Avicennia marina oligosaccharide as well as preparation method and application thereof

Abstract

The invention discloses avicennia marina oligosaccharide as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. An alkaline phosphatase staining test and an alizarin red staining test prove that the avicennia marina oligosaccharide AMO-1 can remarkably improve the activity level of alkaline phosphatase in MC3T3 cells and osteogenic mineralization nodules, and can remarkably 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 invention is high in purity, does not contain impurities such as protein, nucleic acid and the like, is small in toxic and side effects, has a wide application prospect in preparation of low-toxicity and high-efficiency anti-osteoporosis medicines, and is beneficial to further development and utilization of avicennia marina resources.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polysaccharide and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a metabolic bone disease characterized by decreased systemic bone mass and damaged bone tissue microstructure. Osteoporosis easily leads to increased bone brittleness, which in turn increases the risk of fractures in patients, seriously affecting their health and 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 clinical treatment of osteoporosis can be mainly divided into bone resorption inhibitors and bone formation promoting drugs. However, although the existing drugs can alleviate the clinical symptoms of osteoporosis patients to a certain extent, they have problems such as large toxic side effects, high long-term medication costs and poor patient compliance. Therefore, there is still an urgent need to find and discover safe, effective and low-toxic anti-osteoporosis drugs from natural resources to meet clinical needs.

[0003] The fruit of the mangrove plant Avicennia marina (Forssk.) Vierh., belonging to the Verbenaceae family, is called "olive money" because of its resemblance to a green coin. Oalive money is a highly prized medicinal and edible herb widely distributed in Guangdong, Guangxi, Fujian, and other regions of my country. It boasts heat-clearing, diuretic, blood-cooling, and internal heat-clearing properties, and is commonly used by the general public to treat ailments such as colds, sore throats, and dysentery. Annual production in China exceeds 300,000 tons. However, despite its high production and excellent medicinal and edible properties, its development and utilization remains low. In recent years, researchers both domestically and internationally have isolated a variety of chemical components from Oalive money, including carbohydrates, flavonoids, alkaloids, and phenylethanoid glycosides. Oligosaccharides are considered one of the main bioactive components of Oalive money, exhibiting a variety of pharmacological activities, including antibacterial, antioxidant, anti-complementary, and intestinal microbial regulation. However, studies on the anti-osteoporosis activity of Oalive money oligosaccharides have been limited. Summary of the Invention

[0004] In view of this, the main purpose of the present invention is to provide a saccharide of the Chinese medicinal plant, a preparation method thereof and its application in anti-osteoporosis drugs, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] One of the technical solutions of the present invention is a method for preparing oligosaccharides of Lamb's Root, comprising the following steps:

[0007] (1) crushing and defatting the medicinal material of Radix Glehniae and extracting it in a water bath, and concentrating the extract under reduced pressure to obtain a Radix Glehniae crude extract;

[0008] (2) After the crude extract of Amygdalus dulcis is precipitated with alcohol and protein is removed, crude Amygdalus dulcis oligosaccharide (AMO) is obtained;

[0009] (3) The obtained crude oligosaccharide AMO was dissolved in deionized water and centrifuged. The supernatant was subjected to preliminary separation on a DEAE-52 anion exchange column. Gradient elution was performed using deionized water and different concentrations of NaCl solution as eluents. The fractions were collected and tested. The eluted fractions containing sugars were combined, concentrated, dialyzed, and freeze-dried to obtain the secondary fraction AMO-0M.

[0010] (4) The obtained secondary component AMO-0M was dissolved in deionized water and centrifuged. The supernatant was separated by Sephacryl S-200 gel chromatography and eluted with deionized water. The fractions were collected and tested. The eluted fractions containing sugars were combined, concentrated, dialyzed, and freeze-dried to obtain the AMO-1 oligosaccharide.

[0011] Furthermore, in step (1),

[0012] The degreasing is specifically as follows: degreasing by Soxhlet reflux with petroleum ether 2-3 times, and collecting the residue;

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

[0014] The defatted medicinal residue was mixed with water in a mass ratio of 1:30 and extracted in a water bath at a temperature of 80-85°C for 2-3 hours. The extraction was repeated 2-3 times and the extracts were combined.

