COMPOSITIONS COMPRISING HOMOPOLYSACCHARIDES OR DERIVATIVES THEREOF, AND METHODS FOR PREVENTING AND / OR TREATING BONE LOSS USING THE SAME - Patent application

Abstract

The present invention provides compositions and methods for improving bone formation, bone mass, and / or bone strength, and / or treating or preventing bone loss, such as age-related bone loss, in a subject in need of such treatment. Such compositions comprise a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient. The homopolysaccharide is composed primarily of arabinose, galactose, rhamnose, and galacturonic acid as monomer units. Such methods comprise administering an effective amount of such a composition to a subject in need thereof.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Patent Application No. 63 / 389,082, filed July 14, 2022, the contents of which are incorporated herein by reference.

[0002] The present invention relates to compositions having pharmaceutical or functional properties. Specifically, the disclosed subject matter relates to wolfberry extracts, compositions comprising polysaccharides or derivatives thereof, and methods of using them, for example, as pharmaceutical compositions, functional compositions, and / or supplements. [Background technology]

[0003] A recent study on the epidemiology of osteoporosis in the European Union found a 47% prevalence of osteoporosis in women aged 50–55 years and those aged 80 years and older. Among men in the same age groups in the European Union, the prevalence rates were 2.5% and 16%, respectively. Bone loss is not limited to postmenopausal women; osteoporosis in older men is also attracting attention, and its incidence is expected to steadily increase with the aging of the population. Research has revealed that among those aged 50 years and older, one-third of women and one-fifth of men experience bone loss, leading to a sharp increase in the risk of fracture and death.

[0004] Currently, more than 200 million people worldwide suffer from osteoporosis, which is characterized by reduced bone mass and mineral density and destruction of bone microarchitecture. Senile osteoporosis, therefore, poses a new medical and socioeconomic threat due to its high rates of morbidity, disability, and mortality.

[0005] Senile osteoporosis is primarily associated with decreased bone formation and decreased bone metabolism. Age-related bone loss is due to decreased bone formation and increased bone resorption. Bone morphogenetic protein (BMP) signaling plays a fundamental role in skeletal development and bone homeostasis. BMPs are members of the transforming growth factor-β (TGF-β) superfamily and transmit signals by binding to and assembling type I and type II transmembrane serine / threonine receptor kinases. After ligand-induced assembly of two type I and two type II receptors, the constitutively active type II receptor kinase phosphorylates and activates the type I receptor, which is further phosphorylated by Smad or MAPK-mediated signaling, activating the transcription of specific target genes involved in bone differentiation and bone formation.

[0006] Noggin is widely considered a potent BMP inhibitor capable of inhibiting bone function in vivo and in vitro. Noggin has a strong binding affinity for BMPs and can block BMPs from binding to BMP receptors. BMPs play important roles in many stages of bone development. Research has shown that BMP-2 plays a unique role in postnatal human bone formation. Human recombinant bone morphogenetic protein 2 (rhBMP2) is a potent osteogenic growth factor approved by the U.S. Food and Drug Administration and is widely used in bone tissue engineering. Therefore, discovering natural products with BMP-2-mimetic effects that promote bone formation is of great importance. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides compositions and uses thereof for improving skeletal formation, mass and / or strength and / or treating or preventing bone loss in a subject in need thereof. [Means for solving the problem]

[0008] In one aspect, the present invention provides a method for improving bone formation, bone mass, and / or bone strength. The method may be a method for treating or preventing bone loss, such as age-related bone loss, in a subject in need thereof. According to some embodiments, the method comprises administering to a subject in need thereof an effective amount of a composition comprising a homopolysaccharide, wherein the composition comprises a homopolysaccharide or a derivative thereof. The homopolysaccharide is composed primarily of arabinose, galactose, rhamnose, and galacturonic acid as monomer units.

[0009] In some embodiments, the subject is a mammal, preferably a human subject, and may be a healthy human, an elderly adult, or an adult suffering from bone loss.

[0010] The composition may be a pharmaceutical composition, a functional composition, and / or a supplement. In certain embodiments, the composition is administered orally or by gavage. The composition may be in the form of a tablet or a liquid. For example, in certain embodiments, the composition is a pharmaceutical composition in the form of an orally administrable tablet. In certain embodiments, the composition may be a functional composition or in the form of a dry powder. The composition may also be formulated in the form of a sports drink or a snack bar.

[0011] The molecular weight of the homopolysaccharide may be in the range of about 10 kDa to about 150 kDa, for example, about 10 kDa to about 100 kDa, about 10 kDa to about 90 kDa, about 10 kDa to about 60 kDa, or any other suitable range. In the homopolysaccharide, the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units is in the range of 30-70:20-60:0.1-10:0.1-10.

[0012] For example, in the homopolysaccharide designated LBP1C-2 used in the examples of the present invention, the ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units is 49.9:33.6:8.0:8.5. The molecular weight may be approximately a specific value or may be in a narrow range of about 10 kDa to about 150 kDa. Each polysaccharide obtained is homogeneous, with a uniform or narrow molecular weight distribution.

[0013] In some embodiments, the homopolysaccharides described herein are the only polysaccharides in the composition.

[0014] In another embodiment, the composition further comprises other polysaccharides separated from wolfberry extract.Homopolysaccharides account for 15% or more of the total polysaccharide content in the composition.All polysaccharides are derived from wolfberry.For example, the polysaccharide used in the present invention is wolfberry polysaccharide (referred to as LBP).

[0015] In some embodiments, the composition optionally further comprises one or more of a flavonoid, a carotenoid, a polyphenol, a pigment, or any compound isolated from wolfberry extract.

[0016] In some embodiments, the compositions comprise chemically modified derivatives of the homopolysaccharides described herein. For example, such derivatives are pharmaceutically acceptable esters or salts thereof. The pharmaceutically acceptable esters or salts thereof are sulfate ester derivatives of the homopolysaccharides, referred to as sulfated polysaccharides.

[0017] The excipient may be a solvent, co-solvent, colorant, preservative, antimicrobial agent, filler, binder, disintegrant, lubricant, surfactant, emulsifier, suspending agent, or any combination of the above.

[0018] The composition may be administered in any suitable amount. For example, in some embodiments, the effective dose of the composition (expressed herein in terms of the amount of homopolysaccharide) is in the range of 10 mg / kg to 500 mg / kg, based on the total weight of homopolysaccharide per day / the subject's daily body weight. The composition may be administered once a day, twice a day, or more than twice a day.

[0019] In another aspect, the present invention provides a composition for improving bone formation, bone mass, and / or bone strength (as described herein), which can be used to treat or prevent bone loss (such as age-related bone loss) in a subject in need thereof. Such a composition comprises an effective dose of a homopolysaccharide or a derivative thereof and a pharmaceutically acceptable excipient. The homopolysaccharide is composed primarily of arabinose, galactose, rhamnose, and galacturonic acid as monomer units.

[0020] As described herein, the composition may be a pharmaceutical composition, a functional composition, and / or a supplement. For example, in certain embodiments, the composition is in the form of an oral composition and / or a tablet.

[0021] The excipient may be selected from a solvent, a co-solvent, a colorant, a preservative, an antimicrobial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination of the above.

[0022] The molecular weight of the homopolysaccharide is in the range of about 10 kDa to about 150 kDa, for example, about 10 kDa to about 100 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about 60 kDa, or any other suitable range. In the homopolysaccharide, the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units is in the range of 30-70:20-60:0.1-10:0.1-10.

[0023] For example, in certain embodiments, the homopolysaccharide has a ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units of 49.9:33.6:8.0:8.5. The molecular weight may be approximately a specific value or within a narrow range of about 10 kDa to about 150 kDa. Each resulting polysaccharide is homogeneous, with a uniform or narrow molecular weight distribution.

[0024] In some embodiments, the homopolysaccharides described herein are the only polysaccharides in the composition, in some other embodiments, the composition further comprises other polysaccharides isolated from wolfberry extract, and / or the homopolysaccharides account for 15% or more of the total polysaccharide content in the composition.

[0025] In some embodiments, the composition may optionally include flavonoids, carotenoids, polyphenols, pigments, or any combination thereof, isolated from wolfberry extract, hi some other embodiments, the composition does not include flavonoids, carotenoids, or polyphenols isolated from wolfberry extract.

[0026] In some embodiments, the compositions comprise chemically modified derivatives of the homopolysaccharides described herein. For example, such derivatives are pharmaceutically acceptable esters or salts thereof. The pharmaceutically acceptable esters or salts thereof are sulfate ester derivatives of the homopolysaccharides, referred to as sulfated polysaccharides.

