A kind of hydrangea polysaccharide and its preparation method and application

Abstract

The present invention provides a hydrangea polysaccharide, which is polymerized from four monosaccharides, namely, trehalose, galactose, glucose and mannose, and the mass percentages of the monosaccharide components are as follows: trehalose 9.21wt%, galactose 37.29wt%, glucose 34.83wt%, and mannose 18.67%. The hydrangea polysaccharide provided by the present invention is a single polysaccharide active substance, which has a promoting effect on the proliferation of osteoblasts at a relatively low concentration, and can therefore be used as a potential drug for treating osteoporosis.

Description

Technical Field

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

[0002] Osteoporosis is a common chronic disease among the elderly, especially among middle-aged and elderly women. After the disease, osteoporosis pain is likely to occur, which greatly increases the risk of fractures and seriously affects the quality of life of the elderly. The main pathological characteristics of osteoporosis are bone loss and bone microstructure degradation. Osteoblasts are functional cells that play an important role in bone formation.

[0003] At present, the commonly used drugs for the treatment of osteoporosis include anti-bone metabolism drugs, such as estrogen, selective estrogen receptor modulators, bisphosphonates, calcitonins, etc.; bone synthesis drugs, such as parathyroid hormone drugs. Although these drugs have certain therapeutic effects, they also have the risk of side effects, such as: the relationship between estrogen and endometrial cancer and breast cancer is still controversial; conjugated estrogens will significantly increase HDL cholesterol levels; bisphosphonates are prone to produce jaw necrosis, musculoskeletal pain, esophageal cancer, renal failure and other adverse reactions.

[0004] Studies have shown that macrofungi and certain active ingredients have significant effects in improving bone density and relieving osteoporotic pain. In recent years, fungal polysaccharides have attracted extensive attention due to their structural diversity and multiple biological functions. In the prior art, ultrasound-assisted and enzyme-assisted methods are often used to extract macrofungal polysaccharides. Among them, the process of extracting macrofungal polysaccharides by ultrasound-assisted method is as follows: macrofungal powder → ultrasonic treatment and extraction → suction filtration and slag removal → centrifugation (8,000 r / min, 10 min) → supernatant → adding 5% trichloroacetic acid to the supernatant until the extract is not turbid (protein removal)

[18] → centrifugation (8,000 r / min, 20 min) → taking the supernatant and rotary evaporating it at 60°C to about 1 / 4 of its volume → adding 3 times the volume of 95% ethanol and letting it stand for 24 to 48 hours → suction filtration → centrifugation of the filter residue (8,000 r / min, 20 min) and taking the precipitate → washing with anhydrous ethanol and collecting the precipitate → dissolving the precipitate in distilled water and dialyzing for 48 hours → freeze drying → obtaining macrofungal crude polysaccharides. When using ultrasound alone to extract fungal polysaccharides, a higher ultrasonic power is often used to promote the precipitation of polysaccharide components. The local temperature rise and ultrasonic cavitation effect during the ultrasound-assisted extraction process may cause the destruction of the polysaccharide structure, thereby reducing the effective components of the extract, and the results are less reproducible. In addition, it has been reported that ultrasonic treatment causes a decrease in the molecular weight (MW) of polysaccharides by up to 70%. And the resulting polysaccharide is a crude polysaccharide mixture that may contain protein and peptide residues.

