Use of lycium barbarum polysaccharides in the preparation of a medicament for preventing and / or treating bone loss

Abstract

This invention relates to the field of plant polysaccharides, and discloses the use of Lycium barbarum polysaccharide in the preparation of drugs for the prevention and / or treatment of bone loss. The monosaccharides in the Lycium barbarum polysaccharide include detectable arabinose, glucose, galactose, and gluconic acid; wherein the content of polysaccharides with a weight-average molecular weight ≥ 6.5 kDa in the Lycium barbarum polysaccharide is not less than 30 wt%. This invention uses natural substances present in Lycium barbarum as active ingredients, which can be safely used within the experimental dosage range, achieving the prevention or treatment of bone loss. In particular, using Lycium barbarum polysaccharide extracted from Lycium barbarum, it competitively binds to Noggin protein, blocking the Noggin-BMP interaction to relieve bone formation inhibition, and promotes Smad protein phosphorylation to enhance osteogenic differentiation and mineralization, thus having the effect of preventing or treating disuse bone loss.

Description

Technical Field

[0001] This invention relates to the field of plant polysaccharides, and more specifically to the use of a Lycium barbarum polysaccharide in the preparation of a medicament for the prevention and / or treatment of bone loss. Background Technology

[0002] Disuse osteoporosis (DOP) is a secondary osteoporosis caused by lack of mechanical stress or prolonged immobilization, characterized by high incidence and significant clinical harm. Epidemiological surveys show that the incidence of bone loss in spinal cord injury patients is as high as 42.4% within 3 months after injury; astronauts experience a 1.4-1.5% monthly decrease in proximal femoral bone mineral density under microgravity; and patients who undergo fracture fixation may experience up to 50% bone mineral loss within 6 months. Current clinical treatment mainly relies on bisphosphonates and physical interventions, but these have limitations in 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 their implementation is challenging.

[0003] Current research focuses on multi-target combined therapy, precise mechanical intervention, and personalized gene regulation. Bone morphogenetic protein (BMP) signaling plays a fundamental role in bone development and bone homeostasis. While Lycium barbarum polysaccharide (LBP) has been reported to alleviate bone loss, its efficacy still needs further improvement. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide the use of Lycium barbarum polysaccharide in the preparation of a medicine for the prevention and / or treatment of bone loss.

[0005] To achieve the above objectives, the present invention provides the use of Lycium barbarum polysaccharide in the preparation of a medicament for the prevention and / or treatment of bone loss, wherein the monosaccharide in the Lycium barbarum polysaccharide includes detectable arabinose, glucose, galactose and gluconic acid; The content of the polysaccharide with a weight average molecular weight ≥ 6.5 kDa in the wolfberry polysaccharide is not less than 30 wt%.

[0006] This invention uses natural substances found in wolfberry as active ingredients, which can be safely used within the experimental dosage range to prevent or treat bone loss. In particular, it uses wolfberry polysaccharide extracted from wolfberry, which competitively binds to Noggin protein, blocks the interaction between Noggin and BMPs to relieve bone formation inhibition, and promotes Smad protein phosphorylation to enhance osteogenic differentiation and mineralization, thus having the effect of preventing or treating disuse bone loss. Attached Figure Description

[0007] Figure 1Microcomputed tomographic images of the whole femur and trabeculae of mice after different treatments; Figure 2 Colony diagrams of preosteoblasts in mice after different treatments; Figure 3 ALP staining images of mouse preosteoblasts after simulated weightlessness treatment with different treatments; Figure 4 The graph shows the response signals of the interaction between LBPP and Noggin at different concentrations. Detailed Implementation

[0008] The endpoints and any values ​​of the ranges 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 endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

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

[0010] During their research, the inventors of this invention discovered that natural substances present in wolfberry can be safely used as active ingredients within experimental dosage ranges, achieving the prevention or treatment of bone loss. In particular, wolfberry polysaccharides extracted from wolfberry competitively bind to Noggin protein, blocking the interaction between Noggin and BMPs to relieve bone formation inhibition, and promote Smad protein phosphorylation to enhance osteogenic differentiation and mineralization, thus having the effect of preventing or treating disuse bone loss.

