharide and its preparation method and application. BACKGROUND

[0002] Osteoporosis is a metabolic bone disease characterized by decreased bone mass and impaired bone microarchitecture. Osteoporosis is prone to increase bone fragility, and thus exacerbate the risk of fracture in patients, seriously affecting the life and health of patients and the quality of life. The imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption is considered to be the main pathogenesis of osteoporosis. Currently, the drugs used in the treatment of osteoporosis in the clinic can be divided into bone resorption inhibitors and bone formation promoters. However, the existing drugs can alleviate the clinical symptoms of osteoporosis patients to some extent, but there are problems such as large toxic and side effects, high long-term drug costs and poor patient compliance. Therefore, it is still necessary to find and discover safe, efficient and less toxic anti-osteoporosis drugs from natural resources to meet the clinical needs.

[0003] Avicennia marina is the fruit of Avicennia marina (Forssk.) Vierh. of the Acanthaceae mangrove plant white bone soil, which is called "Avicennia marina" because of its shape similar to green coins. Avicennia marina is a good product for both medicine and food, and it is widely distributed in Guangdong, Guangxi, Fujian and other regions of China. Avicennia marina has the effects of clearing heat, diuresis, cooling blood and eliminating fire, and is often used to treat cold, sore throat and dysentery. In China, the annual output of Avicennia marina exceeds 300,000 tons. However, despite the large output of Avicennia marina and its good medicinal and edible functions, its development and utilization rate is still low. In recent years, domestic and foreign scholars have isolated various types of chemical components such as sugars, flavonoids, alkaloids and phenylethanoid glycosides from Avicennia marina. Among them, oligosaccharides are considered to be one of the main bioactive components of Avicennia marina, which has antibacterial, antioxidant, anticomplement and intestinal flora regulating activities, but there is no report on the anti-osteoporosis activity of Avicennia marina oligosaccharide. SUMMARY

[0004] Therefore, the main purpose of the present application is to provide an Avicennia marina oligosaccharide and its preparation method and application in anti-osteoporosis drugs to solve the above-mentioned problems of the prior art.

[0005] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows:

[0006] One of the technical solutions of the present application is a preparation method of Avicennia marina oligosaccharide, comprising the following steps:

[0007] (1) Avicennia marina medicinal materials are crushed, defatted and extracted by water bath, and the extract is concentrated under reduced pressure to obtain Avicennia marina crude extract;

[0008] (2) the crude liquid of A. cochinchinensis is alcohol precipitated and deproteinized to obtain a crude oligosaccharide AMO of A. cochinchinensis;

[0009] (3) the obtained crude oligosaccharide AMO of A. cochinchinensis is dissolved in deionized water, centrifuged, and the supernatant is subjected to preliminary separation by DEAE-52 anion exchange column, gradient elution is performed with deionized water and NaCl solutions with different concentrations as eluents, each fraction is collected and detected, and then the elution fractions containing saccharides are combined, concentrated, dialyzed, and freeze-dried to obtain a secondary component AMO-0M;

[0010] (4) the obtained secondary component AMO-0M is dissolved in deionized water, centrifuged, and the supernatant is subjected to separation by Sephacryl S-200 gel chromatography, elution is performed with a deionized water solution, each fraction is collected and detected, and then the elution fractions containing saccharides are combined, concentrated, dialyzed, and freeze-dried to obtain an oligosaccharide AMO-1 of A. cochinchinensis.

[0011] Further, in step (1),

[0012] The defatting is specifically as follows: the A. cochinchinensis medicinal material is defatted by Soxhlet reflux with petroleum ether for 2-3 times, and the residues are collected.

[0013] The water bath extraction is specifically as follows:

[0014] The defatted residues are mixed with water at a mass ratio of 1:30, and then subjected to water bath extraction at a temperature of 80-85°C for 2-3 h, and the extraction is repeated for 2-3 times, and the extraction solutions are combined.