[0015] Furthermore, in step (1), the temperature of the petroleum ether Soxhlet reflux degreasing is 60-80°C; and the amount of petroleum ether added is 5 to 7 times the weight of the medicinal material Lanqian.

[0016] Furthermore, in step (2), the alcohol precipitation is specifically performed by adding anhydrous ethanol to the crude extract of the Chinese yam, refrigerating and standing overnight, and collecting the precipitate by centrifugation; wherein the amount of anhydrous ethanol added is 3-4 times the volume of the crude extract of the Chinese yam; and the refrigeration temperature is 2-5°C;

[0017] The protein removal method is: using Savag method to remove protein.

[0018] Furthermore, 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, respectively.

[0019] Furthermore, in step (3), the flow rate of the gradient elution is 10 to 14 mL / 15 min;

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

[0021] The second technical solution of the present invention is to provide AMO-1 obtained by any of the above preparation methods, wherein AMO-1 is a neutral oligosaccharide composed of three monosaccharides and has a molecular weight of 666Da;

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

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

[0024] The third technical solution of the present invention is to provide the use of the above-mentioned AMO-1 oligosaccharide in the preparation of drugs for preventing and treating osteoporosis.

[0025] The fourth technical solution of the present invention is to provide a drug for preventing and treating osteoporosis, comprising the above-mentioned AMO-1 oligosaccharide.

[0026] The beneficial effects of the present invention include at least:

[0027] (1) The present invention proposes for the first time that a homogeneous oligosaccharide AMO-1 from Oleracea chinensis has significant anti-osteoporosis activity. Alkaline phosphatase (ALP) staining and Alizarin red (ARS) staining tests have demonstrated that the Oleracea chinensis oligosaccharide AMO-1 can significantly increase the activity of alkaline phosphatase and osteogenic mineralized nodules in MC3T3 cells, and significantly upregulate the expression levels of osteogenic differentiation-related proteins RUNX2, OSX, OCN, and COL1.

[0028] (2) The present invention proposes for the first time that a homogeneous oligosaccharide AMO-1 in Oleander has a significant therapeutic effect on dexamethasone (DEX)-induced osteoporosis mice. Animal experiments have shown that the Oleander oligosaccharide AMO-1 can significantly enhance the cortical bone thickness of the femur and tibia of osteoporosis mice, while significantly enhancing the bone density of trabeculae and restoring their reticular structure. In addition, Oleander oligosaccharide AMO-1 also significantly increased the bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) of osteoporosis mice, and significantly reduced the trabecular separation (Tb.Sp) and trabecular pattern factor (Tb.Pf).

[0029] (3) The oligosaccharide provided by the present invention is of high purity and basically does not contain impurities such as protein and nucleic acid.

[0030] (4) The oligosaccharides provided by the present invention have little toxicity and side effects, and have broad application prospects in the preparation of low-toxic and high-efficiency anti-osteoporosis drugs, which is conducive to the further development and utilization of olive resources.

[0031] (5) The method for preparing oligosaccharides of the present invention is not only simple to operate and low in cost, but also more efficient and practical, and at the same time better protects the biological activity of oligosaccharides, and is worthy of promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is a flow chart for the preparation of AMO-1 oligosaccharide from Lam.

[0034] Figure 2 This is the HPGPC chromatogram of AMO-1 oligosaccharide from A.

[0035] Figure 3 This is the infrared spectrum of AMO-1 oligosaccharide from Lanqian.

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

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

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

[0039] Figure 7 Figure 2. Effect of AMO-1 on alkaline phosphatase activity in MC3T3 cells. A is a representative photograph of alkaline phosphatase staining in each group; B is a quantitative analysis of alkaline phosphatase activity in each group.

[0040] Figure 8 Figure 2. Effects of AMO-1 on the formation of mineralized bone nodules in MC3T3 cells. A is a representative photograph of mineralized bone nodules in each group; B is a quantitative analysis of the number of mineralized bone nodules in each group.