[0027] The present invention also provides the use of a homopolysaccharide or derivative thereof as described herein in the manufacture of a medicament for the treatment of any of the disorders described herein.

[0028] In another aspect, the present invention provides a method for producing the composition or homopolysaccharide. Such a method may include producing or isolating the homopolysaccharide. The method may further include combining the homopolysaccharide with an excipient. The method may include chemical modification, such as sulfation, of the homopolysaccharide. [Brief explanation of the drawings]

[0029] The present invention is best understood by reading the following detailed description in conjunction with the drawings, in which: It is generally understood that the various features in the drawings are not necessarily drawn to scale; conversely, the dimensions of the various features have been arbitrarily increased or decreased for clarity. Like reference numerals refer to like features throughout the specification and drawings. [Figure 1] The trabecular bone structure of control mice and experimental mice administered with wolfberry water extract (LBE), wolfberry crude polysaccharides (LBP), and wolfberry homopolysaccharides (LBP1C-2) is shown. [Figure 2] The bone mineral density (BMD) values (Figure 2A), the ratio of trabecular volume to total bone volume (BV / TV) (Figure 2B), trabecular number (Tb.N) (Figure 2C), trabecular thickness (Tb.Th) (Figure 2D), and trabecular spacing (Tb.Sp) (Figure 2E) of mice in the control group and mice in the experimental groups administered LBE, LBP, and LBP1C-2, respectively, are shown. [Figure 3] The maximum load-bearing force a of mice in the control group and mice in the experimental groups administered with LBE, LBP, and LBP1C-2, respectively, is shown (the maximum load-bearing force of the femur was evaluated by a three-point bending test). [Figure 4] 1 shows the bone formation rates of control mice and experimental mice administered with LBE, LBP, and LBP1C-2, respectively. The abbreviation "BFR / BS" stands for bone formation rate per unit bone surface. [Figure 5] The bone mineralization rate (MAR) of control mice and experimental mice administered LBE, LBP, and LBP1C-2, respectively, is shown. [Figure 6] The contents of osteocalcin in the serum of mice in the control group and mice in the experimental groups administered with LBE, LBP, and LBP1C-2, respectively, are shown. [Figure 7] The content of type I procollagen N-terminal propeptide (PINP) in the serum of control mice and experimental mice administered with LBE, LBP, and LBP1C-2, respectively, is shown. [Figure 8] 1 shows the proliferation of osteoblasts in human mesenchymal stem cells (hMSCs) treated with LBE, LBP, LBP1C-2, and control hMSCs in a CFU (colony forming unit) experiment. [Figure 9] 1 shows the expression levels of osteocalcin in hMSCs treated with LBE, LBP, and LBP1C-2, as well as in control hMSCs. [Figure 10] 1 shows the enzyme activity of alkaline phosphatase (ALP) in hMSCs treated with LBE, LBP, and LBP1C-2, as well as in control hMSCs. [Figure 11] 1 shows the expression levels of genes related to bone formation in the femurs of control mice and experimental mice administered with LBP1C-2. [Figure 12] 1 shows the expression levels of genes related to bone resorption in the femurs of control mice and experimental mice administered with LBP1C-2. [Figure 13-15] 1 shows the expression levels of osteogenesis-related genes Runx2, Col1α, and Bglap in pre-osteoblasts treated with LBP1C-2 and the control group after BMPR1a and BMPR2 gene interference. [Figure 16-17] 1 shows the results of direct interactions between LBP1C-2 and BMPRIA and BMPII in a control group and an experimental group administered with LBP1C-2, as revealed by surface plasmon resonance (SPR) analysis. [Figure 18-19] 1 shows the results of the direct interaction of LBP1C-2 with BMPRIA and BMPRII measured using a fluorescence-based protein thermal shift method. [Figures 20A-20B]Figure 20 shows the phosphorylation levels of Smad1 / 5 / 8 in hMSCs treated with LBP1C-2 and control hMSCs. Figure 20A shows the phosphorylation levels of Smad1 / 5 / 8 and the expression levels of Smad1 / 5 / 8 and GAPDH. Figure 20B analyzes the effect of LBP1C-2 on the phosphorylation levels of Smad1 / 5 / 8 analyzed by Western blotting. GAPDH is used as a loading control. [Figure 21] 1 shows the results of the direct interaction between LBP1C-2 and Noggin measured by surface plasmon resonance (SPR). [Figure 22] 1 shows the release levels of bone morphogenetic protein 2 (BMP2) in hMSCs treated with LBP1C-2 and control hMSCs. [Figures 23A-23B] The levels of early bone formation markers RUNX2 (FIG. 23A) and SP7 (FIG. 23B) in hMSCs treated with LBP1C-2, S-LBP1C-2 (sulfated LBP1C-2), and control hMSCs are shown. [Figure 24A] 1 shows the results of alkaline phosphatase (ALP) staining of hMSCs treated with LBP1C-2, S-LBP1C-2, and control hMSCs (day 7). [Figure 24B] 1 shows ALP enzyme activity in hMSCs from LBP1C-2, S-LBP1C-2, and control groups. [Figure 25A] 1 shows the results of Alizarin Red S staining of hMSCs treated with LBP1C-2, S-LBP1C-2, and control hMSCs (day 21). [Figure 25B] 1 shows the expression levels of osteocalcin in hMSCs treated with LBP1C-2, S-LBP1C-2, and control hMSCs. DETAILED DESCRIPTION OF THE INVENTION

[0030] The range endpoints and any values disclosed herein are not intended to be limited to the precise ranges or values, and these ranges or values should be understood to include values that are close to these ranges or values. In the case of numerical ranges, the range endpoints, the range endpoints and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges. These numerical ranges should be considered to be specifically disclosed herein.

[0031] As used herein, the singular forms "a," "an," and "the" include the plural, and reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. Thus, for example, reference to an "additive" is a reference to one or more such compounds and equivalents known to those skilled in the art, and so forth.

[0032] When values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. As used herein, "about X" (where X is a numerical value) preferably means ±10% of the stated value, including the endpoints. For example, the phrase "about 8" preferably includes values from 7.2 to 8.8, including the endpoints.

[0033] As another example, the phrase "about 8%" preferably (but not always) means values between 7.2% and 8.8%. Where present, all ranges are inclusive and combinable. For example, if a range of "1 to 5" is listed, the listed range should be interpreted as including ranges such as "1 to 4," "1 to 3," "1 to 2," "1 to 2 and 4 to 5," "1 to 3 and 5," "2 to 5," etc. Also, where alternatives are expressly provided, such terms may be interpreted to mean that any alternative may be excluded, for example, by a negative limitation in the claims.

[0034] For example, if a range of "1 to 5" is recited, the recited range can be interpreted as including the negative exclusion of any of 1, 2, 3, 4, or 5. Thus, a recitation of "1 to 5" can be interpreted as "1 and 3 through 5, but not including 2," or simply "but not including 2."

[0035] It is intended that any component, element, attribute, or step explicitly recited herein may be expressly excluded in the claims, regardless of whether such component, element, attribute, or step is listed as an alternative or listed separately. Any component, element, attribute, or step actively recited herein may be expressly excluded in the claims, regardless of whether such component, element, attribute, or step is listed as an alternative or listed separately.

[0036] The terms "subject" and "patient" are used interchangeably herein. As used herein, the term "patient" refers to an animal, preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkeys and humans), preferably a human. In some embodiments, the subject is a non-human animal such as a livestock animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat). In certain embodiments, the subject is a human. In another embodiment, the subject is an adult.

[0037] As used herein, the term "drug" refers to any molecule, compound, method and / or substance for preventing, treating, managing and / or diagnosing a disease or condition.

[0038] As used herein, the term "effective dose" refers to a dose of one therapy that is sufficient to prevent the progression, recurrence or onset of a disease or condition and one or more symptoms thereof, enhance or improve the prophylactic efficacy of another therapy, reduce the severity of a disease or condition, reduce the duration of a disease or condition, alleviate one or more symptoms of a disease or condition, prevent the progression of a disease or condition, cause regression of a disease or condition, and / or enhance or improve the therapeutic efficacy of another therapy.

[0039] The disease or condition (eg, pathology) is associated with bone loss, or is associated with bone formation, bone mass, and / or bone strength.

[0040] As used herein, the phrase "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, and more particularly for use in humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other recognized pharmacopoeias.

[0041] As used herein, the term "therapeutic agent" refers to any molecule, compound and / or substance used in the treatment and / or management of a disease or condition.