[0015] Further, in step (1), the temperature of Soxhlet reflux defatting with petroleum ether is 60-80°C, and the amount of petroleum ether added is 5-7 times the weight of the A. cochinchinensis medicinal material.

[0016] Further, in step (2), the alcohol precipitation is specifically as follows: anhydrous ethanol is added to the crude extract of A. cochinchinensis, and the mixture is refrigerated and left overnight, and then the precipitate is collected by centrifugation; wherein the amount of anhydrous ethanol added is 3-4 times the volume of the crude extract of A. cochinchinensis, and the refrigeration temperature is 2-5°C.

[0017] The deproteinization method is Savag deproteinization.

[0018] Further, in step (3), the NaCl solutions with different concentrations are 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, 1.0 mol / L, and 2.0 mol / L, in sequence.

[0019] Further, in step (3), the flow rate of gradient elution is 10-14 mL / 15 min.

[0020] In step (4), the elution flow rate is 3-5 mL / 15 min.

[0021] The second aspect of the present application provides the above-mentioned any preparation method obtained from the allobaculose AMO-1, wherein the allobaculose AMO-1 is a neutral oligosaccharide composed of three monosaccharides, and the molecular weight is 666 Da.

[0022] The total sugar content of the allobaculose AMO-1 is 98.80%, and the protein content is 1.20%.

[0023] The molar ratio of monosaccharides in the allobaculose AMO-1 is fructose: glucose: galactose = 1: 1: 2.

[0024] The third aspect of the present application provides the use of the above-mentioned allobaculose AMO-1 in the preparation of a drug for preventing and treating osteoporosis.

[0025] The fourth aspect of the present application provides a drug for preventing and treating osteoporosis, comprising the above-mentioned allobaculose AMO-1.

[0026] The present application has at least the following advantages:

[0027] (1) The present application first proposes that a uniform oligosaccharide AMO-1 in allobaculose has significant anti-osteoporosis activity. The alkaline phosphatase (ALP) staining test and alizarin red (ARS) staining test prove that the allobaculose AMO-1 can significantly improve the activity level of alkaline phosphatase in MC3T3 cells and osteogenic mineralization nodules, and can significantly up-regulate the expression levels of osteogenic differentiation related proteins RUNX2, OSX, OCN and COL1.

[0028] (2) The present application first proposes that a uniform oligosaccharide AMO-1 in allobaculose has significant therapeutic effect on dexamethasone (DEX) induced osteoporosis mice. Animal experiments prove that the allobaculose AMO-1 can significantly increase the cortical bone thickness of femur and tibia of osteoporosis mice, and significantly increase the bone density of trabeculae and restore the reticular structure. In addition, the allobaculose AMO-1 also significantly increases the bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N) and trabecular bone thickness (Tb.Th) of osteoporosis mice, and significantly reduces the trabecular bone separation degree (Tb.Sp) and trabecular bone pattern factor (Tb.Pf).

[0029] (3) The allobaculose provided by the present application has high purity and is basically free of impurities such as proteins and nucleic acids.

[0030] (4) The oligosaccharide provided by the present application has small toxic and side effects, and has a wide application prospect in the preparation of low-toxicity and high-efficiency anti-osteoporosis drugs, which is conducive to the further development and utilization of allobaculose resources.

[0031] (5) The method for preparing the oleanolic acid oligosaccharide has simple operation, low cost, high efficiency and practicability, and better protects the biological activity of the oligosaccharide, and is worth popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0033] Figure 1 Preparation flow chart of oleanolic acid oligosaccharide AMO-1.

[0034] Figure 2 HPGPC chromatogram of oleanolic acid oligosaccharide AMO-1.

[0035] Figure 3 Infrared spectrum of oleanolic acid oligosaccharide AMO-1.

[0036] Figure 4 H NMR spectrum of oleanolic acid oligosaccharide AMO-1. 1

[0037] Figure 5 C NMR spectrum of oleanolic acid oligosaccharide AMO-1. 13

[0038] Figure 6 Schematic diagram of cell viability detection of oleanolic acid oligosaccharide AMO-1.