[0005] The process of extracting macrofungal polysaccharides by enzyme-assisted method is as follows: take dried macrofungi and crush them with a crusher until they pass through a 60-mesh sieve; weigh 5g of the crushed macrofungal sample, add citric acid-sodium citrate buffer solution according to a liquid-to-solid ratio of 1:25, then add cellulase, and place it in a water bath constant temperature oscillator for enzymolysis; after enzymolysis, place it in a 121℃ autoclave for 20 minutes for inactivation, centrifuge and take the supernatant to 25.0mL, collect the 2 times of dissolution, use a rotary evaporator to concentrate to 100mL, add 400mL of anhydrous ethanol to a concentration of 80%, place it in a 4℃ refrigerator overnight, and centrifuge to obtain the precipitate, which is the crude macrofungal polysaccharide. The relatively high enzyme cost is one of the main disadvantages of enzyme-assisted extraction. In addition, the scale-up of enzyme-assisted extraction is a challenge because the activity of the enzyme is affected by many factors, such as temperature and dissolved oxygen. The type, concentration, temperature, pH, reaction time and liquid-solid ratio of the enzyme also affect the extraction rate of enzyme-assisted extraction. What is obtained is also crude polysaccharide, which is a polysaccharide mixture and may contain protein and peptide residues.

[0006] Crude polysaccharides are not very effective in increasing bone density and alleviating osteoporosis.

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an application of hydrangea polysaccharide in promoting osteoblast proliferation. Summary of the invention

[0008] The invention provides a hydrangea polysaccharide, which is a single polysaccharide active substance and has the function of effectively promoting the proliferation of osteoblasts.

[0009] The technical solution of the present invention is as follows:

[0010] A hydrangea polysaccharide is polymerized from four monosaccharides, namely trehalose, galactose, glucose and mannose. The chemical formula of the hydrangea polysaccharide is shown in formula (I):

[0011]

[0012] Furthermore, the weight percentages of the monosaccharide components are as follows: trehalose 9.21 wt %, galactose 37.29 wt %, glucose 34.83 wt %, and mannose 18.67 %.

[0013] Furthermore, the molar ratio of sugar chain composition is: t-Fuc(p):t-Man(p):t-Glc(p):t-Gal(p):3-Glc(p):6-Man(p):4-Gal(p):6-Gal(p):3,6-Man(p):4,6-Glc(p):2,6-Gal(p)=9.22:2.38:19.01:3.33:14.35:9.59:11.71:11.59:6.70:3.93:8.19.

[0014] Furthermore, the average molecular weight of the hydrangea polysaccharide is 32249 g / mol.

[0015] The present invention also provides a method for preparing hydrangea polysaccharide, comprising the following steps:

[0016] Step S1, crushing the fruiting bodies of Hydrangea serrata, separating and extracting them by low-power ultrasound-assisted cellulase method, and obtaining crude polysaccharide of Hydrangea serrata by alcohol precipitation;

[0017] Step S2, re-dissolving the crude polysaccharide of Hydrangea macrophylla in distilled water, and then purifying it on a DEAE-52 cellulose column to obtain polysaccharide components F-1, F-2 and F-3;

[0018] Step S3, using 0.2 M / L NaCl as the mobile phase for elution in a gel purification system to obtain the polysaccharide component F-1 with the highest content;

[0019] Step S4, further purifying the polysaccharide component F-1 in a gel purification system to obtain purified hydrangea polysaccharide.

[0020] Based on the hydrangea polysaccharide, the present invention also provides an application of the hydrangea polysaccharide in promoting osteoblast proliferation.

[0021] Furthermore, the concentration of Hydrangea polysaccharide is 6.25-25 μg / ml.

[0022] Based on the hydrangea polysaccharide, the present invention also provides an application of the hydrangea polysaccharide in a drug for treating osteoporosis.

[0023] Compared with the prior art, the hydrangea polysaccharide provided by the present invention and its preparation method and application have the following beneficial effects:

[0024] The hydrangea polysaccharide provided by the present invention is a single polysaccharide active substance, which has a promoting effect on the proliferation of osteoblasts at a relatively low concentration, and can therefore be used as a potential drug for treating osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of 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 creative work.