[0042] As used herein, the terms "therapy" and "treatment" can refer to any method, composition and / or agent for preventing, treating and / or managing a disease or condition, or one or more symptoms thereof. In certain embodiments, the terms "therapy" and "treatment" refer to small molecule therapeutics.

[0043] As used herein, the terms "treatment," "method of treatment," and "treating" refer to the administration of a therapeutic regimen to a subject to reduce or inhibit the progression and / or duration of a disease or condition, reduce or ameliorate the severity of a disease or condition, such as cancer, and / or improve one or more symptoms as a result of the administration of one or more therapeutic regimens.

[0044] The term "excipient" as used herein refers to an inert substance that functions as a carrier or vehicle for a drug or other active substance. Examples of suitable excipients include, but are not limited to, solvents, cosolvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

[0045] As used herein, a "monomer unit" or "monomer units" in a polysaccharide refers to a monoglycosyl unit or single sugar molecule unit or other basic chemical unit. These units are understood to be groups of monosaccharides linked by -O- bonds via condensation reactions between hydroxyl groups.

[0046] Molecular weight is measured by gel permeation chromatography (GPC). GPC is an analytical technique that separates molecules within a polymer by molecular size and provides the molecular weight or molecular weight distribution of the material. The homopolysaccharides described herein contain only one peak in GPC and have a uniform molecular weight. Homopolysaccharides or derivatives thereof are provided and used in the present disclosure.

[0047] Because their molecular weight distributions are uniform (i.e., polydispersity index is 1 or close to 1), their weighted average molecular weights (Mw), number average molecular weights (Mn), or peak molecular weights (Mp) are the same, or approximately the same, or very close. Unless expressly stated otherwise, the molecular weight values and ranges set forth herein may be Mw or Mn. In some examples, the molecular weight values and ranges set forth herein are number average molecular weights (Mn).

[0048] Goji berries, used in traditional Chinese medicine and health foods in China and other countries, belong to the genus Lycium in the Solanaceae family. Goji berries are listed as congener species with medicinal and edible uses. Goji berries have anti-aging and anti-fatigue properties. They have been used to nourish the liver and kidneys and improve eyesight. However, to date, little is known about the molecular mechanisms and functions of goji berries, and their active ingredients remain unknown.

[0049] The inventors have discovered the function of goji berries in muscles. Goji berries contain a variety of components, including polysaccharides, flavonoids, betaine, taurine, vitamins, and fatty acids. The main active ingredient, goji berry polysaccharides, has biological activities such as antioxidant, antiviral, neuroprotective, acute liver damage prevention, and immunomodulatory effects.

[0050] In a recent study, we used a unique separation method to extract and separate aqueous extracts, i.e., wolfberry extract (LBE) and crude polysaccharides (LBP), from wolfberry fruit. A homopolysaccharide designated LBP1C-2 (e.g., yield in dried fruit is 0.02%) was purified from LBP. LBP1C-2 was found to be a pectin. However, it is unknown whether LBP1C-2 affects bone remodeling.

[0051] Wolfberry polysaccharide samples can promote osteoblast proliferation, differentiation, and mineralization. The polysaccharides are extracted from wolfberry fruit and contain six monosaccharides, including galactose, glucose, rhamnose, arabinose, mannose, and xylose. In vitro experiments were performed using human mesenchymal stem cells (hMSCs).

[0052] However, whether goji berry supplementation can improve age-related bone loss, its main chemical basis, mechanism of action, and target of action have yet to be clarified.

[0053] In this study, we investigated the effects of LBE, LBP, and LBP1C-2 on naturally aging mice and determined their target and mechanism of action. LBP1C-2, a homopolysaccharide obtained from wolfberry extract, was found to be the main active ingredient in wolfberry. LBP1C-2 directly binds to BMPRIA and BMPRII, promoting the phosphorylation of Smads and promoting osteogenic differentiation and mineralization. It also directly binds to Noggin, inhibiting the interaction between Noggin and BMPs, thereby increasing bone formation. This study also provides a research foundation for explaining the therapeutic effects of wolfberry extract compositions and provides scientific evidence for their future use as a natural dietary or supplement product to treat age-related bone loss.

[0054] The present invention provides compositions and methods for improving bone formation, bone mass, and / or bone strength, which can be used to treat or prevent bone loss, such as age-related bone loss, in a subject in need thereof.

[0055] According to some embodiments, such compositions comprise a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient, and are administered to a subject in need thereof. The homopolysaccharide is composed primarily of arabinose, galactose, rhamnose, and galacturonic acid as monomer units or molecules. Such methods comprise administering an effective amount of a composition comprising the homopolysaccharide to a subject in need thereof.

[0056] In some embodiments, the subject is a mammal, preferably a human subject, and may be a healthy human, an elderly adult, or an adult with osteoporosis.

[0057] The composition may be a pharmaceutical composition, a functional composition, and / or a supplement. In certain embodiments, the composition is administered orally or by gavage. The composition may be in the form of a tablet or a liquid. For example, in certain embodiments, the composition is an orally administrable pharmaceutical composition in the form of a tablet. In certain embodiments, the homopolysaccharide or its derivative is a therapeutic agent. In certain embodiments, the composition may be a functional composition or may be in the form of a dry powder. The composition may also be formulated in the form of a sports drink or a snack bar.

[0058] The molecular weight of the homopolysaccharide is in the range of about 10 kDa to about 150 kDa, for example, about 10 kDa to about 100 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about 60 kDa, or any other suitable range. In the homopolysaccharide, the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units is in the range of 30-70:20-60:0.1-10:0.1-10.

[0059] For example, in some embodiments, the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units in the homopolysaccharide is 49.9:33.6:8.0:8.5. The molecular weight may be a specific value or a narrow range ranging from about 10 kDa to about 150 kDa. Such molecular weight values may be weight-average molecular weight (Mw) or number-average molecular weight (Mn). Because polysaccharides are homogeneous and have very narrow and uniform molecular weight distributions, the Mw and Mn values are considered to be close. The polydispersity (PD) index, i.e., the ratio of Mw to Mn, may range from about 1 to about 1.3, about 1 to about 1.2, and about 1 to about 1.1.

[0060] In some embodiments, the PD index is close to 1. The molecular weight of polysaccharides may vary depending on factors such as the growing environment and harvest time of the wolfberry raw material. Therefore, the resulting homopolysaccharides may have different molecular weights. However, the polysaccharides obtained from each batch are homogeneous, meaning that their molecular weights are uniform or have a narrow distribution. The molecular weight can be controlled by controlling the quality of the raw material, such as by maintaining the same growing environment and growing time before harvest.

[0061] In some embodiments, the homopolysaccharides described herein are the only polysaccharides in the composition.

[0062] In some embodiments, the homopolysaccharide according to the present invention is LBP1C-2 or a derivative thereof, such as sulfated LBP1C-2.

[0063] In some embodiments, the composition further comprises other polysaccharides isolated from wolfberry extract in addition to LBP1C-2.The homopolysaccharides account for 15% or more of the total polysaccharide content in the composition.All polysaccharides can be derived from wolfberry.For example, the polysaccharide used in the present invention is wolfberry polysaccharide (referred to as LBP).

[0064] In some embodiments, the composition optionally further comprises one or more of a flavonoid, a carotenoid, a polyphenol, a pigment, or any compound isolated from wolfberry extract.

[0065] Wolfberry extract (LBE) can be obtained by extracting Wolfberry with water, and the extract may be in the form of a dry powder. The extract can also be dissolved in water and further purified or separated by fractional purification. Polysaccharides are obtained. These polysaccharides can be further separated by fractional purification and lyophilization to obtain one or more homopolysaccharides in the form of a dry powder.

[0066] In some embodiments, the composition comprises wolfberry extract (LBE). This wolfberry extract comprises polysaccharides (or wolfberry polysaccharides), wolfberry flavonoids, carotenoids, polyphenols, and wolfberry pigments. Each component may be present in only one form, or two or more of the same component may be present. For example, the composition may comprise two or more polysaccharides, two or more wolfberry flavonoids, two or more carotenoids, two or more polyphenols, and / or two or more wolfberry pigments.

[0067] In some embodiments, the polysaccharide content in the wolfberry extract (LBE) is in the range of about 10.0 wt.% to about 70.0 wt.% (e.g., about 50 wt.% to about 70 wt.%), wolfberry flavonoids in the range of about 0.1 wt.% to about 5.0 wt.%, carotenoids in the range of about 0.1 wt.% to about 3.0 wt.%, polyphenols in the range of about 0.1 wt.% to about 8.0 wt.%, and wolfberry pigments in the range of about 0.1 wt.% to about 8.0 wt.%, based on the total dry weight of the extract.