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

[0012] In this invention, preferably, the content of polysaccharides with a weight-average molecular weight ≥ 6.5 kDa in the wolfberry polysaccharide can be 35-90 wt% (for example, it can be any two values ​​formed by 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 values ​​within the range).

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

[0014] In this invention, preferably, the monosaccharide composition detection 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 even more preferably 2.3:5.9:40.7:10.4:16.7:2.9:19.3:1.7. Lycium barbarum polysaccharides with monosaccharide compositions falling within the above range will exhibit better effects in preventing and / or treating bone loss (especially disuse bone loss). The term "monosaccharide composition" used in this invention refers to the composition of the basic units (monosaccharides) that form polysaccharides through glycosidic bonds (dehydration).

[0015] In a preferred embodiment of the present invention, Lycium barbarum polysaccharides can also be compounded with other polysaccharides extracted from Lycium barbarum for use in drugs for the prevention and / or treatment of bone loss, wherein the proportion of other polysaccharide components is not 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 this invention, preferably, the polysaccharide content in the wolfberry polysaccharide is ≥20wt%; more preferably, the polysaccharide content in the wolfberry polysaccharide can be 85-95wt% (for example, it can be any two values ​​formed by 85wt%, 86wt%, 87wt%, 88wt%, 89wt%, 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, and values ​​within the range).

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

[0018] In this invention, the wolfberry polysaccharide is a crude polysaccharide extract. Preferably, the wolfberry polysaccharide may also include flavonoids, carotenoids, polyphenols, pigments, and unavoidable impurities. There may be only one of each of the above components, or there may be two or more of the same type of component.

[0019] In this invention, preferably, the flavonoid content in the wolfberry polysaccharide can be 0.05-5 wt% (for example, it can be a range formed by at least two values ​​from 0.05 wt%, 0.1 wt%, 0.15 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, and values ​​within that range), and the carotenoid content can be 0.05-3 wt% (for example, it can be 0.05 wt%, 0.1 wt%, 0.15 wt%, 0.5 wt%, 0.8 wt%, 1 wt%, 1.2 wt%, 1.5 wt%, 1.8 wt%). The polyphenol content can be 0.05-8 wt% (e.g., it can be a range formed by at least two of the values ​​selected from t%, 2wt%, 2.3wt%, 2.5wt%, 2.7wt%, 3wt%), and the balance is pigments and unavoidable impurities.

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

[0021] In this invention, the wolfberry polysaccharide is derived from the water-extracted and alcohol-precipitated product of wolfberry. Preferably, the preparation method of the wolfberry polysaccharide includes: S1. Mix wolfberries with water and decoct to extract the extract; then perform membrane separation on the obtained decoction. S2. The permeate (LBE) separated by membrane separation is subjected to alcohol precipitation once, and the precipitate is collected to obtain crude polysaccharide of wolfberry (LBP). S3. Dissolve the crude polysaccharide of wolfberry in water and centrifuge at 2000-4000 rpm. Take the supernatant and perform a second alcohol precipitation. Take the precipitate to obtain wolfberry polysaccharide (LBPP).

[0022] The inventors of this invention have discovered that both LBP and LBPP have good efficacy in preventing and / or treating bone loss, but LBPP has a better effect in improving bone microstructure and bone stability to prevent and / or treat disuse bone loss. Therefore, this invention also relates to the use of products (Lycium barbarum polysaccharides, such as LBP and / or LBPP) prepared by the above methods in the preparation of drugs for the prevention and / or treatment of bone loss.

[0023] In this invention, preferably, in step S1, the conditions for decoction extraction may include: the temperature may be 70-100℃ (for example, any two values ​​from 70℃, 72℃, 75℃, 78℃, 80℃, 83℃, 85℃, 87℃, 90℃, 95℃, 100℃, or any value within that range), more preferably 75-85℃; the time may be 1-3h (for example, any two values ​​from 1h, 1.5h, 2h, 2.5h, 3h, or any value within that range), more preferably 1.5-2.5h.