[0041] Figure 9 Figure 3. Effects of AMO-1 on osteogenic differentiation-related proteins. A: Immunoblotting analysis of osteogenic differentiation-related proteins RUNX2, OSX, OCN, and COL1; B: Quantitative analysis of RUNX2 protein; C: Quantitative analysis of OSX protein; D: Quantitative analysis of OCN protein; and E: Quantitative analysis of COL1 protein.

[0042] Figure 10 Figure 2 shows the effects of AMO-1 on DEX-induced osteoporosis in mice. A is an X-ray of the femur and tibia of each mouse group; B is a coronal section of the cortical bone of each mouse group; C is a three-dimensional reconstruction of the cortical bone of each mouse group; D is an H&E staining of the femur of each mouse group; E is a Goldner staining of the femur of each mouse group; FK are quantitative analyses of BMD, BV / TV, Tb.N, Tb.Pf, Tb.Sp, and Tb.Th in the femur of each mouse group. DETAILED DESCRIPTION

[0043] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0044] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0045] The solution proposed by the present invention is described in detail below through specific embodiments:

[0046] Example 1 Preparation of AMO-1

[0047] The detailed preparation process is as follows Figure 1 As shown. Take 10 kg of Lanqian medicinal materials, grind them, pass through a 50-mesh sieve, and defatted by Soxhlet reflux with petroleum ether (the temperature of petroleum ether Soxhlet extraction and defatting is 60-80°C, and the amount of petroleum ether added is 6 times the weight of Lanqian medicinal materials) 3 times, filter, add deionized water to the residue in a ratio of 1:30, extract 3 times in an 80°C water bath, each time for 2 hours, combine the extracts, and concentrate under reduced pressure to obtain a crude Lanqian extract. Add 4 times the volume of anhydrous ethanol to the crude Lanqian extract and let it stand in a refrigerator at 4°C overnight. Centrifuge to remove the supernatant, collect the precipitate, and redissolve it with deionized water. Further use the Savag method to remove protein, centrifuge, discard the precipitate, repeat 5-6 times until there is no precipitate after centrifugation, concentrate under reduced pressure, and freeze-dry to obtain Lanqian crude oligosaccharide AMO (670 g, yield: 6.7%).

[0048] Take crude oligosaccharides from Radix Amygdalus (2.0 g), dissolve in 30 mL of deionized water, centrifuge, and perform preliminary separation on a DEAE-52 anion exchange column. Gradient elution is performed using deionized water and different concentrations of NaCl solution (0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mol / L) as eluents at a flow rate of 12 mL / 15 min to collect each fraction. The absorbance at 490 nm is detected (sulfuric acid-phenol color development). Based on the saccharide color development reaction, the eluted fractions are combined, concentrated, dialyzed, and freeze-dried to obtain a secondary fraction AMO-OM (404 mg, yield: 20.2%).

[0049] AMO-OM (160 mg) was dissolved in deionized water and centrifuged. The supernatant was separated by Sephacryl S-200 gel chromatography. Elution was performed with deionized water at a flow rate of 4 mL / 15 min, and the fractions were collected. The absorbance at 490 nm was measured (sulfuric acid-phenol color development). Based on the saccharide color development reaction, the eluted fractions were combined, concentrated, dialyzed, and freeze-dried to obtain the homogeneous oligosaccharide AMO-1 (124.8 mg, yield: 78%).

[0050] Example 2 Structural Characterization of AMO-1

[0051] (1) Molecular weight determination

[0052] The molecular weight of AMO-1 was determined using high-performance gel permeation chromatography (HPGPC). Chromatographic conditions included separation using a BRT105-103-101 tandem gel column, a flow rate of 0.8 mL / min, an injection volume of 25 μL, a 0.05 mol / L NaCl solution as the mobile phase, a column temperature of 40°C, and a differential refractive index detector (RID-20A).

