Application of lycium barbarum polysaccharide in preparation of medicine for preventing and / or treating bone loss

Abstract

The invention relates to the field of plant polysaccharide, and discloses application of lycium barbarum polysaccharide in preparation of a medicine for preventing and / or treating bone loss, monosaccharide in the lycium barbarum polysaccharide comprises detectable arabinose, glucose, galactose and gluconic acid; wherein the content of polysaccharide of which the weight-average molecular weight is greater than or equal to 6.5 kDa in the lycium barbarum polysaccharide is not less than 30wt%. Natural substances in Chinese wolfberry are adopted as active ingredients and can be safely used in an experimental dose range, bone loss is prevented or treated, particularly, Chinese wolfberry polysaccharide extracted from Chinese wolfberry is used, bone formation inhibition is relieved by competitively combining Noggin protein and blocking Noggin-BMPs interaction, and the effect of preventing or treating bone loss is achieved. The phosphorylation of Smad protein is promoted to enhance osteogenic differentiation and mineralization, and the effect of preventing or treating waste bone loss is achieved.

Description

Technical Field

[0001] The present invention relates to the field of plant polysaccharides, and in particular to use of wolfberry polysaccharide in preparing a medicine for preventing and / or treating bone loss. Background Art

[0002] Disuse osteoporosis (DOP) is a secondary osteoporosis caused by mechanical stress deprivation or prolonged immobilization. Its epidemiological characteristics are characterized by high morbidity and significant clinical harm. Epidemiological surveys show that the incidence of bone loss in patients with spinal cord injury is as high as 42.4% within three months after injury. Astronauts experience a 1.4-1.5% monthly decrease in proximal femoral bone density in microgravity, and patients undergoing fracture immobilization experience up to 50% bone mineral loss within six months. Current clinical treatments primarily rely on bisphosphonates and physical interventions, but these have limited efficacy and side effects. While bisphosphonates can inhibit bone resorption, long-term use may lead to osteonecrosis of the jaw. Physical therapies such as functional electrical stimulation can improve the bone microenvironment but are challenging to implement.

[0003] Current research focuses on multi-target combination therapy, precision mechanical intervention, and personalized gene regulation. Bone morphogenetic protein (BMP) signaling plays a fundamental role in skeletal development and bone homeostasis. While Lycium barbarum polysaccharides (LBP) have been reported to mitigate bone loss, their efficacy still needs to be further improved. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art and to provide a use of Lycium barbarum polysaccharide in the preparation of a medicament for preventing and / or treating bone loss.

[0005] In order to achieve the above object, the present invention provides the use of Lycium barbarum polysaccharide in the preparation of a medicament for preventing and / or treating bone loss, wherein the monosaccharides in the Lycium barbarum polysaccharide include detectable arabinose, glucose, galactose and gluconic acid; The content of polysaccharides with a weight average molecular weight of ≥6.5 kDa in the wolfberry polysaccharide is not less than 30 wt%.

[0006] The present invention uses natural substances present in wolfberry as active ingredients, which can be used safely within the experimental dosage range, and achieves the prevention or treatment of bone loss. In particular, wolfberry polysaccharides extracted from wolfberry are used to competitively bind to Noggin protein, block Noggin-BMPs interaction to relieve bone formation inhibition, promote Smad protein phosphorylation to enhance osteogenic differentiation and mineralization, and have the effect of preventing or treating disuse-induced bone loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1Micro-computed tomography images of whole-body femur and trabecular bone of mice after different treatments; Figure 2 Figure 2 is a picture of preosteoblast colonies in mice after different treatments; Figure 3 ALP staining of mouse preosteoblasts after simulated weightlessness treatment with different treatments; Figure 4 The response signal diagram of the interaction between LBPP and Noggin at different concentrations. DETAILED DESCRIPTION

[0008] The endpoints of the ranges and any values disclosed herein 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 endpoints of each range, the endpoints of each range and 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 to be specifically disclosed herein.

[0009] The present invention provides the use of Lycium barbarum polysaccharide in preparing a medicament for preventing and / or treating bone loss, wherein the monosaccharides in the Lycium barbarum polysaccharide include detectable arabinose, glucose, galactose and gluconic acid; The content of polysaccharides with a weight average molecular weight of ≥6.5 kDa in the wolfberry polysaccharide is not less than 30 wt%.

[0010] During their research, the inventors of the present invention discovered that the natural substances present in wolfberry can be safely used as active ingredients within the experimental dosage range, thereby achieving the prevention or treatment of bone loss. In particular, the wolfberry polysaccharide extracted from wolfberry can competitively bind to Noggin protein, block the Noggin-BMPs interaction to relieve bone formation inhibition, promote Smad protein phosphorylation to enhance osteogenic differentiation and mineralization, and has the efficacy of preventing or treating disuse-induced bone loss.

[0011] It is understood that the “detectable” refers to monosaccharides that can meet the detection limit of HPLC (ie, the amount that can be detected by HPLC).

[0012] In the present invention, preferably, the content of polysaccharide with a weight average molecular weight ≥6.5 kDa in the wolfberry polysaccharide can be 35-90 wt% (for example, it can be a range formed by any two values of 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, and the value within the range).

[0013] In the present invention, preferably, the monosaccharide composition of the Lycium barbarum polysaccharide further includes detectable mannose, rhamnose, xylose and galacturonic acid.