[0068] In some embodiments, the polysaccharides are more preferably about 50.0 wt.% to about 70.0 wt.%. The dry weight is the equivalent weight corresponding to the extract in dry powder form (without water). The extract may contain other trace residues. The extract in dry powder form can be mixed with water to provide the extract in the form of an aqueous solution having the selected concentration described herein.

[0069] In some embodiments, the wolfberry extract (LBE) is in powder form. In some embodiments, the wolfberry extract is dissolved in a solvent, such as water, to provide an aqueous solution having a concentration ranging from 0.1 g / mL to 5 g / mL. In the LBE used in the examples, the polysaccharide content in the dry powder form of the LBE ranges from about 50.0 wt.% to 70.0 wt.%.

[0070] Wolfberry polysaccharides, or polysaccharides (LBP), present in powder form can be further purified from the powdered LBE. For example, the LBE can be dissolved in water and separated through a separation column. Wolfberry polysaccharides (LBP) may contain various polysaccharides that can be further separated.

[0071] The homopolysaccharide LBP1C-2 was isolated from LBE and / or LBP. Based on high-performance gel permeation chromatography (HPGPC) analysis, LBP1C-2 showed a single symmetric peak, indicating its homopolysaccharide identity. Carbohydrate composition analysis revealed that LBP1C-2 is composed of arabinose (Ara), galactose (Gal), rhamnose (Rha), and galacturonic acid in a ratio of 49.9:33.6:8.0:8.5.

[0072] The structure of LBP1C-2 contains a main chain of alternating 1,2-linked α-Rhap and 1,4-linked α-GalpA, and branches of terminal (T)-, 1,3-, 1,6-, and 1,3,6-linked β-Galp, T-, 1,5-, and 1,3,5-linked α-Araf, and T-linked β-Rhap substituted at C-4 of 1,2,4-linked α-Rhap.

[0073] Formulas 1, 2, and 3 show the structure of LBP1C-2, showing the same structure in three different forms.

[0074] Referring to formulas 1-3, LBP1C-2 is composed of Ara, Gal, Rha, and GalA in a molar ratio of 49.9:33.6:8.0:8.5. Structural analysis revealed that LBP1C-2 has a main chain of 1,2-α-Rha and 1,4-α-GalA, with branches including T-α-Ara, 1,5-α-Ara, T-β-Rha, T-β-Gal, 1,3-β-Gal, 1,6-β-Gal, and 1,3,6-β-Gal attached to the C-4 position of the backbone sugar residue of 1,2,4-α-Rha.

[0075] The repeating unit of LBP1C-2 contains the structural moiety shown in Formula 3, which includes a backbone (consisting of 1,2-α-Rha, 1,2,4-α-Rha, and 1,4-α-GalA) and three types of branches including R1, R2, and R3.

[0076] TIFF2025525540000002.tif250170

[0077] TIFF2025525540000003.tif171170

[0078] TIFF2025525540000004.tif201170

[0079] In Equations 1-3, n ranges from 2 to 20. The molecular weight is proportional to the value of n. For example, if the molecular weight of the sample is approximately 13.2 kDa, n is approximately 2. If the molecular weight of the sample is approximately 99.8 kDa, n is approximately 13.

[0080] The present inventors have demonstrated that homopolysaccharide LBP1C-2 is a polysaccharide or active ingredient of LBE or LBP that has the function of improving bone loss, bone mass, and / or bone strength. Homopolysaccharide LBP1C-2 is an active ingredient that can be used to treat or prevent bone loss, such as age-related bone loss, in subjects in need thereof.

[0081] In some embodiments, the composition comprises a chemically modified derivative of a homopolysaccharide described herein. For example, the derivative is a pharmaceutically acceptable ester or salt thereof. The pharmaceutically acceptable ester or salt is a sulfate ester derivative of a homopolysaccharide, called a sulfated polysaccharide. During the synthesis of a sulfated homopolysaccharide, a hydroxyl group in the homopolysaccharide reacts with a modifying agent, such as chlorosulfonic acid, to form an —O—SOH group. The molecular weight after modification is similar to that of the homopolysaccharide described above.

[0082] The degree of sulfate substitution of sulfate ester derivatives of homopolysaccharides ranges from 0.5 to 0.9, e.g., 0.6 to 0.8. The degree of substitution indicates the number of substituents on the sugar unit. For example, a degree of substitution of 0.74 means that the number of sulfate substituents on each hexose or pentose unit is 0.74. Sulfate substitution can be combined with proteins to improve their biological activity.

[0083] The composition can be a pharmaceutical composition, a functional composition, and / or a supplement.For example, the composition can be an orally administrable pharmaceutical composition.In the present invention, the aqueous extract of wolfberry is administered by oral gavage or orally, but is not limited thereto.Any form of administration of the composition to the stomach can be suitable.

[0084] The excipient may be a solvent (such as water or an aqueous solvent), a co-solvent, a colorant, a preservative, an antimicrobial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination thereof.

[0085] The composition may be administered in any suitable amount. For example, in some embodiments, the effective daily amount of the composition (expressed herein in terms of the amount of the homopolysaccharide) is in the range of 10 mg / kg to 500 mg / kg based on the total weight of the homopolysaccharides per subject's body weight. In some embodiments, the dose of wolfberry extract (LBE) or LBP or LBP1C-2 is in the range of 4 mg / kg to 70 mg / kg per day (total daily dry weight of LBP1C-2 per subject's body weight, e.g., human), e.g., 10 mg / kg to 70 mg / kg, 10 mg / kg to 60 mg / kg, 20 mg / kg to 70 mg / kg, 20 mg / kg to 60 mg / kg, 20 mg / kg to 50 mg / kg, or other suitable ranges. The composition may be administered once daily, twice daily, or more than twice daily.

[0086] In some embodiments of the present invention, the dose of wolfberry extract (LBE), or LBP or LBP1C-2 is 40 mg / kg (total daily dry weight of LBP1C-2 / body weight of an animal, such as a mouse) per day. The total daily dose can be administered once or more than once per day. In an example of the present invention, mice are administered LBE, LBP, or LBP1C-2 once per day.

[0087] The dosage and administration instructions provided herein also apply to derivatives of the homopolysaccharide LBP1C-2.

[0088] In some embodiments, these compositions may be ingested together with drinks, foods, or related ingredients. The wolfberry water extract of the present invention is a medicine and food and can be used as a health food. There are no particular limitations on the method of manufacturing the food or health food. For example, it can be made into tablets, drinks, candies, etc. Each food preparation may contain, in addition to the homopolysaccharides, other ingredients commonly used in the art. These other ingredients can be selected by those skilled in the art according to the specific formulation or application.

[0089] In some embodiments, the composition is a food or health product, including sports drinks, protein powders, snack bars, and the like.

[0090] The present invention also provides the use of a homopolysaccharide or derivative thereof as described herein in the manufacture of a medicament for treating any of the disorders described herein.

[0091] In another aspect, the present invention provides a method for preparing the composition or homopolysaccharide. The method may include preparing or isolating the homopolysaccharide. The method may further include combining the homopolysaccharide with an excipient.

[0092] The features and effects of the present invention will be explained by way of examples and test examples, but the following examples and test examples are merely illustrative and do not limit the scope of the present invention.

[0093] Example 1. Material Preparation 1-1. Preparation of wolfberry extract The preparation process for wolfberry extract ("LBE") was as follows. Wolfberry fruits were sourced from Zhongning County, Yinchuan City, Ningxia Hui Autonomous Region, China. Dried wolfberry fruits were washed 3-5 times, then soaked in double-distilled water (pH = 7) at room temperature for 2 hours and crushed. The soaked wolfberry powder was added with 5-8 volumes of neutral water, mixed uniformly, and decoctioned twice at boiling point for 2.0 hours and 1.5 hours, respectively. The combined concentrated decoctions were filtered through a fiber membrane. The filtrates were combined and evaporated under vacuum at 30-55°C to remove water, yielding a concentrate. The resulting concentrate was freeze-dried into powder and stored in a desiccator for subsequent use at the appropriate concentration in experiments.

[0094] An exemplary wolfberry extract was used in the experiments. This exemplary extract was an aqueous extract solution with a concentration of 0.4 g / mL (dry powder or dry weight of extract / volume of extract). This concentration is for illustrative purposes only. The aqueous extract can be adjusted to any suitable concentration, for example, between about 0.1 g / mL and 5 g / mL (e.g., dry weight of extract / volume of extract). In certain embodiments, this aqueous extract can be diluted for use. The aqueous extract can be further diluted for cell experiments.