[0039] Figure 7 Effect of oleanolic acid oligosaccharide AMO-1 on the activity level of alkaline phosphatase in MC3T3 cells. A is a representative photograph of alkaline phosphatase staining in each group; B is a quantitative analysis graph of the activity level of alkaline phosphatase in each group.

[0040] Figure 8 Effect of oleanolic acid oligosaccharide AMO-1 on the osteogenic mineralized nodule in MC3T3 cells. A is a representative photograph of the osteogenic mineralized nodule in each group; B is a quantitative analysis graph of the number of the osteogenic mineralized nodule in each group.

[0041] Figure 9 Effect of oleanolic acid oligosaccharide AMO-1 on the osteogenic differentiation related proteins. A is an immunoblotting analysis graph of the osteogenic differentiation related proteins RUNX2, OSX, OCN and COL1; B is a quantitative analysis graph of the RUNX2 protein; C is a quantitative analysis graph of the OSX protein; D is a quantitative analysis graph of the OCN protein; E is a quantitative analysis graph of the COL1 protein.

[0042] ​​Figure 10 Effects of Almatus Oligosaccharide AMO-1 on DEX-induced osteoporosis mice. A is the X-ray image of femur and tibia of mice in each group; B is the coronal section image of cortical bone of mice in each group; C is the three-dimensional reconstruction image of cortical bone of mice in each group; D is the H&E staining image of femur of mice in each group; E is the Goldner staining image of femur of mice in each group; F-K are the quantitative analysis images of BMD, BV / TV, Tb.N, Tb.Pf, Tb.Sp and Tb.Th of femur of mice in each group. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application.

[0044] In addition, the technical solutions in the embodiments of the present application can be combined with each other, but it must be based on the fact that a person of ordinary skill in the art can realize the combination. When the combination of technical solutions appears to be contradictory or unachievable, it should be considered that the combination of technical solutions does not exist and is not within the protection scope of the present application.

[0045] The technical solutions proposed in the present application will be specifically described below through specific embodiments:

[0046] Example 1 Preparation of Almatus Oligosaccharide AMO-1

[0047] The detailed preparation process is shown in Figure 1 The Almatus medicinal material 10 kg was crushed and passed through a 50-mesh sieve, and was defatted by Soxhlet reflux with petroleum ether (the temperature of Soxhlet extraction with petroleum ether was 60-80℃, and the addition amount of petroleum ether was 6 times the weight of the Almatus medicinal material) for 3 times. Then, the filter was added with deionized water in a proportion of 1:30, and was extracted for 3 times at 80℃ water bath for 2h each time. The extraction liquid was combined and concentrated under reduced pressure to obtain Almatus crude extract. The Almatus crude extract was added with 4 times the volume of anhydrous ethanol, and was placed in a refrigerator at 4℃ overnight. The supernatant was removed by centrifugation, and the precipitate was collected and redissolved with deionized water. Further, the Savag method was used to remove protein, and the precipitate was removed by centrifugation. The above operation was repeated for 5-6 times until there was no precipitate in the centrifugation. The concentrated solution was freeze-dried to obtain Almatus crude oligosaccharide AMO (670g, yield: 6.7%).

[0048] Take the crude oligosaccharide of Canarium album (2.0 g), dissolve in 30 mL of deionized water, centrifuge, and the supernatant is subjected to preliminary separation by DEAE-52 anion exchange column. Gradient elution is performed with deionized water and different concentrations of NaCl solution (0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mol / L) as eluent, and the flow rate is 12 mL / 15 min. The fractions are collected, and the absorbance value at 490 nm is detected (sulfuric acid-phenol coloration). According to the color reaction of saccharides, the elution components are combined, concentrated, dialyzed, and freeze-dried to obtain a secondary component AMO-0M (404 mg, yield: 20.2%).