[0053] Experimental method: Accurately weigh 5 mg each of the AMO-1 oligosaccharide and the Dextrans series standard, dissolve them in 1 mL of mobile phase to make a 5 mg / mL solution, and centrifuge at 12,000 r / min for 10 min. Extract the supernatant, filter it with a 0.22 μm microporous filter membrane, and then detect it, and record the retention time of the polysaccharide molecules. Draw a standard curve with the retention time as the horizontal axis and the logarithm (Log) of the molecular weight of the standard polysaccharide as the vertical axis to obtain the corresponding linear regression equation. Subsequently, the retention time of the oligosaccharide sample to be tested was substituted into the regression equation, and the molecular weight of the oligosaccharide AMO-1 was found to be 666 Da. The results are as follows Figure 2 shown.

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

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

[0056] (3) Monosaccharide composition analysis

[0057] The monosaccharide composition of AMO-1 was determined by ion chromatography (IC). Approximately 5 mg of AMO-1 and 16 monosaccharide standards (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 and hydrolyzed at 120°C for 3 h in 2 mL of TFA (3 mol / L). The hydrolyzate was dissolved in deionized water and the supernatant was centrifuged for IC analysis.

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

[0059] (4) Methylation analysis

[0060] Methylation analysis of AMO-1 oligosaccharide was performed using gas chromatography-mass spectrometry (GC-MS). Approximately 10 mg of AMO-1 was weighed and, under nitrogen, added to 2 mL of anhydrous DMSO. After dissolution, approximately 200 mg of dry NaOH powder was added. In an ice-water bath, 0.5 mL of iodomethane was slowly added dropwise, followed by 1 mL of iodomethane. The reaction was then allowed to proceed in the dark for 90 minutes. After completion of the reaction, 2 mL of deionized water was added to quench the reaction. The methylated oligosaccharide was then extracted with 3 mL of chloroform. The resulting organic phase was concentrated under reduced pressure and dried to obtain the methylated oligosaccharide sample. This was repeated twice until the methylation was complete.

[0061] Take the above sample, add 4 mL of 2M trifluoroacetic acid, and hydrolyze at 110°C for 2 hours. After the reaction is complete, spin dry to remove all TFA, and redissolve the residue in 2 mL of deionized water. Add 30 mg of sodium borohydride and reduce at room temperature for 3 hours with intermittent shaking. After the reaction is complete, neutralize with 25% acetic acid until no bubbles are generated. Add methanol and evaporate to dryness several times. Dry at 100°C for 15 minutes, add 3 mL of acetic anhydride, and continue the reaction for 1.5 hours. After the reaction is complete, cool to room temperature. Repeatedly add toluene to azeotropically dry the product. Extract the acetylated product with chloroform, and wash the organic phase three times with an equal volume of deionized water. Subsequently, dry the organic phase over anhydrous sodium sulfate, concentrate, and analyze by GC-MS.

[0062] The results showed that the main connection pattern of sugar residues in AMO-1 was 2-linked fructose, terminal-linked galactose, 1,6-linked galactose and 1,6-linked glucose, with a ratio of 1:1:1:1.

[0063] (5) Infrared spectroscopy analysis

[0064] The dried KBr powder was mixed with the dried AMO-1 oligosaccharide and ground into 1 mm thick pellets. The FT-IR spectrometer was then used to analyze the particle size at 400-4000 cm -1 Scan and analyze samples within the range.

[0065] The results of the test are as follows Figure 3 As shown, 3395cm -1 The absorption peak at 2919cm is caused by the stretching vibration of –OH; -1 and 1416cm -1 The absorption peak at 1645cm is caused by C–H bending vibration and stretching vibration; -1 The absorption peak at 1244 cm is caused by C=O stretching vibration; -1 and 1045cm -1 The absorption peaks at 500-800 cm are caused by C–O–C and C–O–H stretching vibrations; -1 There are stretching vibration peaks of pyranose ring and furanose ring.

[0066] (5) NMR spectrum analysis

[0067] Take 50 mg of AMO-1 oligosaccharide sample, dissolve it in 0.5 mL of D2O, and analyze the result on Bruker AV-600 ( 1 H: 600MHz, 13 The one-dimensional and two-dimensional NMR spectra were measured on a C: 150 MHz) NMR spectrometer, and the structure of oligosaccharide AMO-1 was confirmed by referring to the NMR spectra. 1 HNMR and 13 The C NMR results are as follows Figure 4 and Figure 5 shown.