[0095] The wolfberry extract (LBE) used in the present invention primarily contains water-soluble wolfberry polysaccharides, wolfberry flavonoids, carotenoids, polyphenols, and pigments. In an exemplary LBE, the polysaccharide content, based on the total equivalent weight of the extract in dry powder form, ranges from about 50.0 wt.% to 70.0 wt.% (e.g., about 54% to 56% or 54%), the wolfberry flavonoid content ranges from about 0.1 wt.% to 5.0 wt.%, the carotenoid content ranges from about 0.1 wt.% to 3.0 wt.%, the polyphenol content ranges from about 0.1 wt.% to 8.0 wt.%, and the wolfberry pigment content ranges from about 0.1 wt.% to 8.0 wt.%. The LBE is in powder form and can be dissolved in water or salt water. An LBE of the same composition was used for comparison.

[0096] 1-2. Separation of wolfberry polysaccharides and homopolysaccharides Homopolysaccharides, such as the examples described herein labeled wolfberry polysaccharides ("LBP") and LBP1C-2, can be isolated from LBE or wolfberry fruit, for example, by the following exemplary method.

[0097] The polysaccharides were extracted as follows: Dried fruit was crushed, 15-30 times the volume of deionized water was added, and the mixture was mixed thoroughly. 3 wt.% cellulase, 1 wt.% amylase, and 0.5 wt.% papain were added, and the mixture was extracted at 55-60°C for 1 hour. The temperature was raised to inactivate the enzymes, and the mixture was then centrifuged. The resulting filtrate was concentrated, dialyzed, and concentrated again. Five times the volume of 95% ethanol was added, and the mixture was centrifuged to obtain a precipitate. This was then washed three times alternately with absolute ethanol and acetone, and dried in vacuo to obtain crude polysaccharides (LBP).

[0098] The above method further includes the following steps: The crude polysaccharide was dissolved in 10-15 times the volume of water, centrifuged, and the supernatant was collected and purified through a diethylaminoethylcellulose (DEAE) anion exchange column. Subsequently, it was eluted with water, 0.05M, 0.1M, and 0.2M sodium chloride, and the fraction eluted with 0.2M sodium chloride was collected, concentrated, dialyzed, and lyophilized to obtain pre-purified wolfberry polysaccharide (LBP1C). The resulting polysaccharide LBP1C was dissolved in 0.2M sodium chloride, centrifuged, and the supernatant was collected. It was then eluted through a Sephacryl-300 (RTM: poly((propenyldextran)-co-N,N'-methylenebisacrylamide)) column. The eluted fraction was collected, concentrated, dialyzed, and lyophilized to obtain the homopolysaccharide LBP1C-2.

[0099] LBP1C-2 has the composition and structure described herein.

[0100] 1-3. Molecular weight detection of LBP1C-2 The molecular weight and homogeneity of LBP1C-2 were measured by high-performance gel permeation chromatography (HP-GPC). The HP-GPC results showed a single symmetrical peak. Based on the dextran standards of known molecular weight used in the HP-GPC measurements, the weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity (PD) index were estimated to be 13,181 Da, 10,750 Da, and 1.22, respectively.

[0101] 1-4. Preparation of sulfated polysaccharide derivatives of LBP1C-2 and measurement of the degree of sulfate substitution (DS) The homopolysaccharide was sulfated according to the chlorosulfonic acid-pyridine method. LBP1C-2 (50 mg) was dissolved in 2.5 mL of dry formamide. 1.5 mL of sulfation reagent consisting of chlorosulfonic acid and pyridine (3:1, v / v) was added under ice bath conditions. The resulting mixture was then stirred at 40 °C for 4 hours, cooled, and neutralized with 5 M NaOH. The solution was first dialyzed against saturated NaHCO3 and then against distilled water. The residue was lyophilized to obtain the sulfated derivative S-LBP1C-2. The sulfation degree was calculated according to the chlorosulfonic acid-pyridine method using Equation (1) as follows: TIFF2025525540000005.tif11170

[0102] Here, DS is the degree of sulfate substitution, and %S is the sulfur content.

[0103] Based on the sulfur content (10%), the degree of sulfate substitution (DS) of S-LBP1C-2 was calculated to be 0.74 by reference to the standard curve of sulfate content detected by the chlorosulfonic acid-pyridine method.

[0104] 2. Biological Experiments 2-1. Animals and groups Healthy wild-type (WT) adult C57BL / 6J mice (male, 2 months old) and naturally aged 14-month-old male C57BL / 6J mice were housed in an SPF barrier facility. Mice were randomly divided into adult groups (15 mice per group) and aged groups (6 mice per group). The adult and aged mice were divided into four groups: control, LBE, LBP, and LBP1C-2. Control mice were gavaged with distilled water (10 mL / kg), and the three extracts were gavaged once daily for 4 months at doses of LBE (40 mg / kg), LBP (40 mg / kg), or LBP1C-2 (40 mg / kg). All animal experiments were conducted in accordance with the ARRIVE guidelines and the UK Animals (Scientific Procedures) Bill 1986 and related guidelines.

[0105] 2-2.Cell culture hMSCs were purchased from ScienCell Research Laboratories. The culture medium used in this study was α-minimum essential medium (α-MEM; Gibco, 12571063) supplemented with 10% fetal bovine serum (FBS; Gibco, 16140071) and 1% penicillin-streptomycin (HyClone, SH40003-12).

[0106] The medium used for differentiation culture was 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 at 37°C in a humidified atmosphere of 5% CO2.

[0107] 2-3. Detection of serum bone formation markers After drug treatment, 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). Osteocalcin and PINP levels were detected using the osteocalcin ELISA kit and PINP ELISA kit, respectively, following the experimental steps of the kit. Osteocalcin and PINP are typical bone formation markers.

[0108] 2-4. Bone analysis using micro-computed tomography CT scanning was performed using a micro-computed tomography (micro-CT) imaging system (Inveon MM system, Siemens, Munich, Germany), and trabecular morphology analysis was performed according to the protocol provided by the manufacturer. The trabecular parameters detected included bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp).

[0109] Briefly, right femur samples were scanned in vivo and ex vivo under the following conditions: effective pixel value of 8.89 μm, voltage of 60 kV, current of 220 μA, exposure time of 1500 ms, exposure of 1° per scan, and 360 exposures throughout. The entire image contained 1536 cross-sectional images, with an effective pixel value of 8.89 μm in all three axial directions. Three-dimensional (3D) visualization images were reconstructed using the 2D images, and parameter analysis was performed using Inveon Research Workplace (Siemens). The region of interest (ROI) for femoral trabecular bone analysis was 1–2 mm below the distal femoral growth plate.

[0110] 2-5. Three-point bending test Three-point bending tests were performed using a bone biomechanical testing device, a small animal bone strength testing machine (Instron 4302, Instron, Norwood, MA). Fresh femurs from each group of mice were harvested and their bone strength was measured immediately after harvest. The three-point bending test used two end support points and one central load point. Biomechanical measurement data were collected from the load-deformation curves.

[0111] 2-6. Surface plasmon resonance (SPR) analysis The binding affinity of LBP1C-2 to the proteins was measured using a BIACORE T200 (GE Healthcare, Stockholm, Sweden). BMPRIA, BMPRII, and Noggin proteins were immobilized on a CM5 sensor chip by amino coupling.

[0112] For interaction measurements, various concentrations of LBP1C-2 were injected into the chip. All runs were performed in HBS-EP running buffer (pH 7.4) containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20. Pharmacokinetic parameters were determined by fitting the data with a 1:1 binding model using Biacore T200 Evaluation Software version 1.0.

[0113] 2-7. Cellular thermal shift assay (CETSA) CETSA experiments were performed according to a previously described protocol (Lie et al., 2017). hMSCs were cultured for 96 hours in control or 4 μM LBP1C-2-containing medium. Cells were then harvested, lysed in RIPA lysis buffer, frozen and thawed three times in liquid nitrogen, and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant from the control group was divided into two portions.

[0114] A portion of the control group was treated with ddH2O2 for 30 minutes, while the other portion of the control group and the 8 μM LBP1C-2-treated group were all treated with control solution for 30 minutes. Soluble proteins were collected by centrifugation at 12,000 g for 15 minutes at 4°C and detected by Western blotting.

[0115] 2-8. Quantitative real-time PCR (RT-PCR) experiments Femoral tissue or 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 in rats from each treatment group were assessed using the 2-ΔΔCq method.