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

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

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

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

[0028] In this invention, preferably, in step S2, the method may further include: in step S2, the permeate separated by membrane separation is dialyzed, and after dialyzing for 48-72 hours, it is centrifuged at 1000-4000 rpm for 10-20 minutes, and the supernatant after centrifugation is subjected to alcohol precipitation once.

[0029] In this invention, preferably, the dialysis method may include: performing dialysis using a dialysis bag or dialysis tubing; wherein 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 may be 3.5-7 kDa; the difference in molecular weight cutoff between the semipermeable membrane and the separating membrane may be 0.2-1 kDa; the thickness of the semipermeable membrane may be 22-30 μm; and the dialysis temperature may be 12-30°C, more preferably 15-20°C.

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

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

[0032] In this invention, preferably, in step S3, the amount of water used relative to each gram of crude Lycium barbarum polysaccharide can be 5-15 mL (for example, it can be any two values ​​from 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, or any value within that range).

[0033] In this invention, preferably, the precipitate is collected after a single alcohol precipitation by centrifuging at 2000-4000 rpm (for example, a range formed by at least two of the values ​​selected from 2000 rpm, 2200 rpm, 2400 rpm, 2600 rpm, 2800 rpm, 3000 rpm, 3200 rpm, 3400 rpm, 3600 rpm, 3800 rpm, and 4000 rpm, and values ​​within that range) for 10-15 min (for example, a range formed by at least two of the values ​​selected from 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min, and values ​​within that range).

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

[0035] In this invention, preferably, the precipitate after secondary alcohol precipitation is collected by centrifugation at 10000-14000 rpm (for example, a range formed by at least two of the values ​​of 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, and 14000 rpm, and values ​​within that range) for 20-30 min (for example, a range formed by at least two of the values ​​of 20 min, 22 min, 24 min, 26 min, 28 min, and 30 min, and values ​​within that range).

[0036] In a preferred embodiment of the present invention, the bone loss is disuse bone loss. Disuse bone loss is a pathological process of rapid bone loss caused by prolonged lack of mechanical load on the bones (such as bed rest, limb immobilization, weightlessness, etc.), leading to osteoporosis. Its core mechanism is an imbalance in the adaptive response of the bones to mechanical stimulation, resulting in reduced bone formation and increased bone resorption. Disuse bone loss is caused by the lack of mechanical stimulation of the bones due to prolonged immobilization (such as paralysis, bed rest, fracture fixation) or weightlessness (astronauts), leading to structural damage to the bone microstructure. Ordinary bone loss, on the other hand, is more often due to physiological or pathological metabolic imbalances. The two have different pathogenic mechanisms, and their treatments are not interchangeable.

[0037] In terms of terminology, "subject" and "patient" are used synonymously in this invention, referring to members of the animal kingdom, with a preference for mammals, specifically encompassing non-primates (such as livestock like cattle, pigs, and horses, as well as laboratory animals like cats, dogs, and rats) and primates (including non-human primates and humans). The most preferred subject is Homo sapiens. Specific implementation examples include farm animals (such as horses, cattle, and pigs) and companion animals (such as dogs and cats), particularly suitable for studies on adult individuals, which aligns closely with the research focus of this invention on bone loss (especially disuse bone loss).

[0038] Regarding the definition of drug-related terms, in this invention, "drug" specifically refers to a system of substances with functions of disease prevention, treatment, management, or diagnosis, encompassing multiple dimensions such as single-molecule compounds, compound formulations, diagnostic reagents, and treatment methods. "Effective dose" refers to a dosage that can produce at least one of the following effects: prevention of the onset or recurrence of a targeted disease, enhancement of the preventive effect of synergistic therapies, reduction of disease severity, shortening of disease duration, relief of clinical symptoms, inhibition of disease progression, promotion of pathological reversal, and enhancement of the therapeutic effect of other therapies. It should be particularly noted that this invention primarily targets interventions for bone loss and related bone metabolism abnormalities. The standard for "pharmaceuticalally acceptable" refers to meeting any of the following conditions: certification by regulatory agencies such as the FDA or EMA, inclusion in international pharmacopoeias such as the USP or EP, or suitability for medical use in mammals.