[0014] In the present invention, preferably, the monosaccharide composition test results of the Lycium barbarum polysaccharide show that the molar ratio of mannose, rhamnose, arabinose, glucose, galactose, xylose, galacturonic acid and gluconic acid can be 0-5:0-8:5-60:1-15:3-20:0-5:0-30:1, more preferably 0.5-3:3-5:20-30:5-8:8-12:1-3:10-15:1, and further preferably 2.3:5.9:40.7:10.4:16.7:2.9:19.3:1.7. Lycium barbarum polysaccharides with a monosaccharide composition falling within the above range will exhibit a better effect in preventing and / or treating bone loss (especially disuse bone loss). The term "monosaccharide composition" as used in the present invention refers to the composition of the basic units (monosaccharides) forming the polysaccharide through glycosidic bonds (dehydration).

[0015] In a preferred embodiment of the present invention, Lycium barbarum polysaccharides can be combined with other polysaccharides extracted from Lycium barbarum for use in a drug for preventing and / or treating bone loss, with the other polysaccharide components accounting for no less than 15 wt % of the total polysaccharide content. In particular, the other polysaccharides extracted from Lycium barbarum include but are not limited to Lycium barbarum polysaccharide LBP1C-2 and / or Lycium barbarum polysaccharide LBP1C.

[0016] In the present invention, preferably, the content of polysaccharides in the wolfberry polysaccharide is ≥20wt%; more preferably, the content of polysaccharides in the wolfberry polysaccharide can be 85-95wt% (for example, it can be a range formed by any two values of 85wt%, 86wt%, 87wt%, 88wt%, 89wt%, 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, and the value within the range).

[0017] In the present invention, preferably, the weight average molecular weight of the polysaccharide in the Lycium barbarum polysaccharide can be 5-800kDa (for example, it can be a range formed by any two values of 5kDa, 5.5kDa, 6kDa, 6.5kDa, 7kDa, 8kDa, 9kDa, 10kDa, 50kDa, 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, 450kDa, 500kDa, 550kDa, 600kDa, 650kDa, 700kDa, 750kDa, 800kDa, and values within the range), the molecular weight can be a specific value or within a narrow range of about 6.5-550kDa. The values of the weight average molecular weight (Mw) and the number average molecular weight (Mn) may be close to each other. The polydispersity (PD) index (i.e., the ratio of Mw to Mn) of the active polysaccharide may range from about 1 to about 1.3, from about 1 to about 1.2, or from about 1 to about 1.1. In certain embodiments, the PD index is close to 1. Because the molecular weight of polysaccharides in the raw wolfberry can vary due to factors such as the growing environment and harvest season, the molecular weight of polysaccharides obtained from different batches may also vary.

[0018] In the present invention, the Lycium barbarum polysaccharide is a crude polysaccharide extract, and preferably, the Lycium barbarum polysaccharide may further include flavonoids, carotenoids, polyphenols, pigments and inevitable impurities, wherein each of the above components may be present in only one form or in two or more forms.

[0019] In the present invention, preferably, the content of flavonoids in the wolfberry polysaccharide can be 0.05-5wt% (for example, it can be a range formed by at least two values of 0.05wt%, 0.1wt%, 0.15wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, and the value within the range), and the content of carotenoids can be 0.05-3wt% (for example, it can be 0.05wt%, 0.1wt%, 0.15wt%, 0.5wt%, 0.8wt%, 1wt%, 1.2wt%, 1.5wt%, 1.8wt%). t%, 2wt%, 2.3wt%, 2.5wt%, 2.7wt%, and 3wt%, and values within the range), the content of polyphenols may be 0.05-8wt% (for example, it may be in the range formed by at least two of the values selected from 0.05wt%, 0.1wt%, 0.15wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, 5.5wt%, 6wt%, 6.5wt%, 7wt%, 7.5wt%, and 8wt%, and values within the range), and the balance may be pigments and unavoidable impurities.

[0020] In the present invention, preferably, the content of flavonoids in the wolfberry polysaccharide may be 0.1-0.15 wt %, the content of carotenoids may be 0.1-0.5 wt %, the content of polyphenols may be 0.1-0.15 wt %, and the balance is pigments and inevitable impurities.

[0021] In the present invention, the Lycium barbarum polysaccharide is derived from the water-extraction and alcohol-precipitation product of Lycium barbarum. Preferably, the preparation method of the Lycium barbarum polysaccharide comprises: S1, mixing wolfberry with water for decoction extraction, and subjecting the obtained decoction to membrane separation; S2, subjecting the membrane-separated permeate (LBE) to an alcohol precipitation step, and obtaining the crude wolfberry polysaccharide (LBP); S3. Dissolve the crude Lycium barbarum polysaccharide in water and centrifuge at 2000-4000 rpm. Take the supernatant for secondary alcohol precipitation to obtain Lycium barbarum polysaccharide (LBPP).

[0022] The inventors of the present invention have discovered that both LBP and LBPP have excellent efficacy in preventing and / or treating bone loss, but LBPP is more effective in preventing and / or treating disuse-related bone loss by improving bone microstructure and stability. Therefore, the present invention also relates to the use of the product (lycium barbarum polysaccharides, such as LBP and / or LBPP) prepared by the above method in the preparation of a medicament for preventing and / or treating bone loss.

[0023] In the present invention, preferably, in step S1, the decoction extraction conditions may include: the temperature may be 70-100°C (for example, it can be a range formed by any two values of 70°C, 72°C, 75°C, 78°C, 80°C, 83°C, 85°C, 87°C, 90°C, 95°C, and 100°C, and a value within the range), and more preferably it can be 75-85°C; the time may be 1-3h (for example, it can be a range formed by any two values of 1h, 1.5h, 2h, 2.5h, and 3h, and a value within the range), and more preferably it can be 1.5-2.5h.