[0049] AMO-0M (160 mg) is dissolved in deionized water, centrifuged, and the supernatant is subjected to separation by Sephacryl S-200 gel chromatography. Elution is performed with deionized water solution, and the flow rate is 4 mL / 15 min. The fractions are collected, and the absorbance value at 490 nm is detected (sulfuric acid-phenol coloration). According to the color reaction of saccharides, the elution components are combined, concentrated, dialyzed, and freeze-dried to obtain uniform oligosaccharide AMO-1 of Canarium album (124.8 mg, yield: 78%).

[0050] Example 2 Structure characterization of Canarium album oligosaccharide AMO-1

[0051] (1) Molecular weight determination

[0052] The molecular weight of Canarium album oligosaccharide AMO-1 is determined by high-performance gel permeation chromatography (HPGPC). The chromatographic conditions are as follows: separation is performed by BRT105-103-101 series gel column, the flow rate is 0.8 mL / min, the injection amount is 25 μL, 0.05 mol / L NaCl solution is used as the mobile phase, the column temperature is 40°C, and the detector is a refractive index detector (RID-20A).

[0053] Experimental method: 5 mg of Canarium album oligosaccharide AMO-1 and Dextrans standard series are accurately weighed, dissolved in 1 mL of mobile phase to prepare a 5 mg / mL solution, and centrifuged at 12,000 r / min for 10 min. The supernatant is extracted, filtered by a 0.22 μm microporous filter, and then detected. The retention time of the polysaccharide molecule is recorded. The standard curve is plotted with the retention time as the abscissa and the logarithmic value (Log) of the molecular weight of the standard polysaccharide as the ordinate, and the linear regression equation is obtained. Subsequently, the retention time of the oligosaccharide sample to be tested is substituted into the regression equation, and the molecular weight of the oligosaccharide AMO-1 is obtained as 666 Da. The results are shown in Table 1. Figure 2

[0054] (2) Determination of total sugar and protein content

[0055] ​The total sugar content of AMO-1 was 98.80% as determined by the phenol-sulfuric acid method; the protein content of AMO-1 was only 1.20% as determined by the bicinchoninic acid method.

[0056] (3) Monosaccharide composition analysis

[0057] Ion chromatography (IC) was used to determine the monosaccharide composition of the olive oligosaccharide AMO-1. About 5 mg of AMO-1 and 16 kinds of monosaccharide standard substances (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, and mannuronic acid) were accurately weighed, 2 mL of TFA (3 mol / L) was added, and hydrolysis was performed at 120°C for 3 h to obtain a hydrolysis product. Deionized water was added to dissolve the product, and centrifugation was performed to obtain the supernatant for IC analysis.

[0058] The results showed that AMO-1 is an oligosaccharide composed of three kinds of monosaccharides. The molar ratio of monosaccharides is fructose: glucose: galactose = 1: 1: 2.

[0059] (4) Methylation analysis

[0060] Gas chromatography-mass spectrometry (GC-MS) was used to perform methylation analysis of the olive oligosaccharide AMO-1. About 10 mg of AMO-1 was weighed, 2 mL of anhydrous DMSO was added under nitrogen protection, and after dissolution, about 200 mg of dry NaOH powder was added. Iodomethane 0.5 mL was slowly added dropwise under an ice water bath, followed by the addition of 1 mL of iodomethane. The reaction was continued in the dark for 90 min. After the reaction was completed, 2 mL of deionized water was added to quench the reaction, and then 3 mL of chloroform was added to extract the methylated oligosaccharide. The obtained organic phase was concentrated and dried under reduced pressure to obtain the methylated oligosaccharide sample. The methylation was repeated twice until it was complete.