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

[0040] In this invention, preferably, the drug may also contain pharmaceutically acceptable excipients, wherein the excipients refer to inactive ingredients that act as carriers or mediators in the formulation, specifically including but not limited to the following categories: solvent systems (such as solvent / cosolvent), stabilizing systems (such as preservatives / antimicrobial agents), solid dosage form excipients (including fillers such as microcrystalline cellulose, binders such as hydroxypropyl methylcellulose, disintegrants such as croscarmellose sodium, lubricants such as magnesium stearate, etc.), and liquid dosage form excipients (such as surfactants polysorbate 80, emulsifiers lecithin, suspending agents xanthan gum, etc.).

[0041] In a preferred embodiment of the present invention, the method of using wolfberry polysaccharide includes: applying wolfberry polysaccharide to a subject or animal cells.

[0042] In this invention, preferably, the wolfberry polysaccharide is the wolfberry polysaccharide described above, and its characteristics are the same as those of the wolfberry polysaccharide described above, and will not be repeated here.

[0043] In some embodiments, the Lycium barbarum polysaccharide described in this invention can be taken with beverages, foods, or related ingredients, or used as a health food. There are no particular limitations on the preparation method of the food or health food. For example, it can be made into tablets, beverages, candies, etc. Each food preparation may include other ingredients used in the art in addition to the polysaccharide component. These other ingredients can be selected by those skilled in the art according to specific formulations or uses.

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

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

[0046] Dried wolfberry fruit was washed five times and soaked in double-distilled water (pH=7) at room temperature for 2 hours, then crushed. The soaked berry powder was added to 8 times its weight of neutral water, mixed thoroughly, and decocted twice at 100°C (after 2 hours of decoction, the concentrated decoction was filtered; the residue was then mixed with 8 times its weight of neutral water, decocted again at 100°C for 1.5 hours, and then filtered). The combined concentrated decoction was filtered through a hollow cellulose membrane, using at least one of regenerated cellulose, polypropylene, polysulfone, or ceramic (the membrane's molecular weight cutoff was 5 kDa, the filtration temperature was 18°C, and the pressure was 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: 100g of LBE was filtered through a dialysis bag (the dialysis bag had a molecular weight cutoff of 6kDa, a temperature of 18℃, was made of regenerated cellulose, had a membrane thickness of 25μm, unfolded into a pleated (stretchable) tube shape, and had an inner diameter of 22mm), and dialyzed with deionized water for 60h. After dialysis, the sample was centrifuged (4000rpm, 15min) and the supernatant was collected. Five volumes of 95% ethanol were added to the supernatant, and the sample was precipitated overnight. The sample was then centrifuged (4000rpm, 10min), and the precipitate was filtered and freeze-dried to obtain 25.6g of crude Lycium barbarum polysaccharide (LBP).

[0048] Dissolve 10g of LBP in 12 times (by mass) of deionized water, stir overnight, centrifuge (4000rpm, 10min), collect the supernatant, add 5 times the volume of 95% ethanol to the supernatant, precipitate overnight, centrifuge (10000rpm, 20min), and take the precipitate as Lycium barbarum polysaccharide (LBPP, dry weight 3.5g).