[0024] In the present invention, preferably, in step S1, the mass ratio of wolfberry to water can be 1:4-10 (for example, it can be a range formed by any two ratios of 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, and a ratio within the range), and more preferably can be 1:5-8.

[0025] In the present invention, preferably, the separation membrane for membrane separation can be at least one selected from a fiber membrane, a polypropylene membrane, a polysulfone membrane and a ceramic membrane.

[0026] In the present invention, preferably, the molecular weight cutoff of the separation membrane for membrane separation can be 3.5-7 kDa (for example, it can be a range formed by any two values of 3.5 kDa, 4 kDa, 4.5 kDa, 5 kDa, 5.5 kDa, 6 kDa, 6.5 kDa, 7 kDa, and a value within the range).

[0027] In the present invention, preferably, the membrane separation conditions may include: temperature may be 12-30°C, more preferably 15-20°C; pressure may be 0.1-10 MPa, more preferably 0.1-0.5 MPa; time may be 48-72h, more preferably 60-72h.

[0028] In the present invention, preferably, in step S2, the method may further include: in step S2, dialyzing the permeate separated by the membrane, centrifuging at 1000-4000 rpm for 10-20 min after dialysis for 48-72 hours, and subjecting the supernatant after centrifugation to alcohol precipitation.

[0029] In the present invention, preferably, the dialysis method may include: dialysis using a dialysis bag or a dialysis tube; wherein the material of the semipermeable membrane used for dialysis is at least one of regenerated cellulose, polypropylene, polysulfone and ceramic; the molecular weight cutoff of the semipermeable membrane may be 3.5-7kDa; the difference in molecular weight cutoff between the semipermeable membrane and the separation membrane of the membrane separation may be 0.2-1kDa; the thickness of the semipermeable membrane may be 22-30μm; the dialysis temperature may be 12-30°C, more preferably 15-20°C.

[0030] In the present invention, the dialysis bag or dialysis tube may be a dialysis method commonly used in the art, for example, the dialysis bag may be a SnakeSkin™ (Thermo Scientific) dialysis bag.

[0031] In a preferred embodiment of the present invention, the dialysis bag is unfolded into a pleated (telescopic) tube form with an inner diameter of 16-35 mm.

[0032] In the present invention, preferably, in step S3, the amount of water used can be 5-15 mL (for example, it can be a range formed by any two values of 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, and 15 mL, and a value within the range) per gram of wolfberry crude polysaccharide.

[0033] In the present invention, preferably, the method of taking the precipitate after the first alcohol precipitation is to centrifuge for 10-15 min (for example, it can be a range formed by at least two values of 2000 rpm, 2200 rpm, 2400 rpm, 2600 rpm, 2800 rpm, 3000 rpm, 3200 rpm, 3400 rpm, 3600 rpm, 3800 rpm, 4000 rpm, and values within the range) under centrifugal conditions.

[0034] In a preferred embodiment of the present invention, the method further comprises: in step S2, dissolving the precipitate (lycium barbarum crude polysaccharide) obtained by centrifugation after the first alcohol precipitation in water, and then evaporating to remove water; the evaporation conditions can be the conditions commonly used in the art for removing water, for example, dehydration under vacuum conditions at 45-70°C.

[0035] In the present invention, preferably, the method of taking the precipitate after the secondary alcohol precipitation is to centrifuge for 20-30 min (for example, it can be a range formed by at least two values of 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm, and a value within the range) under centrifugal conditions.

[0036] In a preferred embodiment of the present invention, the bone loss is disuse bone loss. Disuse bone loss is the rapid loss of bone mass due to a long-term lack of mechanical loading on the skeleton (such as bed rest, immobilization, weightlessness, etc.), which in turn triggers the pathological process of osteoporosis. Its core mechanism is an imbalance in the skeleton's adaptive response to mechanical stimulation, resulting in reduced bone formation and increased bone resorption. Disuse bone loss is the structural destruction of bone microstructure caused by a lack of mechanical stimulation due to long-term immobilization (such as paralysis, bed rest, fracture immobilization) or weightless environments (astronauts). Ordinary bone loss, on the other hand, is more likely to be a physiological or pathological metabolic imbalance. The two have different pathogenic mechanisms and are treated with different medications.

[0037] In terms of the terminology system, "subject" and "patient" are used synonymously in this invention, encompassing members of the animal kingdom, with a preference for mammals, specifically non-primates (such as domestic animals like cattle, pigs, and horses, as well as experimental animals like cats, dogs, and mice) and primates (including non-human primates and humans). The most preferred subject is Homo sapiens. Specific implementation cases include farm animals (such as horses, cattle, and pigs) and companion animals (such as dogs and cats), particularly suitable for research in adult individuals. This is highly consistent with the present invention's research focus on bone loss (particularly disuse-induced bone loss).

[0038] Regarding the definition of drug-related terms, "drug" in the present invention specifically refers to a material system with the function of disease prevention, treatment, management or diagnosis, including multiple dimensions such as single molecule compounds, compound preparations, diagnostic reagents and treatment methods. "Effective dose" refers to the amount of administration that can produce at least one of the following effects: preventing the onset or recurrence of targeted diseases, enhancing the preventive effect of synergistic therapy, reducing the severity of the disease, shortening the duration of the disease, alleviating clinical symptoms, blocking disease progression, promoting pathological reversal, and enhancing the therapeutic effects of other therapies. It should be pointed out in particular that the present invention is mainly aimed at the intervention of bone loss and related symptoms of abnormal bone metabolism. The "pharmaceutically acceptable" standard refers to meeting any of the following conditions: substances certified by regulatory agencies such as FDA or EMA, included in international pharmacopoeias such as USP and EP, or suitable for medical use in mammals.

