Lycium barbarum sulfated polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to Chinese wolfberry sulfated polysaccharide as well as a preparation method and application thereof. The sulfated lycium barbarum polysaccharide is a sulfated derivative of lycium barbarum polysaccharide with the sulfated substitution degree of 0.80, and sulfated sites of the sulfated lycium barbarum polysaccharide comprise the third site and the fifth site of terminal-alpha-arabinose, the second site and the third site of 1, 5-alpha-arabinose, the sixth site of terminal-beta-galactose and the fourth site of 1, 3, 6-beta-galactose. The lycium barbarum sulfated polysaccharide provided by the invention has a huge application prospect in the aspect of candidate drugs for preventing and / or treating aging bone loss, and compared with lycium barbarum sulfated polysaccharide in the prior art, the lycium barbarum sulfated polysaccharide provided by the invention has an effect of inhibiting bone resorption; the reason may be that the sulfated lycium barbarum polysaccharide is superior to lycium barbarum polysaccharide in the aspect of improving the bone phenotype of the aged mouse.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharides, and specifically relates to a sulfated polysaccharide derived from wolfberry, a preparation method thereof, and use thereof in preparing an agent or drug for inhibiting, preventing and / or treating senile bone loss. Background Art

[0002] Population aging is a major issue facing the world today. Senile osteoporosis, a primary osteoporosis, is prevalent in both men and women. Its complications, including fractures, are associated with extremely high rates of disability and mortality, primarily characterized by bone loss and alterations in bone microstructure. According to the 2018 China Osteoporosis Epidemiological Survey Report, the prevalence of osteoporosis in people aged 50 and over was 19.2%, with 32.1% in women and 6.9% in men. The prevalence of osteoporosis in people aged 65 and over was 32.0%, with 51.6% in women and 10.7% in men. Based on these epidemiological data, it is estimated that the number of people with osteoporosis in my country is currently approximately 90 million. In the European Union, the prevalence of osteoporosis in people aged 50-55 is 6% (women) and 2.5% (men), while the prevalence of osteoporotic fractures in people aged 80 and over is 47% (women) and 16% (men). It is worth noting that osteoporotic hip and spine fractures carry a 12-month excess mortality rate of up to 20%. They also increase the risk of complications such as pneumonia and thromboembolic disease caused by chronic immobilization, seriously endangering the health of the elderly. Currently, while commercially available chemical drugs for treating age-related osteoporosis have some efficacy, these drugs are often limited by their inherent toxicity and selectivity, making various systemic and local adverse reactions almost inevitable. Therefore, finding effective preventive and treatment options for age-related osteoporosis is a key scientific issue that needs to be addressed urgently in the medical community.

[0003] Lycium barbarum L., a perennial deciduous shrub in the Solanaceae family, is a traditional and precious Chinese medicinal herb approved by the Ministry of Health as a "dual-purpose food and medicine," or "food and medicine of the same origin." First recorded in the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica), wolfberry is neutral in nature, sweet in taste, and enters the kidney and liver meridians, boasting benefits such as nourishing the kidneys, liver, and lungs. It has multiple benefits, including tonifying the liver and kidneys, strengthening bones and tendons, and anti-aging, making it a commonly used health supplement. Among the many active substances in wolfberry, wolfberry polysaccharides (LBPs) are one of the primary pharmacological components of wolfberry. These polysaccharides have been found to exhibit diverse pharmacological activities, including hypoglycemic, anti-tumor, anti-aging, and anti-fatigue properties. LBP1C-2 has been reported to significantly improve skeletal muscle function and alleviate age-related bone loss in aging mice (Patent No. PCT / B2023 / 000414). There is a need to develop new polysaccharides based on LBPs to further improve their efficacy in inhibiting age-related bone loss. Summary of the Invention

[0004] Based on the problems existing in the prior art, the present invention adopts a simple and effective method for preparing sulfated polysaccharides, and uses wolfberry polysaccharide LBP1C-2 for sulfuric acid derivatization to obtain a sulfate-containing polysaccharide (named: S-LBP1C-2 in this article). In young mice and naturally aging osteoporosis mouse models, the sulfated polysaccharide can significantly increase the mouse bone density, trabecular number and thickness, reduce trabecular separation, increase bone formation rate and bone mineralization rate, and increase the levels of alkaline phosphatase and type I collagen amino terminal peptide in serum. Therefore, the sulfated polysaccharide has the potential to resist aging-related bone loss and is expected to become a potential drug for resisting aging-related bone loss.