[0061] The above sample was taken, 4 mL of 2M trifluoroacetic acid was added, and hydrolysis was performed at 110°C for 2 h. After the reaction was completed, it was spin-dried to remove TFA, and the residue was redissolved in 2 mL of deionized water. 30 mg of sodium borohydride was added, and reduction was performed at room temperature for 3 h with intermittent shaking. After the reaction was completed, it was neutralized with 25% acetic acid until no more bubbles were generated. Methyl alcohol was added and evaporated to dryness several times. It was dried at 100°C for 15 min, 3 mL of acetic anhydride was added, and the reaction was continued for 1.5 h. After the reaction was completed, it was cooled to room temperature. Toluene was repeatedly added to co-distill the product. The acetylated product was extracted with chloroform, and the organic phase was washed with an equal volume of deionized water for 3 times. Subsequently, the organic phase was dried with anhydrous sodium sulfate, concentrated, and analyzed by GC-MS.

[0062] The results showed that the main connection mode of the sugar residues of AMO-1 is: 2-linked fructose, terminal-linked galactose, 1,6-linked galactose, and 1,6-linked glucose, etc. The proportion is 1: 1: 1: 1.

[0063] (5) Infrared spectrum analysis

[0064] Dry KBr powder was mixed with dry Oligosaccharide AMO-1, and grinded into pellets with a thickness of 1 mm. Then, the samples were scanned and analyzed using FT-IR spectrometer in the range of 400-4000 cm -1 .

[0065] The results are shown in Table 1. Figure 3 The absorption peak at 3395 cm -1 was produced by -OH stretching vibration; the absorption peaks at 2919 cm -1 and 1416 cm -1 were produced by C-H bending and stretching vibration; the absorption peak at 1645 cm -1 was produced by C=O stretching vibration; the absorption peaks at 1244 cm -1 and 1045 cm -1 were produced by C-O-C and C-O-H stretching vibration; the stretching vibration peaks of pyranose ring and furanose ring were at 500-800 cm -1 .

[0066] (5) Nuclear magnetic spectrum analysis

[0067] Oligosaccharide AMO-1 sample 50 mg was dissolved in D2O 0.5 mL, and one-dimensional and two-dimensional nuclear magnetic resonance spectra were determined on a Bruker AV-600 1 H: 600 MHz, 13 C: 150 MHz) nuclear magnetic resonance instrument, and the structure of Oligosaccharide AMO-1 was confirmed by referring to the nuclear magnetic spectrum. The results of 1 HNMR and 13 C NMR of AMO-1 are shown in Table 2 and Table 3, respectively. Figure 4 Figure 5

[0068] In the anomeric carbon region of the 13C NMR spectrum of AMO-1, the signals at 93.60-99.72 ppm were assigned to 1,6-α-glucose, 1,6-α-galactose and terminal-α-galactose; the signal at 67.19 ppm was assigned to 2-β-fructose. The signal at 105.17 ppm was assigned to the signal peak of C2 of 2-β-fructose. In the 1 H NMR spectrum, the signals at 5.04-5.48 ppm were the signals of anomeric hydrogens in Oligosaccharide AMO-1, and the signals at 3.50-4.29 ppm were the signals of hydrogens on the sugar ring. According to the integral area ratio of the signals near 5.48, 5.04 and 4.27 ppm, the molar ratio of glucose, galactose and fructose was 1:2:1. ​​

[0069] Based on the above results, it is shown that the structure of Oligomeric Oleanane AMO-1 is T-α-galactose connected to C-6 of 1,6-α-galactose, 1,6-α-galactose is further connected to C-6 of 1,6-α-glucose, and 1,6-α-glucose is further connected to C-2 of 2-β-fructose.

[0070] Example 3 In vitro anti-osteoporosis activity of Oligomeric Oleanane AMO-1

[0071] (1) Cell viability assay of Oligomeric Oleanane AMO-1

[0072] MC3T3 cells were seeded in 96-well plates at a density of 2 x 10 3 After the cells adhered, different concentrations of AMO-1 (12.5 μg / mL, 25 μg / mL, 50 μg / mL) were added to different wells, different experimental groups were divided, and wells without adding compounds were set as blank control group. The 96-well plates were placed in an incubator for continuous culture for 48 h, CCK-8 solution was added, and incubated for 1 h. Subsequently, the absorbance value (OD) of each well was measured at 450 nm by using a multifunctional enzyme label instrument to analyze the changes in cell proliferation or toxicity. The results are shown in Figure 6 Compared with the blank control group, Oligomeric Oleanane AMO-1 had no obvious proliferative effect on MC3T3 cells and had no obvious cytotoxicity.