[0068] In the 13C NMR spectrum of AMO-1, in the anomeric carbon region, the 93.60-99.72ppm signal is attributed to 1,6-α-glucose, 1,6-α-galactose and terminal-α-galactose; the 67.19ppm signal is attributed to 2-β-fructose. The 105.17ppm signal is attributed to the signal peak of 2-β-fructose C2. 1 In the H NMR spectrum, the signals at 5.04-5.48 ppm are those of the anomeric hydrogen in oligosaccharide AMO-1, and those at 3.50-4.29 ppm are those of the hydrogen on the sugar ring. Based on the integrated area ratio of the signals near 5.48, 5.04, and 4.27 ppm, the molar ratio of glucose, galactose, and fructose is 1:2:1.

[0069] The above results show that the structure of AMO-1 is T-α-galactose connected to the C-6 position of 1,6-α-galactose, 1,6-α-galactose is further connected to the C-6 position of 1,6-α-glucose, and 1,6-α-glucose is further connected to the C-2 position of 2-β-fructose.

[0070] Example 3 Determination of the Anti-osteoporosis Activity of AMO-1 in Vitro

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

[0072] MC3T3 cells were cultured at 2×10 3 The cells were inoculated at a density of 100 μg / well in a 96-well plate and cultured. After the cells adhered to the wall, different concentrations of AMO-1 (12.5 μg / mL, 25 μg / mL, 50 μg / mL) were added to different wells to divide the experimental groups, and the wells without compound were set as blank control groups. The 96-well plate was placed in an incubator and cultured for 48 hours. CCK-8 solution was added and incubated for 1 hour. Subsequently, the absorbance value (OD) of each well was measured at 450 nm using a multifunctional microplate reader to analyze changes in cell proliferation or toxicity. The results are shown in Figure 2. Figure 6 As shown in the figure, compared with the blank control group, AMO-1 had no significant proliferation effect on MC3T3 cells and no obvious cytotoxicity.

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

[0074] MC3T3 cells were cultured at 5×10 3 The cells were inoculated at a density of 100 μg / well in a 24-well plate for culture. After the cells adhered to the wall, different concentrations of AMO-1 (12.5 μg / mL, 25 μg / mL, 50 μg / mL) were added to different wells to divide the experimental groups, and the wells without compound were set as blank control groups. After administration to the 7th day, the ALP activity level was measured. First, the culture medium in the 24-well plate was aspirated, and 4% paraformaldehyde was added to fix it for 30 minutes, and then the prepared ALP dye solution was added and stained in the dark. After color development, the stained cells were washed twice with PBS, and the stained cells were observed under a stereomicroscope, and the images were quantitatively analyzed using Image J. The results are shown in the figure. Figure 7 As shown in the figure, compared with the blank control group, the AMO-1 treatment groups at concentrations of 25 μg / mL and 50 μg / mL can significantly increase the activity level of ALP in MC3T3 cells. The above experimental results show that AMO-1 has a significant effect in promoting the osteogenic differentiation of MC3T3 cells.

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

[0076] MC3T3 cells were cultured at 5×10 3 The cells were inoculated at a density of 100 μg / well in a 24-well plate for culture. After the cells adhered to the wall, different concentrations of AMO-1 (12.5 μg / mL, 25 μg / mL, 50 μg / mL) were added to different wells to divide the experimental groups, and the wells without compound were set as blank control groups. After administration to the 14th day, the MC3T3 cell Alizarin Red staining experiment was performed. The culture medium in the well plate was aspirated, washed with PBS, and then fixed with 4% paraformaldehyde for 30 minutes. The cells were then washed two to three times with PBS. After washing, Alizarin Red S staining solution was added and stained overnight in the dark. After washing twice with PBS the next day, the stained cells were observed under a stereomicroscope, and the images were quantitatively analyzed using Image J. The results are shown in the figure. Figure 8 As shown in the figure, compared with the blank control group, the number of osteogenic mineralized nodules in the AMO-1 treatment groups at concentrations of 25 μg / mL and 50 μg / mL increased, and there was a clear concentration-dependent effect. The above experimental results show that AMO-1 has a significant effect on promoting osteogenic mineralization of MC3T3 cells.