[0005] On the one hand, the present invention provides a wolfberry sulfated polysaccharide S-LBP1C-2, which has an RG-I type pectin structure, and its raw sugar has the following structure: a main chain is formed by alternating 1→2 linked α-rhamnose and 1→4 linked α-galacturonic acid, and a branch chain is connected to C-4 of 1→2 linked α-rhamnose, C-6 of 1→3 galactose, C-3 of 1→6 galactose and C-3 of 1→5 arabinose in the main chain, and the branch chain includes a terminal, 1→3 and 1→6 linked β-galactose, and the terminal The invention relates to a sulfated polysaccharide of wolfberry, comprising: a first oligosaccharide ...

[0006] In a specific embodiment, the sulfation sites of the wolfberry sulfated polysaccharide S-LBP1C-2 include positions 3,5 of terminal α-arabinose, positions 2,3 of 1,5-α-arabinose, position 6 of terminal β-galactose and position 4 of 1,3,6-β-galactose.

[0007] In a specific embodiment, the degree of sulfate substitution of the wolfberry sulfated polysaccharide S-LBP1C-2 is 0.97.

[0008] In a specific embodiment, the relative molecular mass of the wolfberry sulfated polysaccharide S-LBP1C-2 is 130.7 kDa.

[0009] In a specific embodiment, the sulfated polysaccharide S-LBP1C-2 mainly contains galactose, arabinose, rhamnose and galacturonic acid in a molar ratio of 33.6:49.9:8.0:8.5.

[0010] In a specific embodiment, in the infrared spectrum of the wolfberry sulfated polysaccharide S-LBP1C-2, 3472.13 cm -1 is the OH stretching vibration absorption peak, 1636.44 cm -1 is the stretching vibration absorption peak of C=O, 1232.90 cm -1 and 599.43cm -1 The absorption peak is the stretching vibration peak of O=S=O, proving the existence of sulfate group, 848.51cm -1 This is the vibration absorption produced by the corresponding C in the polysaccharide after being replaced by sulfate, indicating that the polysaccharide contains sulfate and uronic acid.

[0011] In a specific embodiment, the wolfberry sulfated polysaccharide S-LBP1C-2 13 In the C NMR spectrum, signal peaks are found at the following chemical shifts: anomeric carbon region: δ110.6, δ108.78, δ102.02, δ100.28, δ99.95; non-anomeric carbon region: δ79.65, δ79.06, δ77.75, δ74.49, δ72.59, δ69.95, and δ69.20; the above chemical shifts have a deviation of no more than ±0.5.

[0012] On the other hand, the present invention provides a method for preparing the above-mentioned wolfberry sulfated polysaccharide S-LBP1C-2, the method comprising the following steps:

[0013] S1: Take Lycium barbarum polysaccharide LBP1C-2, add dry formamide, seal with a rubber stopper, and stir to dissolve;

[0014] S2: In another vial, add chlorosulfonic acid to pyridine to prepare a viscous sulfation reagent, and place the sulfation reagent in an oil bath at 50-80°C for reaction; and

[0015] S3: The polysaccharide sample in S1 is added to the sulfation reagent in S2 in a 50-80°C oil bath for reaction.

[0016] In a specific embodiment, in step S1, the concentration of Lycium barbarum polysaccharide LBP1C-2 in formamide is 15-25 mg / mL; in step S2, the volume ratio of chlorosulfonic acid to pyridine is 4:1-2:1, the oil bath temperature is 50-80°C, and the reaction time is 20-60 min; in step S3, the oil bath temperature is 50-80°C, and the reaction time is 2-4 h.

[0017] In a specific embodiment, the Lycium barbarum polysaccharide LBP1C-2 in step S1 has a structure: a main chain is formed by alternating 1→2-linked α-rhamnose and 1→4-linked α-galacturonic acid, and side chains are connected to C-4 of 1→2-linked α-rhamnose, C-6 of 1→3 galactose, C-3 of 1→6 galactose and C-3 of 1→5 arabinose in the main chain, and the side chains include terminal, 1→3 and 1→6-linked β-galactose, terminal-linked α-rhamnose, terminal-linked α-arabinose and 1,5-linked α-arabinose.