[0026] Figure 1 is the anion exchange chromatogram of crude polysaccharide on DEAE-52 cellulose column;

[0027] Figure 2 It is the anion exchange chromatogram of the polysaccharide component F-1 on the gel purification system column;

[0028] Figure 3 It is the HPGPC spectrum of SCPS;

[0029] Figure 4 is the FT-IR spectrum of SCPS;

[0030] Figure 5 is the IC chromatogram of standard monosaccharide and SCPS in the present invention;

[0031] Figure 6 This is a graph showing the effect of the hydrangea polysaccharide SCPS of the present invention on osteoblast viability;

[0032] Figure 7 It is an electron microscope image showing the effect of the hydrangea polysaccharide SCPS of the present invention on the alkaline phosphatase content of osteoblasts;

[0033] Figure 8 It is a bar graph showing the effect of the hydrangea polysaccharide SCPS of the present invention on the alkaline phosphatase content of osteoblasts;

[0034] Fig. 9 It is an electron microscope image of the effect of the hydrangea polysaccharide SCPS of the present invention on the bone mineralization nodules of osteoblasts;

[0035] Fig.10 The invention relates to the influence of the hydrangea polysaccharide SCPS on the promotion rate of osteoblast bone mineralization nodules. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are further described below.

[0037] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0038] In the present invention, Sparassis crispa is a rare medicinal fungus, which is delicious and nutritious. Polysaccharide is one of the main active ingredients of Sparassis crispa (39.3%-43.6%), has multiple biological properties such as anti-tumor, anti-oxidation, anti-virus and prevention of osteoporosis, and can be used to improve immunity. Therefore, the purpose of the present invention is to study the purification of Sparassis crispa polysaccharide and the application of the purified polysaccharide active substance in osteoblast proliferation.

[0039] Example 1

[0040] A method for preparing hydrangea polysaccharide comprises the following steps:

[0041] Step S1, preparing crude polysaccharide of Hydrangea serrata:

[0042] Step S11, after crushing the fruiting bodies of Hydrangea serrata, firstly use 95% ethanol (v / v) to defatted Hydrangea serrata powder, and then add 10 times the amount of deionized water;

[0043] Step S12, adjusting the pH value to 5.2% cellulase, stirring evenly, ultrasonicating at a low power of 475w for 20min, filtering the filtrate, repeating the process twice, and collecting;

[0044] Step S13, after cooling, the mixture was adjusted to pH 8.0 with 0.5 mol / L hydrochloric acid solution, and centrifuged at 10000 g for 10 min;

[0045] Step S14, the supernatant is deproteinized by Sevag method and decolorized by hydrogen peroxide to pH=8;

[0046] Step S15, the extract is dialyzed with deionized water (molecular weight cutoff Mw is 3500Da) for 2 days, and then freeze-dried to obtain crude polysaccharide.

[0047] Step S2, re-dissolving the crude polysaccharide of Hydrangea macrophylla in distilled water, and then purifying it on a DEAE-52 cellulose column to obtain polysaccharide components F-1, F-2 and F-3;

[0048] Specifically, the crude polysaccharide was redissolved in distilled water and purified on a DEAE-52 cellulose column (4.6 cm×50 cm), eluted with distilled water, 0.1 mol / L, 0.3 mol / L, and 0.5 mol / L NaCl at 1 mL / min, respectively. Each component was determined by the phenol-sulfuric acid method, and the main polysaccharide components were collected, dialyzed, concentrated, and freeze-dried.

[0049] See also Figure 1 , is the anion exchange chromatogram of crude polysaccharide on DEAE-52 cellulose column. After purification on DEAE-52 cellulose column, the three main polysaccharide fractions obtained were designated as F-1, F-2 and F-3, among which F-1 refers to the fraction with the highest yield.

[0050] Step S3, using 0.2 M / L NaCl as the mobile phase for elution on a gel purification system column to obtain the polysaccharide component F-1 with the highest content;

[0051] Specifically, the polysaccharide component was eluted on a gel purification system column (3 cm×100 cm) with 0.2 M / L NaCl at a flow rate of 0.1 mL / min to obtain the polysaccharide component F-1 with the highest content.

[0052] Step S4, further purifying the polysaccharide component F-1 in a gel purification system to obtain purified hydrangea polysaccharide, named SCPS; the purification parameters in this step are the same as those in step S3.