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

[0050] Equipped with series Shodex 804 (8.0mm×300mm, resistance limit 4×10) 5 Da) and Shodex 802 pillars (8.0mm × 300mm, resistance limit 1 × 10) 4Homogeneity and molecular weight were measured by high-performance gel permeation chromatography (HP-GPC) on an Agilent 1260 HPLC system (Da), with 0.1 M NaNO3 as the mobile phase and a flow rate of 0.5 mL / min. Samples were prepared into 4 mg / mL solutions using the mobile phase, with 10 μL solutions injected each time. The eluent was monitored using 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.). The HP-GPC results showed that the molecular weight was mainly concentrated in three peaks, with a corresponding weight-average molecular weight of 5.05 × 10⁻⁶. 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: Take 3 mg of each polysaccharide sample (LBE, LBP, LBPP), add 4 mL of 2M trifluoroacetic acid, seal tightly, and hydrolyze in an oven at 110℃ for 4 h. After the reaction is complete, cool, add methanol several times to remove TFA, and redissolve the residue in 200 μL of deionized water. Take 100 μL of the hydrolyzed sample solution and 100 μL of the mixed monosaccharide standard solution (mannose, rhamnose, arabinose, glucose, galactose, xylose, galacturonic acid, and gluconic acid) and add them separately to 2 mL EP tubes. Then add 100 μL of 0.6 M sodium hydroxide solution, mix well, and then add 200 μL of 0.5 M PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution (261.3 mg / 3 mL). Vortex to mix well and react in a 70 °C water bath for 100 min. After the reaction is complete, add 200 μL of 0.3 M hydrochloric acid solution to neutralize, then add 400 μL of deionized water and 1 mL of chloroform for extraction. Vortex and let stand for 2 h or overnight. Extract 850 μL of the upper aqueous phase with 850 μL of chloroform and let stand for 1 h. Extract the remaining 750 μL of the upper aqueous phase with an equal volume of chloroform and let stand to separate the layers. Filter through a 0.22 μM microporous membrane and transfer to a liquid chromatography bottle for HPLC analysis. The types and contents of monosaccharides (characterized by peak area) were determined by comparing the retention time and peak region of the test residues with monosaccharide standards (mannose, rhamnose, arabinose, glucose, galactose, xylose, galacturonic acid, and gluconic acid).

[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. 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. 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.

[0053] 1-5. Determination of the physicochemical properties of LBE, LBP, and LBPP: Total polyphenols and flavonoids were extracted and enriched using polyamide adsorption. 50 g of polyamide (100-200 mesh) packing material was weighed, packed into a column with ethanol, and equilibrated to the initial gradient with 5% ethanol. Each sample group was accurately weighed and dissolved in 10 ml of 5% ethanol before being loaded onto the polyamide column. Elution was then performed sequentially with 500 ml of 5% ethanol and 70% ethanol. Phenolic acids and flavonoids were concentrated in the 70% ethanol fraction.

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

[0055] The polyphenol content of each group of samples was determined according to the Folin-Ciocalteu method, using gallic acid as a standard. After adding the Folin-Ciocalteu reagent, Na₂CO₃ solution was added, and the mixture was shaken well and allowed to stand at room temperature for 60 min. The absorbance was then measured at a wavelength of 760 nm. The polyphenol content in LBE was 3.63 wt%, in LBP it was 0.2 wt%, and in LBPP it was 0.1 wt%.

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

[0057] The polysaccharide content of each sample was determined using the phenol-sulfuric acid colorimetric method. After dissolving each sample, an appropriate amount was mixed with a 5% phenol solution, concentrated sulfuric acid was quickly added, and the reaction was allowed to proceed at room temperature for 20 minutes. The absorbance was then measured at 490 nm, and a standard curve was plotted using glucose as the standard. The polysaccharide content in LBE was 20 wt%, in LBP it was 82%, and in LBPP it was 93 wt%.

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

[0059] 2. Biological Experiments: 2-1. Animals and Grouping Healthy wild-type (WT) adult male C57BL / 6J 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-suspended model group (n=7 per group). The tail-suspended model procedure was as follows: After anesthesia using an inhaled isoflurane anesthesia system, the mice's tails were cleaned, and a custom-made restraint device was attached to both sides of the tail using a thin layer of adhesive. After the restraint device had fully cured, the mice were placed individually in 30×30×30cm cages equipped with a pulley system at the top. After a 6-hour acclimatization period (including free access to food and water and confirmation that the restraint device was completely dry), the tails were raised using the pulley system, suspending the hind limbs off the ground, maintaining the mouse's head tilted downwards at approximately 30°. In this position, the forelimbs could fully support the body weight, and the mice could still move freely within the cage. Throughout the 6-week experimental period, the mice maintained free access to food and water, and their health status was assessed every 12 hours. The suspension height was adjusted as needed to ensure the hind limbs remained off the ground. Mice in the control and model groups were divided into six groups: control group (normal mice), tail-hanging model group, tail-hanging model + LBE group, tail-hanging model + LBP group, tail-hanging model + LBPP group, and tail-hanging model + positive control drug (alendronate sodium, ALN) group. Mice in the control and tail-hanging model groups were administered distilled water (10 mL / kg) orally via gavage. The treatment groups were administered LBE (40 mg / kg), LBP (40 mg / kg), LBPP (40 mg / kg), and ALN (10 mg / kg) once daily via gavage for 6 weeks. All animal experiments complied with the ARRIVE guidelines and the UK Animal (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-minimum basal medium (α-MEM; Gibco, 12571063), supplemented with 10 v / v fetal bovine serum (FBS; Gibco, 16140071) and 1 v / v penicillin-streptomycin (HyClone, SH40003-12). The differentiation culture medium was α-MEM, supplemented with 10 v / v 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 v / v penicillin-streptomycin (HyClone, SH40003-12). The medium was changed every 2 days. Cell cultures were incubated in a humid environment at 37°C with 5 v / v CO2. The experiment was divided into four groups: control group, LBE group, LBP group and LBPP group. The treatment group doses were: LBE (400 μg / ml), LBP (400 μg / ml) and LBPP (400 μg / ml). The control group was treated with an equal volume of double-distilled water.