[0018] In a specific embodiment, in step S1, the concentration of Lycium barbarum polysaccharide LBP1C-2 in formamide is 15-25 mg / mL; in step S2, the volume ratio of chlorosulfonic acid to pyridine is 3:1, the oil bath temperature is 60°C, and the reaction time is 30 minutes; in step S3, the oil bath temperature is 60°C, and the reaction time is 3 hours.

[0019] In a specific embodiment, the method further includes step S4: after the reaction in S3, the pH of the solution is adjusted to 7 with 3-6M, for example, 5M NaOH in an ice-water bath under pH meter monitoring, and then placed in a dialysis bag, dialyzed in a saturated sodium bicarbonate solution for 24 hours, and then dialyzed with deionized water for 72 hours. The obtained dialyzed liquid is concentrated and freeze-dried to obtain the wolfberry sulfated polysaccharide S-LBP1C-2.

[0020] On the other hand, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the wolfberry sulfated polysaccharide S-LBP1C-2 as an active ingredient, and optionally, pharmaceutically acceptable excipients.

[0021] In a specific embodiment, the pharmaceutically acceptable excipients include carriers, excipients, adjuvants and / or diluents.

[0022] In another aspect, the present invention provides use of the aforementioned wolfberry sulfated polysaccharide S-LBP1C-2 or the aforementioned pharmaceutical composition in the preparation of an agent or drug for inhibiting, preventing and / or treating senile bone loss.

[0023] In a specific embodiment, the wolfberry sulfated polysaccharide S-LBP1C-2 or the pharmaceutical composition inhibits aging-related bone loss by promoting bone formation and inhibiting bone resorption.

[0024] Beneficial effects:

[0025] The wolfberry sulfated polysaccharide S-LBP1C-2 prepared in this application can significantly increase the maximum load force and elastic modulus of the femur of naturally aged mice in young mice and naturally aged osteoporotic mice, significantly increase the mineral deposition rate of the femur of naturally aged mice and the alkaline phosphatase content in the serum, and the N-terminal propeptide content of type I procollagen in the serum of mice. Further experiments show that compared with the raw sugar LBP1C-2, the sulfated sample S-LBP1C-2 has the dual effects of promoting bone formation and inhibiting bone resorption, while LBP1C-2 only has the effect of promoting bone formation. The above data show that S-LBP1C-2 has great application prospects in the prevention and / or treatment of aging-related bone loss as a candidate drug.

[0026] The present invention has been described in detail above, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. In addition, the present invention is not limited by any theory described in the above prior art or invention summary or the following examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the characteristic high performance gel permeation chromatogram of the wolfberry sulfated polysaccharide S-LBP1C-2 in Preparation Example 1.

[0028] Figure 2 The characteristics of the sulfated wolfberry polysaccharide S-LBP1C-2 (A) and the original polysaccharide LBP1C-2 (B) in Example 1 are prepared 13 C NMR spectrum.

[0029] Figure 3 Characteristic infrared spectra of the wolfberry sulfated polysaccharide S-LBP1C-2 (A) and the original polysaccharide LBP1C-2 (B) in preparation example 1 (AT: terminal-α-arabinose; A15: 1,5-α-arabinose; the sulfation site is not selective; GT: terminal-β-galactose; G36: 1,3,6-β-galactose).

[0030] Figure 4 Figure 1 shows the effect of the sulfated wolfberry polysaccharide S-LBP1C-2 in inhibiting bone loss in aging mice. A: Micro-CT image of femur; B: Bone density, bone volume fraction, and trabecular number (Tb.N), and reduced trabecular separation (Tb.Sp) in naturally aged mice; C: Maximum load force and elastic modulus of femur in adult and naturally aged mice; D: Calcein and Alizarin Red fluorescence morphology of mouse femur; E: Mineral apposition rate (MAR), osteocalcin content, and type I procollagen N-terminal propeptide (PI NP) content in mouse femur.

[0031] Figure 5The effects of the sulfated wolfberry polysaccharide S-LBP1C-2 in promoting bone formation and inhibiting bone resorption in vivo in Pharmacological Example 2 are shown. A: Fluorescence image of a mouse femoral hard tissue section using calcein-alizarin red fluorescence double labeling, scale bar 20 μm; B: Bone formation rate (BFR / BS, μm 3 / μm 2 / per day), n=6; C: bone mineralization rate (MAR, μm / day), n=6; D: HE staining of femoral tissue, scale bar 200 μm; E: statistics on the number of osteoblasts on the surface of trabeculae, n=6; F: TRAP staining of tibia tissue, scale bar 100 μm; G: statistics on the number of osteoclasts on the surface of trabeculae, n=6; H: osteocalcin (Osteocalcin, ng / mL), n=6; I: amino-terminal propeptide of type I procollagen (PINP, ng / mL), n=6; J: tartrate-resistant acid phosphatase (Trap-5b, ng / mL) derived from osteoclasts, n=6.