[0053] See also Figure 2 and Figure 3 ,in Figure 2 This is the anion exchange chromatogram of the polysaccharide component F-1 on the gel purification system column. Figure 3 This is the HPGPC spectrum of SCPS. Sparassis crispa polysaccharide F-1 was further purified using a gel purification system column, and a single and symmetrical elution peak appeared in the eluent, which was SCPS. The purity and molecular weight of SCPS were determined using the HPGPC system, and there was a single peak on the column, indicating that SCPS was a homogeneous polysaccharide. The average molecular weight of the purified SCPS was 32249 g / mol as determined by GPC-MALS-RI. In addition, the number average molecular weight (Mn) of SCPS was determined to be 23581 g / mol, and the Mw / Mn, i.e., the polydispersity index (PDI), was 1.37, indicating that SCPS has polydispersity in molecular size.

[0054] Example 2

[0055] Structural Characterization of Polysaccharide SCPS from Hydrangea

[0056] (1) FT-IR spectrum of SCPS

[0057] FT infrared spectroscopy is a qualitative analysis method to predict the structure of polysaccharides based on their specific functional groups. Figure 4 As shown. 3600-3200cm -1 The absorption band at 3409cm is the stretching vibration absorption peak of -OH. The absorption peak in this area is the characteristic peak of sugar. -1 OH stretching vibration absorption peak, which is the characteristic peak of sugar; 2925cm -1 There is an absorption peak at 1850cm -1 There is an absorption peak at 1151 cm, which is related to the stretching vibration of C=O; another absorption peak appears at 1151 cm -1 The absorption peak is caused by the stretching vibration of CO; at 1074cm -1 Absorption peaks were also found at 1027 cm-1, which were mainly caused by the OH angle vibration.

[0058] (2) Monosaccharide composition

[0059] The composition of 11 monosaccharides was determined by ion chromatography, such as Figure 5 As shown in (a), Figure 5 (a) shows the IC chromatogram of standard monosaccharides, including 1-trehalose, 2-arabinose, 3-galactose, 4-glucose, 5-xylose, 6-mannose, 7-ribose, 8-galacturonic acid, 9-glucuronic acid, and 10-mannuronic acid (A); Figure 5 (b) shows the IC chromatogram of SCPS. The results show that SCPS is composed of trehalose, galactose, glucose and mannose. The contents of each monosaccharide component are shown in Table 1.

[0060] Table 1: Monosaccharide composition of SCPS

[0061]

[0062] As shown in Table 1, the backbone of SCPS is composed of glucose (34.83%) and galactose (37.29%).

[0063] (3) Sugar chain composition

[0064] After detection, the molar ratio of the sugar chain composition of the hydrangea polysaccharide SCPS of the present invention is: t-Fuc(p):t-Man(p):t-Glc(p):t-Gal(p):3-Glc(p):6-Man(p):4-Gal(p):6-Gal(p):3,6-Man(p):4,6-Glc(p):2,6-Gal(p)=9.22:2.38:19.01:3.33:14.35:9.59:11.71:11.59:6.70:3.93:8.19.

[0065] (4) Nuclear Magnetic Resonance Analysis

[0066] The structure of SCPS was further studied by 1H- and 13C-nmr and 2D-NMR (HSQC, HMBC and COSY spectra). The NMR characterization results of SCPS are shown in Table 2:

[0067] Table 2: NMR characterization results of SCPS

[0068]

[0069] (5) Chemical structure characterization

[0070] After analysis, the chemical structure of the hydrangea polysaccharide SCPS of the present invention is as shown in formula (I):

[0071]

[0072] Example 3

[0073] Activity test of SCPS polysaccharide from Hydrangea macrophylla on osteoblasts

[0074] 1. Experimental Grouping

[0075] Polysaccharide concentration: 1.56, 3.125, 6.25, 12.5, 25 μg / ml;

[0076] At the same time, a positive drug control group (β-estradiol 10 -8 mol / L), negative control DMSO group, and no-drug group, with five replicates in each group.