[0061] 2-3. Detection of serum bone formation markers After drug treatment of mice, the mice were anesthetized, and blood samples were collected from the fundus of the mice. Serum was obtained by centrifugation (2000×g, 20 minutes, 4°C). The osteocalcin content was detected using the osteocalcin ELISA kit (Biovision, catalog number: E4763-100) according to the experimental procedures of the kit. Osteocalcin is a typical marker of bone formation.

[0062] 2-4. Bone analysis using micro-computed tomography (CT) scans. CT scans were performed using a micro-CT imaging system (Inveon MM system, Siemens, Munich, Germany), and trabecular morphology analysis was conducted according to the manufacturer's provided procedures. The detected trabecular parameters included bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). In short, the right femoral sample was scanned in vivo and ex vivo under the following conditions: an effective pixel value of 8.89 μm, a voltage of 60 kV, a current of 220 μA, an exposure time of 1500 ms, one exposure per 1° interval, and a total of 360 exposures. The entire image contains 1536 tomographic images, with an effective pixel value of 8.89 μm in all three axes. The two-dimensional images were reconstructed into three-dimensional (3D) visualizations, and parametric analysis was performed using the 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 testing device (Instron 4302, Instron, Norwood, Massachusetts). Fresh femoral bone strength was assessed immediately after femoral tissue harvesting from mice in each group. The three-point bending test used two end support points and one central loading point. Biomechanical measurements were collected from the load-deformation curves to obtain the maximum load force.

[0064] 2-6. Surface Plasmon Resonance (SPR) Analysis The affinity of LBPP for protein binding 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, different concentrations of LBPP were injected into the chip. All operations were performed in HBS-EP run 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 using a 1:1 binding model with 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 bone formation marker mRNA expression in each treatment group were assessed using the 2-ΔΔCq method.

[0066] 2-8. ALP staining and ALP measurement ALP staining was monitored using the Vector Blue substrate kit (procedure number SK-5300, Vector Laboratories). MC3T3-E1 cells were stained at a rate of 1×10⁻⁶ cells per cell line according to the protocol. 5 MC3T3-E1 cells were seeded at a density of 10 cells per well in 24-well plates and cultured in growth medium for 3 days. On day 7, the cells were incubated with substrate working solution for 30 minutes. Throughout the process, the cells were protected from light. ALP assays were performed according to the kit instructions.

[0067] 3. Results 3-1. LBE, LBP, and LBPP treatments can increase bone mass and bone stability. Microcomputed tomography (μCT) was used to investigate whether LBE, LBP, and LBPP affected trabecular bone structure parameters in mice. μCT was used to quantify the morphological parameters of the femur in both the treated and control groups. Compared to the tail-hanging model group, LBE, LBP, and LBPP treatments showed better bone morphology (e.g., ...). Figure 1 As shown in the figure, LBE, LBP, LBPP and ALN treatments improved tail-induced bone loss in mice. Mice treated with LBE, LBP, LBPP and ALN showed higher BMD (bone mineral density), higher number of trabeculae (Tb.N), thicker trabecular bone thickness (Tb.Th) and smaller trabecular bone separation (Tb.Sp).