[0073] (2) Effect of Oligomeric Oleanane AMO-1 on osteogenic differentiation of MC3T3 cells

[0074] MC3T3 cells were seeded in 24-well plates at a density of 5 x 10 3 After the cells adhered, different concentrations of AMO-1 (12.5 μg / mL, 25 μg / mL, 50 μg / mL) were added to different wells, different experimental groups were divided, and wells without adding compounds were set as blank control group. After drug administration for 7 days, the ALP activity level was determined. First, the culture medium in the 24-well plate was aspirated, 4% paraformaldehyde was added for fixation for 30 min, then ALP staining solution was added and stained in the dark. After color development, the cells were washed twice with PBS, and the stained cells were observed under a stereoscope, and the images were quantitatively analyzed by using Image J. The results are shown in Figure 7 Compared with the blank control group, AMO-1 treatment groups with concentrations of 25 μg / mL and 50 μg / mL could significantly improve the ALP activity level in MC3T3 cells. The above experimental results show that Oligomeric Oleanane AMO-1 has a significant effect on promoting osteogenic differentiation of MC3T3 cells.

[0075] (3) Effect of Oligomeric Oleanane AMO-1 on osteogenic mineralization of MC3T3 cells

[0076] MC3T3 cells were loaded at 5×10 3 Cells were seeded at specific densities in 24-well plates and cultured. After cell attachment, different concentrations of AMO-1 (12.5 μg / mL, 25 μg / mL, and 50 μg / mL) were added to different wells to create different experimental groups. Wells without the compound were set up as a blank control group. On day 14, Alizarin Red staining was performed on MC3T3 cells. The culture medium was aspirated from the wells, washed with PBS, and then fixed with 4% paraformaldehyde for 30 min. Cells were then washed two to three times with PBS, followed by Alizarin Red S staining solution and staining overnight in the dark. The next day, after washing twice with PBS, the stained cells were observed under a stereomicroscope, and the images were quantitatively analyzed using ImageJ. The results are as follows: Figure 8 As shown, compared with the blank control group, the osteogenic mineralization nodules in the AMO-1 treatment groups at concentrations of 25 μg / mL and 50 μg / mL were increased, and the increase was significant in a concentration-dependent manner. These experimental results indicate that the oligosaccharide AMO-1 has a significant effect on promoting osteogenic mineralization of MC3T3 cells.

[0077] (4) Effects of AMO-1 oligosaccharide on the expression of osteogenic differentiation-related proteins

[0078] MC3T3 cells were fed at a rate of 1×10 5 Cells were seeded at a density of 1 / well in 6-well plates and cultured. After cell attachment, different concentrations of AMO-1 (12.5 μg / mL, 25 μg / mL, 50 μg / mL) were added for treatment. After 7 days of treatment, the supernatant was discarded, cell lysis buffer was added, and cells were lysed in an ice bath for 30 min. Protein was collected. Subsequently, the cells were centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was used to determine the protein content using a BCA protein concentration kit. An appropriate amount of protein was subjected to gel electrophoresis, transfer to a membrane, and blocking. After incubation with primary antibody at 4 °C overnight, the membrane was washed three times with TBST for 10 min each time. After incubation with secondary antibody at room temperature for 2 h, the membrane was washed three times with TBST for 10 min each time. The protein was visualized and analyzed using an ECL detection system, and the imaging results were analyzed using ImageJ software. The results are as follows: Figure 9 As shown, compared with the normal control group, the oligosaccharide AMO-1 significantly upregulated the expression levels of osteogenic differentiation-related proteins RUNX2, OSX, OCN and COL1.