[0039] In terms of treatment system terminology, "therapeutic agent" specifically refers to active substances with disease intervention functions; "therapy" includes but is not limited to various forms such as small molecule drug treatment plans, biological agent intervention methods, and combination treatment plans; and the technical effects of "treatment" mainly include inhibiting disease progression, shortening the pathological process, alleviating clinical symptoms, and improving physiological indicators.

[0040] In the present invention, preferably, the drug may further contain a pharmaceutically acceptable excipient, wherein the excipient refers to an inactive ingredient that acts as a carrier or medium in the preparation, specifically including but not limited to the following categories: solvent system (such as solvent / co-solvent), stabilizing system (such as preservative / antimicrobial agent), solid preparation excipient (including fillers such as microcrystalline cellulose, binders such as hypromellose, disintegrants such as croscarmellose sodium, lubricants such as magnesium stearate, etc.) and liquid preparation excipient (such as surfactant polysorbate 80, emulsifier lecithin, suspending agent xanthan gum, etc.).

[0041] In a preferred embodiment of the present invention, the method of using Lycium barbarum polysaccharide comprises: administering Lycium barbarum polysaccharide to a subject or animal cells.

[0042] In the present invention, preferably, the wolfberry polysaccharide is the above-mentioned wolfberry polysaccharide, and its characteristics are consistent with the above-mentioned wolfberry polysaccharide, which will not be repeated here.

[0043] In some embodiments, the Lycium barbarum polysaccharides of the present invention can be taken with beverages, foods, or related ingredients, or used as health foods. There are no particular restrictions on the preparation methods of the food or health foods. For example, it can be made into tablets, beverages, candies, etc. In addition to the polysaccharide component, each food preparation may also include other ingredients used in the art. Other ingredients can be selected by those skilled in the art based on the specific formulation or application.

[0044] The present invention will be described in detail below through examples.

[0045] Example 1. Material preparation and testing 1-1. Preparation of wolfberry extract: The preparation process of wolfberry extract (LBE) was as follows: wolfberry fruits were collected from Zhongning County, Yinchuan, Ningxia, China.

[0046] Dried wolfberry fruits were washed five times, soaked in double-distilled water (pH 7) at room temperature for 2 hours, and then crushed. The soaked berry powder was added to 8 times the weight of neutral water, mixed evenly, and decocted twice at 100°C. (After 2 hours of decoction, the concentrated decoction was filtered. The filtered residue was then added to 8 times the weight of neutral water, mixed evenly, and decocted at 100°C for 1.5 hours before filtering.) The combined concentrated decoction was filtered through a hollow cellulose membrane made of at least one of regenerated cellulose, polypropylene, polysulfone, or ceramic (with a molecular weight cutoff of 5 kDa, and the filtration temperature and pressure were 18°C and 0.1 MPa). The filtrates were combined and evaporated under vacuum at 45°C to remove water, yielding a concentrate.

[0047] 1-2. Separation of Lycium barbarum polysaccharide (LBP) and active polysaccharide (LBPP) components: 100 g of LBE was filtered through a dialysis bag (molecular weight cutoff, 6 kDa, temperature, 18°C, regenerated cellulose, membrane thickness, 25 μm, unfolded to form a corrugated (telescopic) tube with an inner diameter of 22 mm) and dialyzed against deionized water for 60 h. After dialysis, the mixture was centrifuged (4000 rpm, 15 min) and the supernatant recovered. Five volumes of 95% ethanol were added to the supernatant, and the mixture was precipitated overnight. The resulting mixture was then centrifuged (4000 rpm, 10 min). The precipitate was filtered and freeze-dried to yield 25.6 g of crude Lycium barbarum polysaccharide (LBP).

[0048] 10 g of LBP was dissolved in 12 times (mass) of deionized water, stirred overnight, and then centrifuged (4000 rpm, 10 min). The supernatant was recovered, and 5 times the volume of 95% ethanol was added to the supernatant. The mixture was precipitated overnight and centrifuged (10000 rpm, 20 min). The precipitate was taken as Lycium barbarum polysaccharide (LBPP, dry basis weight is 3.5 g).

[0049] 1-3. Molecular weight detection of LBPP: The molecular weight of LBPP was measured by high-performance gel permeation chromatography (HP-GPC).

[0050] In a series Shodex 804 (8.0 mm × 300 mm, exclusion limit 4 × 10 5 Da) and Shodex 802 column (8.0 mm × 300 mm, exclusion limit 1 × 10 4Homogeneity and molecular weight were measured by high-performance gel permeation chromatography (HP-GPC) on an Agilent 1260 HPLC system equipped with a 1.5-Da (1.5-Da) HPLC system. 0.1 M NaNO₃ was used as the mobile phase at a flow rate of 0.5 mL / min. Samples were prepared at 4 mg / mL using the mobile phase, and 10 μL of the solution was injected each time. The eluent was monitored with RI and UV detectors, and the column temperature was maintained at 25°C. (Jin, C., Du, Z., Lin, L., Zhou, L., Li, S., Liu, Q., & Ding, K. (2017). Structural Characterization of Mannoglucan from Dendrobium nobile Lindl and the Neuritogenesis-Induced Effect of Its Acetylated Derivative on PC-12 Cells. Polymers , 9 (12), 399.). In the HP-GPC results, the main peaks are concentrated in three peaks, and the corresponding weight-average molecular weight is 5.05×10 5 Da, 5.82×10 4 Da and 6.50×10 3 Da.