[0068] Next, the effects of LBE, LBP, and LBPP on bone biomechanical properties were investigated. The results showed that, compared to the tail-hanging model group, mice treated with LBE, LBP, and LBPP exhibited a significant increase in maximum load-bearing capacity.

[0069] Table 1 shows the femoral bone mineral density, number of trabeculae, trabecular thickness, trabecular separation, and maximum load force for each group.

[0070] Table 1

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

[0072] 3-2. LBE, LBP, and LBPP treatments promote bone formation. This study analyzed the rate of bone formation (BFR) and bone mineralization deposition rate (or bone mineralization rate) within the femur. BFR represents the amount of new bone formed per unit time, typically measured by quantifying the amount of mineralized bone matrix or osteogenic matrix (unmineralized bone) formed in a specific bone surface region. BFR specifically focuses on the mineralization rate of newly formed bone matrix, measuring the rate at which mineral crystals, primarily hydroxyapatite, are deposited onto the osteogenic matrix surface during bone formation, determined using a calcein-alizarin red fluorescence double labeling method. BFR and bone mineralization rate were assessed using mouse femoral hard tissue sections using the calcein-alizarin red fluorescence double labeling method. Specifically, mice were intraperitoneally injected with calcein and alizarin red S (10 mg / kg) 10 days and 2 days prior to euthanasia. Undecalcified femoral sections were harvested for histological analysis. Unstained 5 μm sections were examined using a fluorescence microscope, and statistical analysis was performed using the Osteomeasure analysis system. Bone mineralization rate (MAR) was calculated by dividing the distance between fluorescent markers by the dosing interval; the mineralized surface area was further calculated, and the bone formation rate (BFR) was the product of bone mineralization rate (MAR) and the mineralized surface area. Results showed that LBE, LBP, LBPP, and ALN treatments significantly increased bone formation rate (BFR) and bone mineralization deposition rate (MAR) in mice. Serum osteocalcin levels were significantly increased compared to the tail-hanging model. These results indicate that LBE, LBP, and LBPP supplementation can regulate bone mass by promoting bone formation and osteoblast mineralization.

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

[0074] Table 2

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

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

[0077] To investigate whether LBE, LBP, and LBPP affect osteoblast differentiation and to further understand the effects of these specific compounds, MC3T3-E1 cells were used to analyze osteoblast proliferation, differentiation, and mineralization. Results showed that LBE, LBP, and LBPP significantly promoted the formation of CFU-F (fibroblast colony-forming units) colonies (e.g., ...). Figure 2 As shown in the figure, LBE, LBP, and LBPP significantly promoted osteoblast differentiation and mineralization, promoting MC3T3-E1 cell proliferation. Similarly, LBE, LBP, and LBPP significantly promoted osteoblast differentiation and mineralization. Furthermore, in cells treated with LBE, LBP, and LBPP, the expression levels of two osteoblast differentiation markers, Bglap and ALP enzyme activity, were significantly increased. All these results indicate that LBE, LBP, and LBPP treatment enhances osteoblast differentiation and mineralization.

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

[0079] Table 3

[0080] * p <0.05, ** p <0.01 (compared to 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 rotary device to simulate weightlessness. Treatment conditions: 20 rpm / min, 37˚C, 48 h. ALP staining results (e.g.) Figure 3 As shown in Table 4, LBPP can promote the differentiation of MC3T3-E1 cells under simulated weightlessness, and ALP enzyme activity is significantly increased (ALP enzyme activity is shown in Table 4).

[0082] Table 4

[0083] ** p <0.01 (compared to the control group); # p <0.05, ## p <0.01 (compared to 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. Quantitative SPR detection (e.g.) Figure 4 As shown in the figure, LBPP binds to Noggin in a concentration-dependent manner within the range of 100-400 μg / mL, and the binding signal increases with increasing LBPP concentration. Based on the average molecular weight of LBPP (249.25 kDa), the equilibrium dissociation constant (KD) for this interaction is calculated to be 0.60 μmol / L, confirming that LBPP can directly bind to Noggin. Furthermore, to determine whether this binding inhibits the interaction between Noggin and BMP2, the concentration of BMP2 in the supernatant of LBPP-treated MC3T3-E1 cells was measured. The results showed that a certain amount of BMP2 was detected in the cell supernatant under Noggin conditions, and a large amount of BMP2 was detected under LBPP conditions. The LBPP-induced upregulation of BMP-2 levels was also inhibited by Noggin. These data indicate that LBPP interacts with Noggin and inhibits the binding of Noggin to BMP2 (BMP2 release is shown in Table 5).