[0079] Example 4: The therapeutic effect of AMO-1 oligosaccharide on DEX-induced osteoporosis in mice.

[0080] C57BL / 6 male mice 40 (body weight: 24-25g), according to the body weight was randomly divided into 5 groups, namely A, B, C, D, E group, 8 in each group.

[0081] Among them, group A is the normal control group (Control), the mice in this group are given distilled water by gavage every day, and physiological saline is injected intraperitoneally every day.

[0082] Group B is the algarov oligosaccharide AMO-1 treatment group, the mice in this group are given AMO-1 (200mg / kg) by gavage every day, and physiological saline is injected intraperitoneally every day.

[0083] Group C is the model group, the mice in this group are given distilled water by gavage every day, and DEX (30mg / kg) is injected intraperitoneally every day.

[0084] Group D is the algarov oligosaccharide AMO-1 administration group, the mice in this group are given AMO-1 (200mg / kg) by gavage every day, and DEX (30mg / kg) is injected intraperitoneally every day.

[0085] Group E is the positive control calcitriol (Cal) administration group, the mice in this group are given Cal (60ng / kg) by gavage every day, and DEX (30mg / kg) is injected intraperitoneally every day.

[0086] After 63 days of treatment, the body weight of mice in each group was measured, and tissue samples such as femur and tibia of mice were taken for X-ray analysis, μCT analysis and H&E staining analysis, etc.

[0087] (1) The effect of algarov oligosaccharide AMO-1 on bone loss in DEX-induced osteoporotic mice.

[0088] DEX is a glucocorticoid, long-term use will have adverse effects on bone tissue microstructure and impair the differentiation ability of osteoblasts. Therefore, DEX is currently commonly used to construct osteoporosis mouse models, and then evaluate the in vivo anti-osteoporosis activity of drugs. The results of X-ray analysis are shown in Figure 10 A, compared with the normal control group, the cortical bone thickness of the femur and tibia of the model group mice was reduced, while the algarov oligosaccharide AMO-1 treatment group significantly reversed these pathological changes. The results of μCT analysis are shown in Figure 10 B and 10C, compared with the normal control group, the trabecular bone of the model group mice not only became shorter and thinner, but also the reticular structure was loose and poorly interconnected. While after administration of algarov oligosaccharide AMO-1, the bone density of trabecular bone was significantly increased, and the reticular structure was restored, which was consistent with the results of X-ray analysis. In addition, as Figure 10As shown in F-K, compared with the model group, AMO-1 also significantly increased the bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N) and trabecular bone thickness (Tb.Th) of the osteoporosis mice, and significantly reduced the trabecular bone separation (Tb.Sp) and trabecular bone pattern factor (Tb.Pf). In addition, H&E staining Figure 10 D) and Goldner staining Figure 10 E) further confirmed that the reticular structure of the trabecular bone could be restored after administration of AMO-1.

[0089] In summary, the present application provides an AMO-1, which has a molecular weight of 666 Da and a total sugar content of 98.80%, and contains substantially no protein. The AMO-1 comprises fructose, glucose and galactose. The ALP staining test and ARS staining test confirmed that the AMO-1 can significantly increase the activity level of ALP in MC3T3 cells and the osteogenic mineralization nodule, and can significantly up-regulate the expression levels of the osteogenic differentiation related proteins RUNX2, OSX, OCN and COL1. The animal experiment confirmed that the AMO-1 can significantly alleviate the bone loss of the osteoporosis mice induced by DEX, and can be used for preparing a medicine for preventing and treating osteoporosis. The oligosaccharide provided by the present application has high purity, contains no protein, nucleic acid and other impurities, and has small toxic and side effects, and therefore has a wide application prospect in the preparation of low-toxicity and high-efficiency anti-osteoporosis medicines, and is conducive to the further development and utilization of the resources of amomum villosum.