[0051] 1-4. Determination of Monosaccharide Composition of LBE, LBP, and LBPP: 3 mg of each polysaccharide sample (LBE, LBP, and LBPP) was added to 4 mL of 2 M trifluoroacetic acid, sealed, and hydrolyzed in a 110°C oven for 4 h. After the reaction, the mixture was cooled and methanol was added several times to remove all TFA. The residue was then redissolved in 200 μL of deionized water. 100 μL of the hydrolyzed sample solution and 100 μL of a mixed monosaccharide standard solution (mannose (Man), rhamnose (Rha), arabinose (Ara), glucose (Glc), galactose (Gal), xylose (Xyl), galacturonic acid (GalA), and gluconic acid (GlcA)) were added to a 2 mL EP tube. 100 μL of 0.6 M sodium hydroxide solution was added and mixed thoroughly. Then, 200 μL of a 0.5 M methanol solution of PMP (1-phenyl-3-methyl-5-pyrazolone) (261.3 mg / 3 mL) was added. After vortexing, the mixture was reacted in a 70°C water bath for 100 min. After completion of the reaction, 200 μL of 0.3 M hydrochloric acid solution was added for neutralization. Extraction was then performed with 400 μL of deionized water and 1 mL of chloroform. The mixture was vortexed and allowed to stand for 2 h or overnight. 850 μL of the upper aqueous phase was aspirated and extracted with 850 μL of chloroform. The mixture was allowed to stand for 1 hour. The upper aqueous phase (750 μL) was then extracted with an equal volume of chloroform and allowed to stand for separation. After filtration through a 0.22 μM microporous filter, the mixture was transferred into a liquid chromatography vial and analyzed by HPLC. The type and content of monosaccharides (indicated by peak area) were determined by comparing the retention time and peak area of the test residue with those of monosaccharide standards (mannose (Man), rhamnose (Rha), arabinose (Ara), glucose (Glc), galactose (Gal), xylose (Xyl), galacturonic acid (GalA), and gluconic acid (GlcA)).

[0052] The results showed that LBE contained Ara, Glc, Gal, Xyl, GalA, and GlcA in molar ratios of 3.3:92.1:1.3:0.4:1.8:1.4, respectively. LBP contained Rha, Ara, Gal, Glc, Man, and GlcA in molar ratios of 5.07:28.45:35.60:29.47:28.89:1.84, respectively. LBPP contained Man, Rha, Ara, Glc, Gal, Xyl, GalA, and GlcA in molar ratios of 2.3:5.9:40.7:10.4:16.7:2.9:19.3:1.7, respectively.

[0053] 1-5. Determination of physicochemical properties of LBE, LBP and LBPP: The total polyphenols and flavonoids were extracted and enriched using a polyamide adsorption method. 50 g of polyamide (100-200 mesh) was weighed and loaded onto a column with ethanol. Equilibrated to an initial gradient with 5% ethanol, each sample was accurately weighed and dissolved in 10 ml of 5% ethanol before loading onto the polyamide column. The column was then eluted with 500 ml of 5% ethanol and then 70% ethanol. Phenolic acids and flavonoids are concentrated in the 70% ethanol fraction.

[0054] The flavonoid content of each sample was determined using aluminum salt colorimetric spectrophotometry. Rutin was used as the standard. A 5% sodium nitrite solution was added, followed by a 10% aluminum nitrate solution. The mixture was shaken and allowed to stand for 6 minutes. Then, 10 ml of sodium hydroxide solution was added, and the absorbance was measured at 510 nm. The flavonoid content in LBE was 0.97 wt%, in LBP 0.2 wt%, and in LBPP 0.1 wt%.

[0055] The polyphenol content of each sample was determined using the Folin-phenol method, using gallic acid as the standard. After adding the Folin-phenol reagent, a NaCO solution was added. The samples were shaken and allowed to stand at room temperature for 60 minutes. The absorbance was measured at 760 nm. The polyphenol content in LBE was 3.63 wt%, in LBP 0.2 wt%, and in LBPP 0.1 wt%.

[0056] Carotenoids were extracted and enriched using petroleum ether. The absorbance was measured at 470 nm using β-carotene as a standard. The carotenoid content in LBE, LBP, and LBPP was 1.21 wt%, 0.1 wt%, and 0.1 wt%, respectively.

[0057] The polysaccharide content of each sample was determined using the phenol-sulfuric acid colorimetric method. After each sample was dissolved, an appropriate amount was mixed with a 5% phenol solution. Concentrated sulfuric acid was then added rapidly. After reacting at room temperature for 20 minutes, the absorbance was measured at 490 nm. A standard curve was constructed using glucose as the standard. The polysaccharide content in LBE was 20% by weight, in LBP 82%, and in LBPP 93% by weight.

[0058] The remainder of LBE, LBP and LBPP is pigments and unavoidable impurities.