[0116] 2-9. Western blot analysis Femoral tissues or cells were prepared for analysis and lysed using RIPA buffer, which contains 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 5 mM EDTA, and protease inhibitor cocktail solution (Roche).

[0117] Next, 40 mg of protein from each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to an NC membrane, which was then blocked for 1 hour in TBST solution (TBST: 10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4) containing 5% nonfat dry milk.

[0118] After the blocking step, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used herein included anti-p-Smad1 / 5 / 8 (1:1000), anti-Smad1 / 5 / 8 (1:1000), and anti-GAPDH (1:2000). The membranes were then washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000 dilution, Zhongshan Jinqiao Co., Ltd., Beijing, China) at room temperature for 1 hour. The membranes were washed three times with TBST. To visualize the protein signals, each membrane was treated with ECL solution (Thermo Scientific, Waltham, USA), and the signals were detected using a molecular imaging system, ChemiDoc XRS+ (Bio-Rad).

[0119] 2-10. ALP staining and ALP assay ALP staining was monitored using a Vector Blue substrate kit (program number SK-5300, Vector Laboratories). hMSCs were plated at 1 × 10 per well in a 24-well plate according to the protocol. 5 The cells were seeded at a density of 1000 μg / ml and cultured in growth medium for 3 days. On day 7, the hMSCs were incubated with the substrate working solution for 30 minutes. Throughout this process, the cells were protected from light. The ALP assay was performed according to the steps previously described.

[0120] 2-11. Alizarin Red S staining hMSCs were plated in a 24-well plate at 1 x 10 per well. 5 The cells were seeded at a density of 100 μg / ml and cultured in growth medium for 3 days. Osteoblast differentiation was induced by culturing in differentiation medium. On day 14 after osteoblast differentiation induction, the cells were fixed with 4% paraformaldehyde for 15 minutes, treated with 0.1% Triton X-100 for 10 minutes, and then stained with 2% (w / v) Alizarin Red S solution (pH 4.2) for 10 minutes.

[0121] 3.Results In the following experiments, LBE, LBP, and LBP1C-2 solutions were prepared by dissolving the powders in distilled water and administered once daily. Control mice were administered distilled water by oral gavage, while treatment groups received LBE (40 mg / kg), LBP (40 mg / kg), or LBP1C-2 (40 mg / kg) by oral gavage once daily for 4 months. The effective dose of LBP1C-2 (based on the amount of homopolysaccharide described herein) was 40 mg / kg, calculated based on the total weight of homopolysaccharide divided by subject body weight per day. The dose of LBE and LBP (40 mg / kg) was calculated as the total dry weight of LBE or LBP divided by subject body weight.

[0122] 3-1. Increased bone mass and stability with LBE, LBP, and LBP1C-2 treatment Aging is significantly associated with bone loss and is related to the deterioration of bone microarchitecture. Therefore, we investigated whether LBE, LBP, and LBP1C-2 affected trabecular bone structural parameters in mice using micro-computed tomography (μCT). Bone histomorphological parameters of femurs from treated and control mice were quantified using μCT. LBE, LBP, and LBP1C-2 treatment improved bone histomorphology compared with the adult and aged control groups (Figure 1).

[0123] Compared with the adult and aged control groups, LBE, LBP, and LBP1C-2 treatment ameliorated age-related bone loss, as mice treated with LBE and LBP exhibited higher BMD (Figure 2A), higher bone volume fraction (BV / TV) (Figure 2B), greater trabecular number (Tb.N) (Figure 2C), greater trabecular thickness (Tb.Th) (Figure 2D), and smaller trabecular spacing (Tb.Sp) (Figure 2E). *p<0.05, **p<0.01 (compared to the adult control group); #p<0.05, ##p<0.01 (compared to the aged control group).

[0124] We next investigated whether LBE, LBP, and LBP1C-2 could regulate bone biomechanical properties. Experimental results showed that the maximum load force of mice treated with LBE, LBP, and LBP1C-2 was significantly increased compared to the adult and aged control groups (Figure 3).

[0125] 3-2. Promotion of bone formation by LBE, LBP, and LBP1C-2 treatment The bone formation rate and bone mineralization rate of femoral bones in vivo were analyzed. The bone formation rate (BFR) indicates the amount of new bone formed per unit time. It is typically measured by quantifying the amount of mineralized bone matrix or osteogenic matrix (non-mineralized bone) formed within a specific bone surface area. The bone mineralization rate (MAR) specifically focuses on the mineralization rate of newly formed bone matrix.

[0126] This measures the rate at which mineral crystals, primarily hydroxyapatite, are deposited on the surface of the osteogenic matrix during bone formation. The bone formation rate (BFR, Figure 4) and bone mineralization rate (MAR, Figure 5) of mice treated with LBE, LBP, and LBP1C-2 were significantly improved, as measured by the calcein-alizarin red fluorescent double labeling method. Serum levels of the bone formation markers osteocalcin (Figure 6) and P1NP (a bone formation marker, Figure 7) were significantly increased compared to the control group.

[0127] The effect of LBP1C-2 was even more pronounced. As mentioned above, these results indicate that supplementation with LBE, LBP, and LBP1C-2 can regulate bone mass by promoting bone formation and osteoblast mineralization. *p<0.05, **p<0.01 (compared to adult control group); #p<0.05, ##p<0.01 (compared to aged control group).

[0128] 3-3. Promotion of osteoblast proliferation, differentiation, and mineralization by LBE, LBP, and LBP1C-2 To gain further understanding of the specific compounds mentioned above, we investigated whether LBE, LBP, and LBP1C-2 affect the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts. hMSCs were utilized to analyze osteoblast proliferation, differentiation, and mineralization. The results showed that LBE, LBP, and LBP1C-2 significantly promoted the formation of CFU-F (colony-forming unit-of-fibroblast) colonies and enhanced the proliferation of hMSCs (Figure 8).

[0129] Similarly, LBE, LBP, and LBP1C-2 significantly promoted osteoblast differentiation and mineralization. Furthermore, two osteoblast differentiation markers, Bglap expression levels (Figure 9) and ALP enzyme activity (Figure 10), were significantly increased in cells treated with LBE, LBP, and LBP1C-2. All these results indicate that treatment with LBE, LBP, and LBP1C-2 promoted osteoblast differentiation and mineralization of hMSCs.

[0130] 3-4. LBP1C-2 promotes bone formation and has little effect on bone resorption. To elucidate the mechanism of action of LBP1C-2, we analyzed the expression of bone formation and bone resorption genes by RT-PCR. In cells treated with LBP1C-2, the mRNA expression of osteogenic differentiation markers, such as Col1α, Runx2, Bglap, and Opn, was significantly upregulated (Figure 11). To further confirm the effect of LBP1C-2 on bone resorption, we measured the mRNA expression of osteoclast markers, including Ocstamp, Oscar, Opg, and Rankl, in femurs. No significant differences were observed (Figure 12).

[0131] These results indicate that LBP1C-2 promotes bone formation without affecting bone resorption.

[0132] 3-5. LBP1C-2 promotes BMPRIA- and BMPRII-dependent bone formation Because BMPR is a key receptor for osteogenic differentiation, we investigated whether its expression was associated with LBP1C-2-induced osteogenic differentiation. Various doses of BMPR-siRNA were used to confirm the specific inhibitory effect on BMPRIA and BMPRII. Quantitative real-time PCR analysis showed that the expression levels of downstream genes Col1α, Runx2, and Bglap were significantly increased.

[0133] Furthermore, silencing of BMPRIA and BMPRII resulted in downregulation of Runx2 (Figure 13), Col1α (Figure 14), and Bglap (Figure 15), indicating that the osteogenic differentiation and mineralization induced by LBP1C-2 treatment were inhibited by downregulation of BMPRIA and BMPRII. Therefore, the results indicate that LBP1C-2 is dependent on BMPRIA and BMPRII for osteogenic differentiation and mineralization.

[0134] Direct binding of LBP1C-2 to BMPRIA and BMPRII and their massive activation To investigate whether LBP1C-2 directly interacts with BMPR and to address this issue, we measured their interaction using SPR. The results showed that this interaction was dose-dependent. The equilibrium dissociation constant (Kd) of LBP1C-2 and BMPRIA was approximately 3.6 μmol / L (Figure 16), and the Kd of BMPRII was 5.6 μmol / L (Figure 17).

[0135] The direct interaction between LBP1C-2 and BMPR was further confirmed by a fluorescence-based protein thermal shift assay. LBP1C-2 dose-dependently increased the melting temperature (Tm) of BMPRIA (Figure 18) and BMPRII (Figure 19) by more than 4°C before reaching a plateau. The BMP / Smad signaling pathway is a key pathway regulating the differentiation of BMSCs into osteoblasts.