[0077] 2. Experimental steps

[0078] 2.1 Take mouse skull preosteoblasts (MC3T3-E1), count the cells, and adjust the cell concentration to 5×10 4 Pieces / ml;

[0079] 2.2 Prepare 100uL (5000 cells) / well of cell suspension in a 96-well plate. Place the plate in an incubator at 37°C, 5% CO2 for 24 hours;

[0080] 2.3 According to different groups, add polysaccharides or drugs with different concentrations and continue to incubate at 37°C for 24 hours;

[0081] 2.4 After the culture, wash twice with PBS, remove the PBS, and add 10uL CCK-8 solution to each well;

[0082] 2.5 Incubate the culture plate in the incubator for 1 hour;

[0083] 2.6 Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0084] 3. Experimental results

[0085] Changes in MC3T3-E1 cell viability in different groups Figure 6 As shown by Figure 6 It can be seen that the hydrangea polysaccharide SCPS of the present invention has no effect on the activity of osteoblasts MC3T3-E1.

[0086] Example 4

[0087] Extracellular alkaline phosphatase detection

[0088] 1. Instruments and reagents

[0089]

[0090] 2. Experimental Grouping

[0091] Blank control group, positive drug (17β-estradiol, 10 -8 mol / L) administration group and polysaccharide sample group (the concentrations were 1.56, 3.125, 6.25, 12.5, and 25 μg / ml, respectively).

[0092] 3. Experimental steps

[0093] 3.1 MC3T3-E1 cells in the logarithmic growth phase were seeded in 96-well plates at 5000 cells / well, and the cell culture medium was replaced with osteogenic conditioned medium (containing mineralization inducer (1x10 -5 α-MEM (1% P / S and 10% FBS) with 50 ug / mL of β-glycerophosphate sodium and 50 ug / mL of ascorbic acid);

[0094] 3.2 According to the above grouping, add drugs to each group and culture for 14 days, during which fresh drug-containing culture medium is replaced every 2 to 3 days;

[0095] 3.3 Collect the cell supernatant culture fluid from each group;

[0096] 3.4 According to the instructions of the ALP kit, use the kit to detect the changes of extracellular ALP;

[0097] 3.5 The OD value of each well was measured at a wavelength of 520 nm using an enzyme reader, converted into King's units, and recorded and analyzed.

[0098] Please refer to Figure 7 and Figure 8 ,in Figure 7 This is an electron microscope image showing the effect of the hydrangea polysaccharide SCPS of the present invention on the alkaline phosphatase content of osteoblasts. Figure 8 This is a bar graph showing the effect of the hydrangea polysaccharide SCPS of the present invention on the alkaline phosphatase content of osteoblasts. Figure 8 * indicates significant changes compared with the control group (P<0.05). Alkaline phosphatase (ALP) is an important marker for the differentiation of bone marrow mesenchymal stem cells into osteoblasts. Figure 7 and Figure 8 It can be seen that compared with the control group, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL, and 25μg / mL SCPS significantly increased the ALP activity of osteoblasts MC3T3-E1 (P<0.05). The results showed that a certain concentration of SCPS can significantly promote the ALP activity and the formation of mineralized nodules in osteoblasts MC3T3-E1.

[0099] Example 5

[0100] Bone mineralized nodule detection

[0101] 1. Instruments and reagents

[0102]

[0103]

[0104] 2. Experimental Grouping

[0105] Blank control group, positive drug (17B-estradiol, 10 -8 mol / L) administration group and polysaccharide sample group (the concentrations were 6.25, 12.5, and 25 μg / ml, respectively).

[0106] 3. Experimental steps

[0107] 3.1 MC3T3-E1 cells were seeded in a 96-well plate at a cell density of 1x105 cells per well. After the cells were cultured until they grew to more than 80%, fresh osteogenic differentiation induction medium was replaced for induction culture of mineralized calcium nodules.