[0059] 2. Biological experiments: 2-1. Animals and Grouping Healthy wild-type (WT) adult C57BL / 6J male mice (8 weeks old) were housed in an SPF barrier facility. Mice were randomly divided into a control group (n=7 per group) and a tail-suspension model group (n=7 per group). The tail-suspension model was performed as follows: After inhaled isoflurane anesthesia, the mouse's tail was cleaned and a custom-made restraint was affixed to each side of the tail using a thin layer of adhesive. After the restraint had fully cured, the mouse was individually placed in a 30×30×30 cm cage equipped with a pulley system. After a 6-hour acclimatization period (including free access to food and water and confirmation that the restraint was completely dry), the tail was raised using the pulley system to suspend the hind limbs off the ground, maintaining the mouse in a head-down tilted position of approximately 30°. In this position, the forelimbs could fully support the body, and the mouse could still move freely within the cage. Free access to food and water was maintained throughout the 6-week experimental period. Health status was assessed every 12 hours, and the suspension height was adjusted as necessary to ensure the hind limbs remained off the ground. Mice in the control and model groups were divided into six groups: control (normal mice), tail-suspended model group, tail-suspended model + LBE group, tail-suspended model + LBP group, tail-suspended model + LBPP group, and tail-suspended model + positive drug (alendronate sodium, ALN) group. Mice in the control and tail-suspended model groups were orally administered distilled water (10 mL / kg) via gavage. The treated groups received LBE (40 mg / kg), LBP (40 mg / kg), LBPP (40 mg / kg), and ALN (10 mg / kg) orally via gavage once daily for 6 weeks. All animal experiments were conducted in accordance with the ARRIVE guidelines and the UK Animals (Scientific Procedures) Act 1986 and related guidelines.

[0060] 2-2. Cell Culture MC3T3-E1 cells were purchased from ATCC (American Type Culture Collection). The culture medium used in this study was α-minimal essential medium (α-MEM; Gibco, 12571063) supplemented with 10% fetal bovine serum (FBS; Gibco, 16140071) and 1% penicillin-streptomycin (HyClone, SH40003-12). Differentiation culture was performed using α-MEM supplemented with 10% FBS, L-ascorbic acid (50 μg / ml; Sigma, A8960-5G), 0.1 μM dexamethasone (Sigma, D8893-1MG), 10 mM β-glycerophosphate (Sigma, G9422-10G), and 1% penicillin-streptomycin (HyClone, SH40003-12). The culture medium was changed every 2 days. Cell cultures were incubated in a humidified atmosphere at 37°C with 5% CO₂. The experiment was divided into 4 groups: control group, LBE group, LBP group and LBPP group. The dosages of the treatment groups were: LBE (400 μg / ml), LBP (400 μg / ml), LBPP (400 μg / ml). The control group was given an equal volume of double-distilled water.

[0061] 2-3. Detection of serum bone formation markers After drug treatment, mice were anesthetized, blood samples were collected from the fundus, and serum was obtained by centrifugation (2000 × g, 20 minutes, 4°C). Osteocalcin, a typical bone formation marker, was measured using an osteocalcin ELISA kit (Biovision, Cat. No. E4763-100) according to the kit's protocol.

[0062] 2-4. Bone analysis using micro-computed tomography CT scans were performed using a micro-computed tomography (micro-CT) imaging system (Inveon MM system, Siemens, Munich, Germany), and trabecular bone morphology analysis was performed according to the manufacturer's protocol. Trabecular bone parameters measured included bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Briefly, right femoral specimens were scanned in vivo and ex vivo using an effective pixel size of 8.89 μm, a voltage of 60 kV, a current of 220 μA, an exposure time of 1500 ms, and 360 exposures per 1° of exposure throughout the scan. The total image contained 1536 slices with an effective pixel size of 8.89 μm in all three axes. Two-dimensional images were reconstructed into three-dimensional (3D) visualization, and parameter analysis was performed using Inveon Research Workplace (Siemens). The region of interest (ROI) for femoral trabecular analysis was 1–2 mm below the distal femoral growth plate.

[0063] 2-5. Three-point bending test Three-point bending tests were performed using a small animal bone strength tester (Instron 4302, Instron, Norwood, Massachusetts). Fresh femoral bone strength was measured immediately after sampling from each group of mice. The three-point bending test used two end supports and a central loading point. Biomechanical measurements were collected from the load-deflection curves to determine the maximum load.

[0064] 2-6. Surface Plasmon Resonance (SPR) Analysis The affinity of LBPP binding to proteins was measured using a BIACORE T200 (GE Healthcare, Stockholm, Sweden). Noggin protein was immobilized on a CM5 sensor chip via amino coupling. For interaction measurements, varying concentrations of LBPP were injected into the chip. All runs were performed in HBS-EP running buffer (pH 7.4), containing 0.01 M HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), 0.15 M NaCl (sodium chloride), 3 mM EDTA (ethylenediaminetetraacetic acid), and 0.005% surfactant P20. Kinetic parameters were determined by fitting the data to a 1:1 binding model using Biacore T200 Evaluation Software version 1.0.

[0065] 2-7. Quantitative Real-Time PCR (RT-PCR) Experiment MC3T3-E1 cells were harvested, and total RNA was extracted using TRIzol reagent (87803, Invitrogen, USA) according to the manufacturer's protocol (Invitrogen). mRNA levels were measured by qRT-PCR using a 7500 Real-Time PCR System (Applied Biosystems) as previously described. Changes in mRNA expression of bone formation markers in each treatment group were assessed by the 2-ΔΔCq method.

[0066] 2-8. ALP staining and ALP determination ALP staining was monitored using the Vector Blue Substrate Kit (Procedure No. SK-5300, Vector Laboratories). MC3T3-E1 cells were plated at 1 × 10 5 Cells were seeded at a density of 100 μg / well in 24-well plates and cultured in growth medium for 3 days. On day 7, MC3T3-E1 cells were incubated with the substrate working solution for 30 minutes. Throughout this process, cells were protected from light. ALP assays were performed according to the kit's instructions.