[0136] To verify whether LBP1C-2 in hMSCs regulates osteogenic differentiation of hMSCs via the BMP / Smad signaling pathway, we measured the phosphorylation levels of Smad1 / 5 / 8 in hMSCs 14 days after osteoblast differentiation. Western blot and quantitative analysis showed that LBP1C-2 treatment significantly increased the phosphorylation levels of Smad1 / 5 / 8 in hMSCs (Figures 20A-20B).

[0137] These data indicate that LBP1C-2 can directly bind to and activate BMPRIA and BMPRII, significantly increasing the phosphorylation levels of downstream Smad1 / 5 / 8. Thus, LBP1C-2 upregulated bone formation by binding to BMPRIA and BMPRII and regulating the BMP / Smad signaling pathway.

[0138] Blockade of the interaction between Noggin and BMP by LBP1C-2 LBP1C-2 can directly activate BMPRIA and BMPRII, and activation of BMPRIA and BMPRII ultimately promotes the phosphorylation of downstream effectors Smad1 / 5 / 8, thereby promoting osteogenic differentiation and mineralization. Noggin is known to play a role in inhibiting the BMP signaling pathway. Because the interaction of Noggin with BMP inhibits osteogenic differentiation, we investigated whether LBP1C-2 activates this signaling pathway by binding to Noggin.

[0139] As shown in Figure 21, the effect of LBP1C-2 on Noggin was detected after treatment, and the interaction between LBP1C-2 and Noggin was measured by SPR analysis. The results showed that LBP1C-2 has a strong binding affinity with Noggin, with an equilibrium dissociation constant (Kd) of 0.313 μM. To further investigate whether this binding inhibits the interaction between Noggin and BMP2, the BMP2 concentration in the supernatant of hMSCs treated with LBP1C-2 was measured.

[0140] The results showed that a certain amount of BMP2 was detected in the cell supernatant under Noggin conditions, whereas a large amount of BMP2 was detected in the cell supernatant under LBP1C-2 conditions. The upregulation of BMP-2 levels induced by LBP1C-2 was also inhibited by Noggin. These data indicate that LBP1C-2 interacts with Noggin and inhibits the binding of Noggin to BMP2 (Figure 22).

[0141] Age-related bone loss occurs equally in men and women, and osteoporotic fractures are a major cause of morbidity and mortality in the elderly. In this study, we demonstrated for the first time the bone-protecting effects of wolfberry in a natural aging model. The LBE, LBD, and LBP1C-2 supplements described herein effectively ameliorated age-related osteoporosis in mice by improving overall bone microarchitecture. Furthermore, LBP1C-2 was found to be the basis for the main active ingredient.

[0142] Mechanistic studies have revealed that LBE, LBD, and LBP1C-2 regulate bone formation, not bone resorption. LBP1C-2, the main component of wolfberry, can directly bind to BMPRIA and BMPRII, promoting the phosphorylation of downstream effector Smads and promoting osteogenic differentiation and mineralization. Meanwhile, LBP1C-2 can also directly bind to Noggin and inhibit the interaction between Noggin and BMPs. This binding further upregulates the BMP / Smad signaling pathway, increasing bone formation.

[0143] These results indicate that the compositions described herein promote bone formation similarly to BMPs and can be used as supplements or pharmaceutical compositions for treating or preventing age-related bone loss.

[0144] Although the experimental period is relatively long, the natural aging animal model is currently the most suitable animal model for simulating age-related bone loss, and our study of goji berries in the natural aging mouse model has clear advantages. The natural supplementation method of LBE, LBP, and LBP1C-2 described in this invention effectively improved age-related bone loss in adult and elderly mice.

[0145] Silencing of BMPR resulted in downregulation of osteogenic markers Col1α1, Runx2, and Bglap in preosteoblasts treated with LBP1C-2. We further examined whether LBP1C-2 directly interacts with BMPR using SPR, protein thermal shift assays, and CETSA.

[0146] We found that LBP1C-2 directly binds to BMPRIA and BMPRII and significantly enhances the phosphorylation of downstream effectors Smad1 / 5 / 8. Furthermore, we discovered for the first time that LBP1C-2 not only binds to BMPRIA and BMPRII, but also binds more strongly to Noggin, blocking the interaction between Noggin and BMP. These interactions allow LBP1C-2 to promote osteogenic differentiation, indicating that LBP1C-2 mimics the BMPR ligand and regulates osteogenic differentiation via the BMP / Smad signaling pathway.

[0147] There are currently no drugs specifically designed to treat senile osteoporosis, and existing osteoporosis medications have certain limitations, with the effectiveness of long-term use unknown. Clinically, osteoporosis treatment options are primarily divided into the following strategies: Antiresorptive drugs such as denosumab, odanacatib, and saracatinib are commonly used to treat osteoporosis, but inhibiting bone resorption can also impede bone remodeling and potentially inhibit bone formation. These osteoporosis medications often cause serious side effects, such as osteonecrosis of the jaw and atypical fractures.

[0148] Teriparatide, a recombinant parathyroid hormone, is currently the primary drug for promoting bone formation in the treatment of osteoporosis, administered by daily subcutaneous injection at appropriate doses. Against this background, natural products may be an excellent alternative or treatment for osteoporosis due to their minimal side effects. Wolfberry is used as a traditional Chinese medicine and health food to treat aging-related diseases.

[0149] In this study, we demonstrated that LBP1C-2, a homopolysaccharide extracted from wolfberry, significantly increased bone mass and strength in adult and elderly mice after long-term administration (4 months in mice), without any adverse reactions. Therefore, LBP1C-2 may be a potentially effective and safe compound suitable for the development of anti-osteoporosis drugs in both pathological and non-pathological conditions.

[0150] As described above, the present study demonstrates that LBP1C-2, a homopolysaccharide derived from wolfberry, can be used to treat or prevent age-related bone loss. Osteoporosis is an emerging medical and socioeconomic threat due to its high morbidity, disability, and mortality rates. Wolfberry, a traditional Chinese medicine and food, has many health benefits. However, it remains unclear whether wolfberry can improve age-related bone loss and what its main active components are.

[0151] In the present invention, research results have shown that three wolfberry extracts, namely, wolfberry aqueous extract (LBE), wolfberry polysaccharide (LBP, isolated from LBE), and homopolysaccharide LBP1C-2 (isolated from LBP), are effective in treating or preventing bone loss.

[0152] Supplementation with LBE, LBP, and LBP1C-2 significantly increased bone mass and strength in adult and aged mice. LBE, LBP, and LBP1C-2 also promoted the proliferation, differentiation, and mineralization of osteoblasts in hMSCs and in treated mice (in vivo). Furthermore, mechanistically, the main active ingredient, LBP1C-2, directly binds to the BMP receptors BMPRIA and BMPRII, and activation of BMPRIA and BMPRII leads to phosphorylation of downstream effector Smads, promoting osteogenic differentiation and mineralization.

[0153] On the other hand, Noggin is a potent inhibitor of BMP, and LBP1C-2 also directly binds to Noggin, inhibiting the interaction between Noggin and BMP. This binding further upregulates the BMP / Smad signaling pathway and promotes bone formation. These results suggest that wolfberry extract can prevent age-related bone loss, and that its main polysaccharide component, LBP1C-2, ameliorates age-related bone loss through BMPRIA, BMPRII, and Noggin in the BMP / Smad signaling pathway. This study also provides a basis for further research into LBE, LBP, and LBP1C-2 as functional foods for intervention in senile osteoporosis.

[0154] 3-8. Results of sulfated derivative of LBP1C-2 (S-LBP1C-2) A sulfated derivative of LBP1C-2 was prepared according to the method described above and is called S-LBP1C-2. To investigate the ability of sulfated LBP1C-2 (S-LBP1C-2) to promote osteoblast differentiation and mineralization, we used human mesenchymal stem cells (hMSCs) to detect the effect of S-LBP1C-2 on the expression of early osteogenic markers involved in bone formation. RUNX2 and SP7 (also known as Osterix) are two transcription factors that play important roles in bone formation and osteoblast differentiation. RUNX2 is thought to be the master regulator of osteoblast differentiation.

[0155] RUNX2 is essential for the differentiation of mesenchymal stem cells into osteoblasts and their subsequent osteoblast maturation. RUNX2 regulates the expression of multiple genes related to osteoblast differentiation, extracellular matrix formation, and mineralization. Meanwhile, SP7 functions downstream of RUNX2 and is considered a marker of mature osteoblasts. It is a transcription factor primarily expressed in osteoblasts and is involved in osteoblast differentiation and function.