[0032] ** indicates P < 0.01, # Indicates P<0.05. DETAILED DESCRIPTION

[0033] Material:

[0034] The Chinese herbal medicine wolfberry was purchased from Ningxia Zhongning, and its origin is Ningxia.

[0035] For the preparation of LBP1C-2, refer to international application WO2024 / 013562.

[0036] equipment:

[0037] High-performance gel permeation chromatography (HPLC) was performed using Shodex SUGAR KS-804 (8.0 mm × 300 mm, Agilent, USA) and Shodex SUGAR KS-802 (8.0 mm × 300 mm, Agilent, USA) columns in series. A standard curve was prepared using T-series standard dextran with different molecular weights.

[0038] High performance liquid chromatography (HPLC) was performed using an Agilent 1260Seri high performance liquid chromatography system (Agilent, USA);

[0039] Infrared analysis was performed using a Perkin-Elmer 599B infrared spectrophotometer (Perkin-Elmer, USA);

[0040] Nuclear magnetic resonance analysis was performed using a Brucker AM-500 nuclear magnetic resonance spectrometer (Brucker, Germany).

[0041] Preparation Example 1: Preparation and Structural Characterization of Lycium barbarum Sulfated Polysaccharide S-LBP1C-2

[0042] (1) Sulfation of Lycium barbarum polysaccharides:

[0043] Take 100 mg of dried polysaccharide LBP1C-2 and add dry formamide to a 20 mg / mL solution using a syringe. Seal with a rubber stopper, add a stir bar, and stir to dissolve at room temperature. In another vial, add pyridine and slowly add chlorosulfonic acid to pyridine (v / v = 3:1) dropwise at room temperature. This will release a large amount of white smoke, resulting in a viscous sulfation reagent. The sulfation reagent is quickly placed in a 60°C oil bath and allowed to react for 30 minutes. Next, the formamide solution of the polysaccharide is added dropwise to the sulfation reagent in a 60°C oil bath (melt it first if it solidifies) and allowed to react for 3 hours. After the reaction, adjust the pH of the solution to 7 with 5M NaOH in an ice-water bath while monitoring with a pH meter. The solution is then placed in a dialysis bag (cutoff 3500 Da) and dialyzed against saturated sodium bicarbonate solution for 24 hours, changing the solution 2-3 times to ensure saturation. The solution is then dialyzed against deionized water for 72 hours. The dialyzed liquid was concentrated and freeze-dried to obtain the sulfated derivative S-LBP1C-2.

[0044] (2) The relative molecular mass of the sulfated polysaccharide S-LBP1C-2 was determined to be 130.7 kDa by high performance gel permeation chromatography (HPGPC). Figure 1 ).

[0045] The monosaccharide composition of a completely hydrolyzed sample of the wolfberry sulfated polysaccharide S-LBP1C-2 was determined by PMP derivatization. The main components of the sulfated polysaccharide S-LBP1C-2 were galactose, arabinose, rhamnose, and galacturonic acid, with a molar ratio of 8.0:8.5:33.6:49.9. A calibration curve was constructed using a standard sulfate solution using the barium chloride-gelatin method. The degree of substitution (DS) of the sulfate groups in S-LBP1C-2 was 0.97, calculated using the formula (1.62 × %S) / (32 - 1.02 × %S).

[0046] (3) Analysis of structure and sulfation substitution sites: The structural unit of the wolfberry sulfated polysaccharide S-LBP1C-2 is composed of 1→2 linked α-rhamnose and 1→4 linked α-galacturonic acid alternately linked to form a main chain, and the side chains are connected to C-4 of 1→2 linked α-rhamnose, C-6 of 1→3 galactose, C-3 of 1→6 galactose and C-3 of 1→5 arabinose in the main chain, and the side chains include 1→, 1→3 and 1→6 linked β-galactose; terminal linked α-rhamnose, terminal linked α-arabinose and 1,5 linked α-arabinose; nuclear magnetic resonance showed that its sulfation sites are located at the 3,5 positions of terminal-α-arabinose and the 2,3 positions of 1,5-α-arabinose, respectively. There is also sulfation at the 6 position of terminal-β-galactose and sulfation at the 4 position of 1,3,6-β-galactose (such as Figure 2 ).