[0090] It should be noted that in this document, the terms "comprising", "containing", or any other variant thereof are intended to cover non-exclusive inclusions, such that a process, method, article or apparatus that comprises a list of elements does not only include those elements, but also other elements not explicitly listed, or other elements inherent to such a process, method, article or apparatus. Without more limitations, the element defined by the phrase "comprising a" does not exclude the presence of additional identical elements in the process, method, article or apparatus that includes the element.

[0091] The above-mentioned sequence numbers of the embodiments of the present application are only for description, and do not represent the advantages or disadvantages of the embodiments.

[0092] The above embodiments are only used to illustrate the technical solutions of the present application and not to limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. A method for preparing an oligosaccharide of oleanolic acid, characterized by, It comprises the following steps: (1) Take the semen canarii medicinal materials after crushing, defatting, water bath extraction, and the extract is reduced pressure concentrated to obtain the semen canarii crude extract; (2) The semen canarii crude extract is alcohol precipitated and deproteinized to obtain the semen canarii crude oligosaccharide AMO; (3) The obtained semen canarii crude oligosaccharide AMO is dissolved in deionized water, centrifuged, and the supernatant is subjected to preliminary separation by DEAE-52 anion exchange column, gradient elution is carried out with deionized water and different concentrations of NaCl solution as eluent, and after detection, the elution fractions containing saccharides are combined, concentrated, dialyzed, and freeze-dried to obtain the secondary component AMO-0M; (4) The obtained secondary component AMO-0M is dissolved in deionized water, centrifuged, and the supernatant is separated by Sephacryl S-200 gel chromatography, eluted with deionized water, and after detection, the elution fractions containing saccharides are combined, concentrated, dialyzed, and freeze-dried to obtain the semen canarii oligosaccharide AMO-1; The semen canarii oligosaccharide AMO-1 is a neutral oligosaccharide composed of three monosaccharides, and the molecular weight is 666 Da; The total sugar content of the semen canarii oligosaccharide AMO-1 is 98.80%, and the protein content is 1.20%; The molar ratio of monosaccharides in the semen canarii oligosaccharide AMO-1 is fructose: glucose: galactose = 1: 1:

2.

2. The production method according to claim 1, characterized by, In step (1), The defatting is specifically: 2-3 times of petroleum ether Soxhlet reflux defatting, and the drug residue is collected; The water bath extraction is specifically: After the drug residue is defatted, it is mixed with water at a mass ratio of 1:30, and then water bath extraction is carried out, the temperature is 80-85℃, the time is 2-3h, and after repeated extraction for 2-3 times, the extract is combined.

3. The preparation method according to claim 2, characterized in that, The temperature of petroleum ether Soxhlet reflux defatting is 60-80℃; the addition amount of petroleum ether is 5-7 times the weight of semen canarii medicinal materials.

4. The method of claim 1, wherein, In step (2), the alcohol precipitation is specifically: anhydrous ethanol is added to the semen canarii crude extract, and the mixture is refrigerated and placed overnight, and then the precipitate is collected by centrifugation; wherein the addition amount of anhydrous ethanol is 3-4 times the volume of the semen canarii crude extract; the refrigeration temperature is 2-5℃; The method for removing protein is: Savag method is used to remove protein.

5. The preparation method according to claim 1, characterized in that, In step (3), the different concentrations of NaCl solution are 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, 1.0 mol / L, and 2.0 mol / L, respectively.

6. The preparation method of claim 1, wherein In step (3), the flow rate of gradient elution is 10-14 mL / 15 min; In step (4), the elution flow rate is 3-5 mL / 15 min.

7. The use of the semen canarii oligosaccharide AMO-1 of claim 1 in the preparation of a drug for preventing and treating osteoporosis.

8. A medicament for preventing and treating osteoporosis, characterized by comprising the compound of claim 1. It comprises the semen canarii oligosaccharide AMO-1 of claim 1.

9. The medicament according to claim 8, characterized in that, It also comprises a pharmaceutically acceptable excipient.

Images