[0067] 3. Results 3-1. LBE, LBP, and LBPP treatments increase bone mass and stability Microcomputed tomography (μCT) was used to investigate whether LBE, LBP, and LBPP affect trabecular structural parameters in mice. Using μCT, bone histomorphometric parameters were quantified in the femurs of treated and control mice. Compared with the tail-suspended model group, LBE, LBP, and LBPP treatments showed improved bone histomorphology (e.g., Figure 1 As shown in Figure 3 ), LBE, LBP, LBPP, and ALN treatment ameliorated tail-hanging-induced bone loss in mice. Mice treated with LBE, LBP, LBPP, and ALN showed higher BMD (bone mineral density), higher trabecular number (Tb.N), thicker trabecular thickness (Tb.Th), and smaller trabecular separation (Tb.Sp).

[0068] Next, they examined whether LBE, LBP, and LBPP could modulate bone biomechanical properties. The results showed that the maximum load force of mice treated with LBE, LBP, and LBPP was significantly increased compared to the tail-suspension model group.

[0069] The femoral bone density, trabecular number, trabecular thickness, trabecular separation and maximum load of each group are shown in Table 1.

[0070] Table 1

[0071] *p<0.05, ** p < 0.01 (compared with the control group); # p<0.05, ## p<0.01 (compared with the model group), n=7.

[0072] 3-2. LBE, LBP, and LBPP treatment promote bone formation The bone formation rate and bone mineral apposition rate (or bone mineralization rate) were analyzed within the femur. The bone formation rate (BFR) represents the amount of new bone formed per unit time and is typically measured by quantifying the amount of mineralized bone matrix or osteogenic matrix (unmineralized bone) formed within a specific bone surface area. The bone mineral apposition rate (MAR) specifically focuses on the mineralization rate of newly formed bone matrix. It measures the rate at which mineral crystals, primarily hydroxyapatite, are deposited onto the surface of the osteogenic matrix during bone formation and is determined using a calcein-alizarin red fluorescence double-labeling assay. Bone formation rate and bone mineralization rate were assessed using calcein-alizarin red fluorescence double-labeling in hard tissue sections of mouse femurs. Mice were intraperitoneally injected with calcein and Alizarin Red S (10 mg / kg) 10 and 2 days before euthanasia. Undecalcified femurs were then histologically analyzed. Unstained 5 μm sections were examined using fluorescence microscopy, and statistical analysis was performed using the Osteomeasure analysis system. The bone mineralization rate (MAR) was calculated by dividing the distance between fluorescent markers by the dosing interval. The mineralized surface ratio was further calculated, and the bone formation rate (BFR) was the product of the bone mineralization rate (MAR) and the mineralized surface ratio. The results showed that the bone formation rate (BFR) and bone mineral apposition rate (MAR) were significantly increased in mice treated with LBE, LBP, LBPP, and ALN. Serum osteocalcin levels were significantly increased compared to the tail-suspension model. These results suggest that supplementation with LBE, LBP, and LBPP can regulate bone mass by promoting bone formation and osteoblast mineralization.

[0073] The bone formation rate, bone mineralization rate and serum osteocalcin of each group are shown in Table 2.

[0074] Table 2

[0075] * p<0.05, ** p < 0.01 (compared with the control group); # p<0.05, ## p<0.01 (compared with the model group), n=7.

[0076] 3-3. LBE, LBP and LBPP can promote the proliferation, differentiation and mineralization of osteoblasts.

[0077] To determine whether LBE, LBP, and LBPP affect osteoblast differentiation and to further understand the specific compounds mentioned above, MC3T3-E1 cells were used to analyze osteoblast proliferation, differentiation, and mineralization. The results showed that LBE, LBP, and LBPP significantly promoted the formation of CFU-F (fibroblast colony forming unit) colonies (e.g., Figure 2 LBE, LBP, and LBPP significantly promoted the proliferation of MC3T3-E1 cells. Similarly, LBE, LBP, and LBPP significantly promoted osteoblast differentiation and mineralization. Furthermore, the expression levels of two osteoblast differentiation markers, Bglap, and ALP enzyme activity, were significantly increased in LBE-, LBP-, and LBPP-treated cells. Together, these results suggest that LBE, LBP, and LBPP treatment enhances osteoblast differentiation and mineralization.

[0078] The relative mRNA expression levels of CFU-F and Bglap and ALP activity of each group are shown in Table 3 .

[0079] Table 3

[0080] * p <0.05, ** p <0.01 (compared with the control group), n=3.

[0081] 3-4. LBPP promotes osteogenic differentiation of MC3T3-E1 cells under simulated weightlessness To elucidate the effect of LBPP on MC3T3-E1 cells under simulated weightlessness, MC3T3-E1 cells were treated with a rotator at 20 rpm / min, 37°C, for 48 h. Figure 3 The results show that LBPP can promote the differentiation of MC3T3-E1 cells under simulated weightlessness, and the ALP enzyme activity is significantly increased (ALP enzyme activity is shown in Table 4).

[0082] Table 4

[0083] ** p <0.01 (compared with the control group); # p <0.05, ## p <0.01 (compared with the model group), n=7.