[0108] 3.2 According to the above grouping, add drugs to each group and culture for 15 days. During this period, fresh drug-containing culture medium is replaced every 2 to 3 days until the appearance of mineralized calcium nodules is observed and the culture is terminated;

[0109] 3.3 Discard the old culture medium and gently wash twice with 0.5mL PBS solution, then add 300uL of 4% paraformaldehyde solution to each well for fixation for 15 minutes;

[0110] 3.4 After each well is washed once with 0.5 mL PBS, add 0.5 mL 1% Alizarin Red S staining solution and stain for 10 minutes;

[0111] 3.5 Rinse repeatedly with double distilled water until the background color is washed away, wait for the cell plate to dry and take photos for qualitative observation and comparison;

[0112] 3.6 Add 500uL of 0.5M HCl-5% SDS solution to each well and dissolve the mineralized calcium nodules by continuous shaking for 30 minutes;

[0113] 3.7 Pipette 200uL of dissolved solution from each well and transfer to a 96-well plate. Set the wavelength to 415nm in the microplate reader to detect the OD value and calculate the promotion rate.

[0114] Please refer to Fig. 9 and Fig.10 ,in Fig. 9 This is an electron microscope image of the effect of the hydrangea polysaccharide SCPS of the present invention on the mineralized bone nodules of osteoblasts. Fig.10 The effect of the hydrangea polysaccharide SCPS of the present invention on the promotion rate of osteoblast bone mineralization nodules, Fig.10 The * indicates a significant change compared with the control group (P<0.05). The formation of calcified nodules is one of the main signs of osteoblast maturation. Fig. 9 and Fig.10 It can be seen that the three different concentrations of SCPS (6.25, 12.5, and 25 μg / ml) can significantly promote the mineralization of osteoblasts MC3T3-E1 (P<0.05).

[0115] In summary, the hydrangea polysaccharide SCPS obtained by separation and purification by DEAE-52 cellulose and gel purification system column in the present invention has a promoting effect on the proliferation of osteoblasts at a relatively low concentration and can be used as a potential drug for treating osteoporosis.

[0116] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations made to these embodiments without departing from the principles and spirit of the present invention are still within the protection scope of the present invention.

Claims

1. An application of hydrangea polysaccharide in the preparation of a drug for promoting osteoblast proliferation, characterized in that: The hydrangea polysaccharide is polymerized from four monosaccharides, namely fucose, galactose, glucose and mannose. The chemical formula of the hydrangea polysaccharide is shown in formula (I): 。 2. The use according to claim 1, characterized in that: The weight percentages of the monosaccharide components are as follows: fucose 9.21 wt %, galactose 37.29 wt %, glucose 34.83 wt %, and mannose 18.67 %.

3. The use according to claim 1, characterized in that: The molar ratio of sugar chain composition is: t-Fuc(p): t-Man(p): t-Glc(p): t-Gal(p): 3-Glc(p): 6-Man(p): 4-Gal(p): 6-Gal(p): 3,6-Man(p): 4,6-Glc(p): 2,6-Gal(p) = 9.22: 2.38: 19.01: 3.33: 14.35: 9.59: 11.71: 11.59: 6.70: 3.93: 8.

19.

4. The use according to claim 1, characterized in that: The average molecular weight of the hydrangea polysaccharide is 32249 g / mol.