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

[0078] MC3T3 cells were cultured at a rate of 1×10 5 The cells were seeded at a density of 100 μg / well in a 6-well plate for culture. After the cells adhered to the wall, different concentrations of AMO-1 (12.5 μg / mL, 25 μg / mL, 50 μg / mL) were added for treatment. After 7 days of administration, the supernatant was discarded, and cell lysis solution was added. The cells were lysed in an ice bath for 30 minutes to collect the protein. Subsequently, the cells were centrifuged at 4°C and 12,000 rpm for 30 minutes. The supernatant was taken and the protein content was determined using a BCA protein concentration kit. An appropriate amount of protein was taken for gel electrophoresis, transfer, and blocking. After incubation with the primary antibody at 4°C overnight, the membrane was washed 3 times with TBST for 10 minutes each time. After incubation with the secondary antibody at room temperature for 2 hours, the membrane was washed 3 times with TBST for 10 minutes each time. The protein was visualized using the ECL detection system, and finally the imaging results were analyzed using Image J software. The results are shown in Figure 2. Figure 9 As shown in the results, compared with the normal control group, AMO-1 could significantly upregulate the expression levels of osteogenic differentiation-related proteins RUNX2, OSX, OCN and COL1.

[0079] Example 4 Therapeutic Effects of AMO-1 on DEX-Induced Osteoporosis in Mice

[0080] Forty C57BL / 6 male mice (weight: 24-25 g) were randomly divided into five groups according to their body weight, namely groups A, B, C, D, and E, with 8 mice in each group.

[0081] Among them, group A was the normal control group (Control), and the mice in this group were gavaged with distilled water every day and intraperitoneally injected with normal saline every day.

[0082] Group B was the AMO-1 treatment group. The mice in this group were orally gavaged with AMO-1 (200 mg / kg) daily and intraperitoneally injected with normal saline daily.

[0083] Group C was the model group. The mice in this group were gavaged with distilled water daily and intraperitoneally injected with DEX (30 mg / kg) daily.

[0084] Group D was the AMO-1 administration group. The mice in this group were orally gavaged with AMO-1 (200 mg / kg) daily and intraperitoneally injected with DEX (30 mg / kg) daily.

[0085] Group E was the positive control calcitriol (Cal) administration group. The mice in this group were orally gavaged with Cal (60 ng / kg) daily and intraperitoneally injected with DEX (30 mg / kg) daily.

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

[0087] (1) Effect of AMO-1 oligosaccharide on bone loss in DEX-induced osteoporosis mice.

[0088] DEX is a glucocorticoid. Long-term use of DEX can adversely affect bone microstructure and impair the differentiation ability of osteoblasts. Therefore, DEX is commonly used to construct osteoporosis mouse models and then evaluate the anti-osteoporosis activity of drugs in vivo. Figure 10 As shown in 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 AMO-1 treatment group significantly reversed these pathological changes. Figure 10 As shown in Figures B and 10C, compared with the normal control group, the trabeculae in the model group mice were not only shorter and thinner, but also had a loosely arranged reticular structure and poor interconnection. However, after administration of AMO-1, the trabecular bone density was significantly enhanced and its reticular structure was restored, which was consistent with the results of X-ray analysis. In addition, Figure 10As shown in FK, compared with the model group, AMO-1 also significantly increased the bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) of osteoporosis mice, and significantly reduced the trabecular separation (Tb.Sp) and trabecular pattern factor (Tb.Pf). In addition, H&E staining ( Figure 10 D) and Goldner staining ( Figure 10 E) It was further confirmed that AMO-1 can restore the trabecular bone network structure after administration.