[0084] 3-5. LBPP blocks the interaction between Noggin and BMP Since the interaction between Noggin and BMP inhibits osteogenic differentiation, the inventors investigated whether LBPP activates this signaling pathway by binding to Noggin. Surface plasmon resonance (SPR) technology was used to analyze the direct molecular binding between the two. SPR quantitative detection (e.g. Figure 4 LBPP binding to Noggin exhibited concentration-dependent binding within the 100-400 μg / mL concentration range, with the binding signal increasing with increasing LBPP concentration. Based on the average molecular weight of LBPP, 249.25 kDa, the equilibrium dissociation constant (KD) for this interaction was calculated to be 0.60 μmol / L, confirming that LBPP directly binds to Noggin. Furthermore, to determine whether this binding inhibits the interaction between Noggin and BMP2, BMP2 concentrations were measured in the supernatants of MC3T3-E1 cells treated with LBPP. The results showed that a certain amount of BMP2 was detected in the cell supernatant under Noggin conditions, and a significant amount of BMP2 was detected in the cell supernatant under LBPP conditions. The LBPP-induced upregulation of BMP-2 levels was also inhibited by Noggin. These data demonstrate that LBPP interacts with Noggin and inhibits the binding of Noggin to BMP2 (the amount of BMP2 released is shown in Table 5).

[0085] Table 5

[0086] ** p <0.01 (compared with the control group); # p <0.05, ## p <0.01 (compared with the LBPP group), n=3.

[0087] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. Use of Lycium barbarum polysaccharide in the preparation of a medicament for preventing and / or treating bone loss, characterized in that: The monosaccharide composition of the Lycium barbarum polysaccharide includes detectable arabinose, glucose, galactose and glucuronic acid; The content of polysaccharides with a weight average molecular weight of ≥6.5 kDa in the wolfberry polysaccharide is not less than 30 wt%.

2. The use according to claim 1, wherein The content of polysaccharides with a weight average molecular weight of ≥6.5 kDa in the wolfberry polysaccharide is 35-90 wt %.

3. The use according to claim 1 or 2, wherein The monosaccharide composition of the wolfberry polysaccharide also includes detectable mannose, rhamnose, xylose and galacturonic acid; Preferably, the monosaccharide composition test results of the wolfberry polysaccharide show that the molar ratio of mannose, rhamnose, arabinose, glucose, galactose, xylose, galacturonic acid and gluconic acid is 0-5:0-8:5-60:1-15:3-20:0-5:0-30:1, more preferably 0.5-3:3-5:20-30:5-8:8-12:1-3:10-15:

1.

4. The use according to claim 1, wherein The content of polysaccharide in the wolfberry polysaccharide is ≥20wt%; Preferably, the content of polysaccharide in the wolfberry polysaccharide is 85-95wt%.

5. The use according to claim 1 or 2, wherein The weight average molecular weight of the polysaccharide in the wolfberry polysaccharide is 5-800 kDa, preferably 6.5-550 kDa.

6. The use according to claim 1 or 2, wherein The wolfberry polysaccharide also includes flavonoids, carotenoids, polyphenols, pigments and inevitable impurities.

7. The use according to claim 6, wherein The content of flavonoids in the wolfberry polysaccharide is 0.05-5wt%, the content of carotenoids is 0.05-3wt%, the content of polyphenols is 0.05-8wt%, and the balance is pigments and inevitable impurities; Preferably, the content of flavonoids in the wolfberry polysaccharide is 0.1-0.15 wt %, the content of carotenoids is 0.1-0.5 wt %, the content of polyphenols is 0.1-0.15 wt %, and the balance is pigments and inevitable impurities.

8. The use according to any one of claims 1 to 7, wherein The preparation method of the wolfberry polysaccharide comprises: S1, mixing wolfberry with water for decoction extraction, and subjecting the obtained decoction to membrane separation; S2, subjecting the membrane-separated permeate to alcohol precipitation, and obtaining the crude wolfberry polysaccharide from the precipitate; S3. Dissolve the crude wolfberry polysaccharide in water and centrifuge at 2000-4000 rpm, collect the supernatant for secondary alcohol precipitation, and collect the precipitate to obtain wolfberry polysaccharide.

9. The use according to claim 8, wherein In step S1, the decoction extraction conditions include: temperature of 70-100°C; time of 1-3 hours, preferably 1.5-2.5 hours; And / or, in step S1, the mass ratio of wolfberry to water is 1:4-10, preferably 1:5-8; and / or, the separation membrane for membrane separation is selected from at least one of a fiber membrane, a polypropylene membrane, a polysulfone membrane and a ceramic membrane; and / or, the separation membrane for membrane separation has a molecular weight cutoff of 3.5-7 kDa; And / or, the membrane separation conditions include: temperature of 12-30°C, preferably 15-20°C; pressure of 0.1-10 MPa, preferably 0.1-0.5 MPa; time of 48-72 h, preferably 60-72 h; And / or, the method further comprises: in step S2, dialyzing the permeate separated by the membrane, centrifuging at 1000-4000 rpm for 10-20 minutes after dialysis for 48-72 hours, and subjecting the supernatant after centrifugation to alcohol precipitation; wherein the dialysis conditions include: the semipermeable membrane used for dialysis is made of at least one of regenerated cellulose, polypropylene, polysulfone and ceramic; the molecular weight cutoff of the semipermeable membrane is 3.5-7 kDa; the difference in molecular weight cutoff between the semipermeable membrane and the separation membrane of the membrane separation is 0.2-1 kDa; the thickness of the semipermeable membrane is 22-30 μm; and the dialysis temperature is 12-30° C., preferably 15-20° C.; And / or, in step S3, the amount of water used is 5-15 mL per gram of crude wolfberry polysaccharide; and / or, the precipitate after the first alcohol precipitation is collected by centrifugation at 2000-4000 rpm for 10-15 min; And / or, after the secondary alcohol precipitation, the precipitate is collected by centrifugation at 10,000-14,000 rpm for 20-30 minutes.

10. The use according to claim 1, wherein The bone loss is disuse bone loss.

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