5. The use according to claim 1, characterized in that: The preparation method of the hydrangea polysaccharide comprises the following steps: Step S1, preparing crude hydrangea polysaccharide: Step S11, after crushing the hydrangea fruiting bodies, firstly defatting the hydrangea powder with 95% ethanol (v / v), and then adding 10 times the amount of deionized water; Step S12, adjusting the pH value to 5.2% cellulase, stirring evenly, ultrasonicating at a low power of 475w for 20min, filtering the filtrate, repeating the process twice, and collecting; Step S13, after cooling, the pH of the mixture was adjusted to 8.0 with 0.5 mol / L hydrochloric acid solution, and centrifuged at 10000 g for 10 min; Step S14, the supernatant is deproteinized by Sevage method and decolorized by hydrogen peroxide to pH=8; Step S15, the extract is dialyzed with deionized water for 2 days, and then freeze-dried to obtain crude polysaccharide, wherein the dialysis molecular weight cut-off Mw is 3500 Da; Step S2, re-dissolving the crude polysaccharide of Hydrangea sphaerocephala in distilled water, and then purifying it on a DEAE-52 cellulose column, eluting with distilled water, 0.1 mol / L, 0.3 mol / L, and 0.5 mol / L NaCl at 1 mL / min, to obtain polysaccharide fractions F-1, F-2, and F-3, wherein F-1 refers to the fraction with the highest yield; Step S3, using 0.2 M / L NaCl as the mobile phase for elution on a gel purification system column to obtain the polysaccharide component F-1 with the highest content; Step S4, further purifying the polysaccharide component F-1 in a gel purification system to obtain the hydrangea polysaccharide as claimed in claim 1.

6. Use of hydrangea polysaccharide in the preparation of a drug for treating osteoporosis, characterized in that: The hydrangea polysaccharide is polymerized from four monosaccharides, namely fucose, galactose, glucose and mannose. The chemical formula of the hydrangea polysaccharide is shown in formula (I): 。 7. The use according to claim 6, characterized in that: The weight percentages of the monosaccharide components are as follows: fucose 9.21 wt %, galactose 37.29 wt %, glucose 34.83 wt %, and mannose 18.67 %.

8. The use according to claim 6, characterized in that: The molar ratio of sugar chain composition is: t-Fuc(p): t-Man(p): t-Glc(p): t-Gal(p): 3-Glc(p): 6-Man(p): 4-Gal(p): 6-Gal(p): 3,6-Man(p): 4,6-Glc(p): 2,6-Gal(p) = 9.22: 2.38: 19.01: 3.33: 14.35: 9.59: 11.71: 11.59: 6.70: 3.93: 8.

19.

9. The use according to claim 6, characterized in that: The average molecular weight of the hydrangea polysaccharide is 32249 g / mol.

10. The use according to claim 6, characterized in that: The preparation method of the hydrangea polysaccharide comprises the following steps: Step S1, preparing crude hydrangea polysaccharide: Step S11, after crushing the hydrangea fruiting bodies, firstly defatting the hydrangea powder with 95% ethanol (v / v), and then adding 10 times the amount of deionized water; Step S12, adjusting the pH value to 5.2% cellulase, stirring evenly, ultrasonicating at a low power of 475w for 20min, filtering the filtrate, repeating the process twice, and collecting; Step S13, after cooling, the pH of the mixture was adjusted to 8.0 with 0.5 mol / L hydrochloric acid solution, and centrifuged at 10000 g for 10 min; Step S14, the supernatant is deproteinized by Sevage method and decolorized by hydrogen peroxide to pH=8; Step S15, the extract is dialyzed with deionized water for 2 days, and then freeze-dried to obtain crude polysaccharide, wherein the dialysis molecular weight cut-off Mw is 3500 Da; Step S2, re-dissolving the crude polysaccharide of Hydrangea sphaerocephala in distilled water, and then purifying it on a DEAE-52 cellulose column, eluting with distilled water, 0.1 mol / L, 0.3 mol / L, and 0.5 mol / L NaCl at 1 mL / min, to obtain polysaccharide fractions F-1, F-2, and F-3, wherein F-1 refers to the fraction with the highest yield; Step S3, using 0.2 M / L NaCl as the mobile phase for elution on a gel purification system column to obtain the polysaccharide component F-1 with the highest content; Step S4, further purifying the polysaccharide component F-1 in a gel purification system to obtain the hydrangea polysaccharide as claimed in claim 1.

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