[0047] (4) IR spectrum of S-LBP1C-2 (such as Figure 3 ):3389.81cm in the infrared spectrum of Lycium barbarum polysaccharide LBP1C-2 -1 is the OH stretching vibration absorption peak, 29355.10cm -1 is the CH stretching vibration absorption peak, 1618.00 cm -1 The IR spectrum of the sulfated wolfberry polysaccharide S-LBP1C-2 shows that the 3472.13 cm -1 is the OH stretching vibration absorption peak, 1636.44 cm -1 is the stretching vibration absorption peak of C=O, 1232.90 cm -1 and 599.43cm -1 The absorption peak is the stretching vibration peak of O=S=O, proving the existence of sulfate group, 848.51cm -1 This is the vibration absorption produced by the corresponding C in the sugar after being replaced by sulfate, indicating that the polysaccharide contains sulfate and uronic acid.

[0048] Pharmacological Example 1: Lycium barbarum sulfated polysaccharide S-LBP1C-2 promotes bone formation in naturally aging mice

[0049] To determine the effect of the sulfated polysaccharide S-LBP1C-2 on bone formation in mice, a natural aging-induced bone loss mouse model (14-month-old male mice) was established. Adult C57BL / 6J mice (2-month-old male mice) served as controls. The aging mice were randomly divided into three groups: an aging model group, an LBP1C-2-treated group, and an S-LBP1C-2-treated group. The treated groups were gavaged daily with a defined dose of LBP1C-2 (40 mg / kg) and S-LBP1C-2 (40 mg / kg) for four months. The control and model groups were given an equal volume of distilled water.

[0050] Bone histomorphometric parameters, including bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp), were measured in each group of mice using an Inveon MM CT (SIEMENS, Munich, Germany) micro-CT bone phenotyping instrument. Tube voltage and rotation center calibration were performed before use. The femurs or vertebrae, fixed with fixative, were then removed, blotted dry with absorbent paper, and placed in the micro-CT specimen chamber for scanning. Scanning parameters were: voltage 60 kV, current 220 μA, exposure time 1500 ms, effective pixel size 8.89 μm, and 360 exposures per 1° exposure. The total image contained 1536 slices with an effective pixel size of 17.78 μm. Two-dimensional images were reconstructed into three-dimensional images using the commercial Cobra EXXIM software package. The scanned and reconstructed data were then imported into the Inveon Research Workplace analysis software for analysis. The femoral region of interest (ROI) analysis starts 0.5 mm below the lowest point of the second growth plate, with a step size of 0.5 mm. The analysis results include both graphical and statistical data. The graphical representations include three-dimensional planar images of the femur, a stereoscopic image of trabecular bone within the ROI, and a coronal stereoscopic image of the femur (0.3 mm thick). In addition to line segment and area measurements, the data also allows for the separation of trabecular bone from bone marrow by setting a threshold, thereby calculating three-dimensional bone morphological parameters.

[0051] The results showed that compared with the aged control group mice, the administration of LBP1C-2 and S-LBP1C-2 significantly increased the bone density, bone volume fraction and trabecular number (Tb.N) of naturally aged mice, and reduced the trabecular separation (Tb.Sp) ( Figure 4 In conclusion, both the Lycium barbarum polysaccharide LBP1C-2 and the Lycium barbarum sulfated polysaccharide S-LBP1C-2 can significantly improve the bone tissue morphological parameters of naturally aged mice, and the effect of S-LBP1C-2 is better than that of LBP1C-2.

[0052] The bone strength of each group of mice was tested by a three-point bending test using an Instron electronic universal material testing machine (Instron, Canton, Mass., USA) bone biomechanical testing equipment. Immediately after sampling, the three-point bending test was used to test the bone strength of fresh femurs of each group of mice. The three-point bending test uses two end support points and one central loading point. Biomechanical measurement data were collected from the load-deformation curve. The three-point bending test uses two end support points and one central loading point. Biomechanical measurement data were collected from the load-deformation curve. Compared with the aged control group mice, both LBP1C-2 and S-LBP1C-2 treatment significantly increased the maximum load force and elastic modulus of the femur of adult and naturally aged mice ( Figure 4 C), in which S-LBP1C-2 was more effective than LBP1C-2.