[0156] First, we examined the levels of key osteogenic markers RUNX2 and SP7. qPCR detection showed that S-LBP1C-2 enhanced the mRNA levels of RUNX2 and SP7 after 7 days of treatment with hMSCs (Figures 23A and 23B). These experimental results indicated that S-LBP1C-2 has the ability to promote osteogenic differentiation.

[0157] Furthermore, at equivalent concentrations of 4 μmol, LBP1C-2 upregulated RUNX2 by 2.13-fold, whereas S-LBP1C-2 upregulated RUNX2 by 3.01-fold. Similarly, LBP1C-2 upregulated SP7 by 1.68-fold, whereas S-LBP1C-2 upregulated SP7 by 2.01-fold. Therefore, the effect of S-LBP1C-2 was superior to that of LBP1C-2.

[0158] BALP staining, also known as alkaline phosphatase staining, is a technique used to evaluate the enzymatic activity of alkaline phosphatase (ALP) during osteoblast differentiation. ALP is a major enzyme expressed in osteoblasts and is involved in various aspects of bone formation. During osteoblast differentiation, ALP expression and activity increase. ALP staining can be used to observe and quantify ALP activity, providing an indirect method for assessing osteoblast differentiation and maturation.

[0159] Next, we evaluated the effects of S-LBP1C-2 on ALP staining (Figure 24A) and ALP activity (Figure 24B). We observed that S-LBP1C-2 enhanced ALP secretion and activity. The promotion of ALP secretion and activity by S-LBP1C-2 exceeded that by LBP1C-2, indicating that S-LBP1C-2 has a stronger ability to promote osteoblast differentiation. Alizarin Red S staining is a widely used technique for detecting and quantifying calcium mineralization in bone and mineralized tissues.

[0160] This is typically used to evaluate the degree of mineralized matrix deposition during bone formation by osteoblasts. Furthermore, Alizarin Red S staining of S-LBP1C-2 (Figure 25A) and the expression levels of osteocalcin (BGLAP) (Figure 25B) in hMSCs were detected. Alizarin Red S staining results indicated that S-LBP1C-2 promotes bone formation and mineralization. BGLAP, also known as osteocalcin, is a protein that plays an important role in bone formation and mineralization. It is synthesized and secreted primarily by osteoblasts, the cells responsible for bone formation. BGLAP is considered a marker of osteoblast activity.

[0161] Furthermore, S-LBP1C-2 increased the mRNA levels of BGLAP, the gene encoding osteocalcin, after 21 days of hMSC treatment. Similar to previous findings, at an equivalent concentration of 4 μmol, LBP1C-2 was observed to upregulate BGLAP by 2.21-fold, whereas S-LBP1C-2 upregulated BGLAP by 2.51-fold, indicating that S-LBP1C-2 had a greater effect on BGLAP expression than LBP1C-2.

[0162] 4. Experimental and Control Samples Four homopolysaccharides were isolated from wolfberry extract. Of these, only LBP1C-2 exhibited the functions described here. The sugar composition and structure of three of the four homopolysaccharides, including LBP1A1-1, LBP1B-S-2, and LBP1C-2, were analyzed.

[0163] Referring to Formula 4, LBP1A1-1 is composed of rhamnose (Rha), arabinose (Ara), glucose (Glc), and galactose (Gal) in a molar ratio of 1.2:47.8:1.4:49.8. Structural analysis has shown that LBP1A1-1 is primarily composed of 1,4-α-Glc, 1,3-β-Gal, and 1,6-β-Gal. Its branches primarily include terminal (T)-β-Rha, T-β-Gal, T-α-Ara, T-β-Ara, and 1,5-α-Ara. These branches are connected to the C-6 position of the main chain sugar 1,3-β-Gal residue and the C-3 position of the 1,6-β-Gal residue.

[0164] TIFF2025525540000006.tif220170

[0165] Referring to formula 5, LBP1B-S-2 is composed of Rha, GlcA (glucuronic acid), Gal, and Ara in a molar ratio of 3.13:3.95:39.37:53.55. Structural analysis revealed that LBP1B-S-2 is primarily composed of 1,3-β-Gal and 1,6-β-Gal, with branches primarily containing 1,4-β-GlcA, T-β-Rha, T-β-Gal, T-α-Ara, T-β-Ara, 1,5-α-Ara, and a portion of 1,6-β-Gal. These branches are connected to the C-6 position of the main chain sugar residue 1,3-β-Gal and the C-3 position of the main chain sugar residue 1,6-β-Gal.

[0166] TIFF2025525540000007.tif199170

[0167] Among the four structurally distinct homopolysaccharides, only LBP1C-2 showed the most potent function and activity. In some embodiments, LBP1C-2, its derivatives, such as sulfate derivatives, and compositions comprising LBP1C-2 or its derivatives are preferred compositions for improving bone formation, bone mass, and / or bone strength in subjects in need thereof, and for treating or preventing bone loss, such as senile osteoporosis.

[0168] While the present invention has been described through exemplary embodiments, it is not intended to be limited thereto, but rather the appended claims should be interpreted broadly to include other variations and embodiments that may occur to those skilled in the art.

Claims

1. 1. A method for treating or preventing bone loss and / or improving bone formation, bone mass or bone strength in a subject in need thereof, comprising: administering to a subject in need thereof an effective dose of a composition comprising a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient; The homopolysaccharides are composed primarily of arabinose, galactose, rhamnose, and galacturonic acid as monomer units.

2. The method of claim 1 , wherein the subject is a mammal.

3. 10. The method of claim 1, wherein the subject is a human subject and / or the bone loss is age-related bone loss.

4. The method of claim 1 , wherein the composition is administered orally.

5. 2. The method of claim 1, wherein the molecular weight of the homopolysaccharide or derivative thereof is in the range of about 10 kDa to about 150 kDa, and / or the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units in the homopolysaccharide is in the range of 30-70:20-60:0.1-10:0.1-10.

6. 2. The method of claim 1, wherein the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units in the homopolysaccharide is 49.9:33.6:8.0:8.

5.

7. 2. The method of claim 1, wherein the composition further comprises other polysaccharides isolated from wolfberry extract, and / or the homopolysaccharides account for 15% or more of the total polysaccharides in the composition.

8. 10. The method of claim 1, wherein the composition further comprises one or more of a flavonoid, a carotenoid, a polyphenol, a pigment, or any compound isolated from wolfberry extract.

9. The method of claim 1 , wherein the homopolysaccharide is the only polysaccharide in the composition.

10. 10. The method of claim 1, wherein the pharmaceutically acceptable ester or salt is a sulfate ester derivative of the homopolysaccharide.

11. 10. The method of claim 1, wherein the excipient is selected from a solvent, a co-solvent, a colorant, a preservative, an antimicrobial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination thereof.

12. 2. The method of claim 1, wherein the effective daily amount of the composition is 10 mg / kg to 500 mg / kg based on the total weight of the homopolysaccharide or its pharmaceutically acceptable ester or salt per subject's body weight.

13. 1. A composition for improving bone formation, bone mass or bone strength, and / or treating or preventing bone loss in a subject in need thereof, comprising: an effective amount of a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination of the above, and a pharmaceutically acceptable excipient; Homopolysaccharides are composed primarily of arabinose, galactose, rhamnose, and galacturonic acid as monomer units.

14. The composition of claim 13, wherein the pharmaceutically acceptable ester or salt is a sulfate ester derivative of the homopolysaccharide.

15. The composition according to claim 14, wherein the degree of substitution of the sulfate group in the sulfate ester derivative of the homopolysaccharide is in the range of 0.5 to 0.

9.

16. The composition of claim 13, wherein the composition is a pharmaceutical composition, a functional composition and / or a supplement.

17. The composition of claim 13, wherein the composition is an oral composition and / or is in the form of a tablet.

18. 14. The composition of claim 13, wherein the excipient is selected from a solvent, a co-solvent, a colorant, a preservative, an antimicrobial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination thereof.

19. the molecular weight of the homopolysaccharide or derivative thereof is in the range of about 10 kDa to about 150 kDa; 14. The composition of claim 13, wherein the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units is in the range of 30-70:20-60:0.1-10:0.1-10.

20. 14. The composition of claim 13, wherein the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units in the homopolysaccharide is 49.9:33.6:8.0:8.

5.

21. 14. The composition of claim 13, wherein the homopolysaccharide is the only polysaccharide in the composition.

22. 14. The composition of claim 13, which is a pharmaceutical composition for treating age-related bone loss in humans.

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