[0089] In summary, the present invention provides a kind of AMO-1 oligosaccharide with a molecular weight of 666Da and a total sugar content of 98.80%, which is basically protein-free and composed of fructose, glucose and galactose. ALP staining test and ARS staining test confirmed that AMO-1 oligosaccharide can significantly increase the activity level of ALP and osteogenic mineralized nodules in MC3T3 cells, and can significantly upregulate the expression levels of osteogenic differentiation-related proteins RUNX2, OSX, OCN and COL1; animal experiments confirmed that AMO-1 can significantly alleviate bone loss in DEX-induced osteoporosis mice, and can be used to prepare drugs for preventing and treating osteoporosis. The oligosaccharide provided by the present invention has high purity, does not contain impurities such as protein and nucleic acid, and has low toxic and side effects. It has broad application prospects in the preparation of low-toxic and efficient anti-osteoporosis drugs, which is conducive to the further development and utilization of AMO resources.

[0090] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0091] The serial numbers of the above embodiments of the present invention are for description only 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 invention and are not intended to limit the present invention. Although the present invention 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 invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing scutellaria oligosaccharide, characterized in that: The following steps are involved: (1) crushing and defatting the medicinal material of Radix Glehniae and extracting it in a water bath, and concentrating the extract under reduced pressure to obtain a Radix Glehniae crude extract; (2) After the crude extract of Amygdalus dulcis is precipitated with alcohol and protein is removed, crude Amygdalus dulcis oligosaccharide (AMO) is obtained; (3) The obtained crude oligosaccharide AMO was dissolved in deionized water and centrifuged. The supernatant was subjected to preliminary separation on a DEAE-52 anion exchange column. Gradient elution was performed using deionized water and different concentrations of NaCl solution as eluents. The fractions were collected and tested. The eluted fractions containing sugars were combined, concentrated, dialyzed, and freeze-dried to obtain the secondary fraction AMO-0M. (4) The obtained secondary component AMO-0M was dissolved in deionized water and centrifuged. The supernatant was separated by Sephacryl S-200 gel chromatography and eluted with deionized water. The fractions were collected and tested. The eluted fractions containing sugars were combined, concentrated, dialyzed, and freeze-dried to obtain the AMO-1 oligosaccharide.

2. The preparation method according to claim 1, characterized in that In step (1), The degreasing is specifically as follows: degreasing by Soxhlet reflux with petroleum ether 2-3 times, and collecting the residue; The water bath extraction is specifically as follows: The defatted medicinal residue was mixed with water in a mass ratio of 1:30 and extracted in a water bath at a temperature of 80-85°C for 2-3 hours. The extraction was repeated 2-3 times and the extracts were combined.

3. The preparation method according to claim 2, characterized in that In step (1), the temperature of petroleum ether Soxhlet reflux degreasing is 60-80° C.; the amount of petroleum ether added is 5-7 times the weight of the medicinal material Lanqian.

4. The preparation method according to claim 1, characterized in that In step (2), the alcohol precipitation is specifically performed as follows: adding anhydrous ethanol to the crude extract of the Chinese yam, refrigerating and standing overnight, and collecting the precipitate by centrifugation; wherein the amount of anhydrous ethanol added is 3-4 times the volume of the crude extract of the Chinese yam; and the refrigeration temperature is 2-5°C; The protein removal method is: using Savag method to remove protein.

5. The preparation method according to claim 1, characterized in that 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, respectively.

6. The preparation method according to claim 1, characterized in that 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 AMO-1 oligosaccharide prepared according to any one of claims 1 to 6, characterized in that: The AMO-1 is a neutral oligosaccharide composed of three monosaccharides with a molecular weight of 666Da. The total sugar content of the AMO-1 oligosaccharide is 98.80%, and the protein content is 1.20%; The monosaccharide molar ratio in the AMO-1 is fructose: glucose: galactose = 1:1:

2.

8. Use of the AMO-1 oligosaccharide according to claim 7 in the preparation of a medicament for preventing and treating osteoporosis.

9. A drug for preventing and treating osteoporosis, characterized in that: Including the AMO-1 oligosaccharide according to claim 7.

10. The drug according to claim 9, characterized in that Pharmaceutically acceptable excipients are also included.

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