[0053] Whether the improvement in bone mass and bone stability was due to the promotion of bone formation by LBP1C-2 and S-LBP1C-2 was evaluated by calcein-alizarin red fluorescence double-labeling of mouse femoral hard tissue sections. Mice were intraperitoneally injected with calcein and alizarin red S (10 mg / kg) 10 days and 2 days before euthanasia. The femurs of mice were obtained for undecalcified histological analysis, and unstained 5 μm sections were examined using a fluorescence microscope. Statistical analysis was performed using the Osteomeasure analysis system. The experimental results showed that LBP1C-2 and S-LBP1C-2 treatment significantly increased the bone mineralization deposition rate and bone formation rate ( Figure 4 D), in which S-LBP1C-2 was more effective than LBP1C-2.

[0054] The effects of LBP1C-2 and S-LBP1C-2 on the levels of bone formation markers bone alkaline phosphatase (BALP) and type I procollagen amino-terminal peptide (PINP) in serum were further examined using BALP ELISA kit and PINP ELISA kit. Compared with the aging model group, the levels of BALP and PINP in the serum of mice treated with LBP1C-2 and S-LBP1C-2 were significantly increased ( Figure 4 These results indicate that LBP1C-2 and S-LBP1C-2 can improve aging-related bone loss by promoting bone formation, with S-LBP1C-2 showing a superior effect compared to LBP1C-2.

[0055] Pharmacological Example 2: Effects of Lycium barbarum sulfated polysaccharide S-LBP1C-2 on promoting bone formation and inhibiting bone resorption in vivo

[0056] Based on the significant improvement in the bone phenotype of naturally aged mice by S-LBP1C-2, the regulatory role of S-LBP1C-2 on bone formation and resorption in vivo in naturally aged mice was further explored. Calcein-alizarin red fluorescence double-labeling assays assessed bone formation in vivo. Results showed that both LBP1C-2 and S-LBP1C-2 significantly increased the bone formation rate and bone mineralization rate in aged mice compared with the aging model group (Figures A, B, and C). HE staining of femoral tissues revealed a significant increase in the number of osteoblasts on the trabecular surface of mice treated with LBP1C-2 and S-LBP1C-2 (Figures D and E). To further evaluate the regulatory role of S-LBP1C-2 on bone resorption, TRAP staining revealed that S-LBP1C-2 significantly decreased the number of osteoclasts on the trabecular surface of aged mice, whereas no significant changes were observed in the LBP1C-2-treated group (Figures F and G). Analysis of serum markers showed significant increases in serum bone formation markers (osteocalcin and PINP) in the LBP1C-2 and S-LBP1C-2 treatment groups (Figures H, I). Furthermore, serum bone resorption marker (Trap-5b) levels were significantly decreased in the S-LBP1C-2 treatment group, whereas no significant changes were observed in the LBP1C-2 treatment group (Figure J). These results suggest that S-LBP1C-2 has dual effects of promoting bone formation and inhibiting bone resorption, while LBP1C-2 only promotes bone formation. This may explain why S-LBP1C-2 is superior to LBP1C-2 in improving the bone phenotype in aging mice.

[0057] In summary, the results of the above pharmacological examples show that the wolfberry sulfated polysaccharide S-LBP1C-2 of the present invention can significantly improve bone loss in aging mice and the effect is better than LBP1C-2. Therefore, the sulfated polysaccharide component S-LBP1C-2 can become a potential carbohydrate drug for preventing and / or treating aging-related bone loss.

[0058] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced with equivalents, without departing from the spirit and substance of the claims of the present invention; and such modifications or replacements remain within the scope of the claims of the present invention.