[0085] Table 5

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

[0087] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. The use of Lycium barbarum polysaccharides in the preparation of medicaments for the prevention and / or treatment of bone loss, characterized in that, The monosaccharide composition of the Lycium barbarum polysaccharide includes arabinose, glucose, galactose, glucuronic acid, mannose, rhamnose, xylose and galacturonic acid; The content of polysaccharides with a weight-average molecular weight ≥6.5kDa in the wolfberry polysaccharide is 35-90wt%. The monosaccharide composition test results show that the molar ratio of mannose, rhamnose, arabinose, glucose, galactose, xylose, galacturonic acid and glucuronic acid is 0.5-3:3-5:20-30:5-8:8-12:1-3:10-15:

1. The weight-average molecular weight of the polysaccharides in the wolfberry polysaccharide is 6.5-550kDa. The bone loss mentioned above refers to disuse-related bone loss.

2. The use according to claim 1, wherein, The polysaccharide content in the wolfberry polysaccharide is 85-95 wt%.

3. The use according to claim 1, wherein, The wolfberry polysaccharide also includes flavonoids, carotenoids, polyphenols, pigments, and unavoidable impurities.

4. The use according to claim 3, 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 remainder is pigments and unavoidable impurities.

5. The use according to claim 3, wherein, The content of flavonoids in the wolfberry polysaccharide is 0.1-0.15wt%, the content of carotenoids is 0.1-0.5wt%, the content of polyphenols is 0.1-0.15wt%, and the remainder is pigments and unavoidable impurities.

6. The use according to any one of claims 1-5, wherein, The preparation method of the wolfberry polysaccharide includes: S1. Mix wolfberries with water and decoct to extract the extract; then perform membrane separation on the obtained decoction. S2. The permeate from the membrane separation was subjected to alcohol precipitation once, and the precipitate was collected to obtain crude polysaccharide from wolfberry. S3. Dissolve the crude polysaccharide of wolfberry in water and centrifuge at 2000-4000 rpm. Take the supernatant and perform a second alcohol precipitation. Take the precipitate to obtain wolfberry polysaccharide.

7. The use according to claim 6, wherein, In step S1, the conditions for decoction extraction include: a temperature of 70-100℃ and a time of 1-3 hours; And / or, in step S1, the mass ratio of the goji berries to water is 1:4-10; And / or, the separation membrane of the membrane separation is selected from at least one of fiber membranes, polypropylene membranes, polysulfone membranes and ceramic membranes; and / or, the molecular weight cutoff of the separation membrane of the membrane separation is 3.5-7 kDa; And / or, the membrane separation conditions include: a temperature of 12-30°C; a pressure of 0.1-10 MPa; and a time of 48-72 h; And / or, the method further includes: in step S2, dialysis of the membrane-separated permeate, followed by centrifugation at 1000-4000 rpm for 10-20 min after dialysis for 48-72 h, and alcohol precipitation of the supernatant after centrifugation; wherein the dialysis conditions include: 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 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℃; And / or, in step S3, the amount of water used is 5-15 mL relative to each gram of crude polysaccharide from wolfberry; And / or, after one alcohol precipitation, the precipitate is collected by centrifugation at 2000-4000 rpm for 10-15 min; And / or, after secondary alcohol precipitation, the precipitate is collected by centrifugation at 10000-14000 rpm for 20-30 min.

8. The use according to claim 6, wherein, In step S1, the conditions for decoction extraction include: a time of 1.5-2.5 hours; And / or, in step S1, the mass ratio of the goji berries to water is 1:5-8; And / or, the membrane separation conditions include: a temperature of 15-20°C; a pressure of 0.1-0.5 MPa; and a time of 60-72 h; And / or, the method further includes: in step S2, dialysis of the membrane-separated permeate at a dialysis temperature of 15-20°C.

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