Claims

1. A wolfberry sulfated polysaccharide S-LBP1C-2, which has an RG-I type pectin structure, wherein the raw sugar has the following structure: a main chain formed by alternating 1→2 linked α-rhamnose and 1→4 linked α-galacturonic acid, with side chains connected to C-4 of the 1→2 linked α-rhamnose, C-6 of the 1→3 galactose, C-3 of the 1→6 galactose, and C-3 of the 1→5 arabinose of the main chain, and the side chains include terminal, 1→3, and 1→6 linked β-galactose; the terminal linked α-rhamnose, terminal-linked α-arabinose and 1,5-linked α-arabinose, the sulfation site of the wolfberry sulfated polysaccharide S-LBP1C-2 is selected from one or more of the following: 3,5 positions of terminal-α-arabinose, 2,3 positions of 1,5-α-arabinose, 6 positions of terminal-β-galactose and 4 position of 1,3,6-β-galactose, and the sulfate substitution degree of the wolfberry sulfated polysaccharide S-LBP1C-2 is 0.80-1.

10.

2. The wolfberry sulfated polysaccharide S-LBP1C-2 according to claim 1, wherein the sulfation sites include positions 3 and 5 of the terminal α-arabinose, positions 2 and 3 of 1,5-α-arabinose, position 6 of the terminal β-galactose and position 4 of 1,3,6-β-galactose; and / or the degree of sulfate substitution of the wolfberry sulfated polysaccharide S-LBP1C-2 is 0.

97. 3 . The wolfberry sulfated polysaccharide S-LBP1C-2 according to claim 1 , having a relative molecular mass of 130.7 kDa. The wolfberry sulfated polysaccharide S-LBP1C-2 according to claim 1 , comprising galactose, arabinose, rhamnose and galacturonic acid in a molar ratio of 33.6:49.9:8.0:8.

5.

5. A method for preparing the wolfberry sulfated polysaccharide S-LBP1C-2 according to any one of claims 1 to 4, comprising the following steps: S1: Take Lycium barbarum polysaccharide LBP1C-2, add dry formamide, seal with a rubber stopper, stir and dissolve, The Lycium barbarum polysaccharide LBP1C-2 has the following structure: a main chain is formed by alternating 1→2 linked α-rhamnose and 1→4 linked α-galacturonic acid, with side chains connected to C-4 of 1→2 linked α-rhamnose, C-6 of 1→3 galactose, C-3 of 1→6 galactose, and C-3 of 1→5 arabinose of the main chain, and the side chains include terminal, 1→3 and 1→6 linked β-galactose, terminal linked α-rhamnose, terminal linked α-arabinose, and 1,5 linked α-arabinose; S2: In another vial, add chlorosulfonic acid to pyridine to prepare a viscous sulfation reagent, and place the sulfation reagent in an oil bath at 50-80°C for reaction; and S3: The polysaccharide sample in S1 is added to the sulfation reagent in S2 in a 50-80°C oil bath for reaction.

6. The method according to claim 5, wherein: In step S1, the concentration of Lycium barbarum polysaccharide LBP1C-2 in formamide is 15-25 mg / mL; in step S2, the volume ratio of chlorosulfonic acid to pyridine is 4:1-2:1, the oil bath temperature is 50-80°C, and the reaction time is 20-60 min; in step S3, the oil bath temperature is 50-80°C, and the reaction time is 2-4 h.

7. The method according to claim 5, wherein: In step S1, the concentration of Lycium barbarum polysaccharide LBP1C-2 in formamide is 15-25 mg / mL; in step S2, the volume ratio of chlorosulfonic acid to pyridine is 3:1, the oil bath temperature is 60°C, and the reaction time is 30 minutes; in step S3, the oil bath temperature is 60°C, and the reaction time is 3 hours.

8. The method according to claim 5, wherein The method further includes step S4: after the reaction in S3, adjusting the pH of the solution to 7 using 3-6M, for example, 5M, NaOH in an ice-water bath under pH meter monitoring, placing the solution in a dialysis bag, dialyzing it in a saturated sodium bicarbonate solution for 24 hours, and then dialyzing it with deionized water for 72 hours. The dialyzed liquid obtained is concentrated and freeze-dried to obtain the wolfberry sulfated polysaccharide S-LBP1C-2.

9. A pharmaceutical composition comprising a therapeutically effective amount of the sulfated wolfberry polysaccharide S-LBP1C-2 according to any one of claims 1 to 4 as an active ingredient, and optionally, pharmaceutically acceptable excipients.

10. Use of the wolfberry sulfated polysaccharide S-LBP1C-2 according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 9 in the preparation of an agent or medicament for inhibiting, preventing and / or treating senile bone loss. Preferably, the wolfberry sulfated polysaccharide S-LBP1C-2 or the pharmaceutical composition inhibits senile bone loss by promoting bone formation and inhibiting bone resorption.

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