Compositions comprising homogeneous polysaccharides or their derivatives and methods thereof for preventing and / or treating bone loss

Abstract

This invention provides a composition and method for improving bone formation, bone mass, and / or strength in subjects requiring this therapy, and / or treating or preventing bone loss, such as age-related bone loss. The composition comprises a homogeneous polysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient. The homogeneous polysaccharide is primarily composed of arabinose, galactose, rhamnose, and galacturonic acid as monomeric units. The method involves administering an effective dose of this composition to the subject in need.

Description

[0001] Priority and cross-reference

[0002] This application claims the benefit of U.S. Patent Application 63 / 389,082, filed July 14, 2022, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a composition having pharmaceutical or functional properties. Specifically, the disclosed subject matter relates to wolfberry extract, resulting compositions containing polysaccharides or derivatives thereof, and methods of using them as, for example, pharmaceutical compositions, functional compositions, and / or dietary supplements. Background Technology

[0004] A recent study on the epidemiology of osteoporosis in the European Union found that the prevalence of osteoporosis was 47% in women aged 50-55 and 80 and over. For EU men in the same age groups, the prevalence was 2.5% and 16%, respectively. Bone loss is not limited to postmenopausal women; increasing attention is also being paid to osteoporosis in older men, and this number is expected to steadily increase as the population ages. Studies have shown that among people over 50, one-third of women and one-fifth of men experience a rapidly increasing risk of fractures and mortality due to bone loss. Currently, more than 200 million people worldwide suffer from osteoporosis. Osteoporosis is characterized by decreased bone mass and bone density, along with deterioration of bone microstructure. Therefore, osteoporosis in the elderly has become an emerging medical and socioeconomic threat due to its high prevalence, high disability rate, and high mortality rate.

[0005] Osteoporosis in the elderly is primarily associated with decreased bone formation and reduced 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 with type I and type II transmembrane serine / threonine receptor kinases. Following ligand-induced assembly of the two type I and two type II receptors, the resident active type II receptor kinase phosphorylates and activates the type I receptor. The type I receptor is further phosphorylated, leading to signal transduction via Smads or MAPKs, which in turn activates the transcription of specific target genes involved in osteogenic differentiation and bone formation. The head protein noggin is widely considered a potent inhibitor of BMPs, inhibiting osteogenic function in vivo and in vitro. Noggin has a strong affinity for BMPs, preventing BMPs from binding to BMP receptors. BMPs play important roles in many steps of bone development. Studies have shown that BMP-2 plays a unique role in bone formation after birth in humans. Human recombinant bone morphogenetic protein-2 (rhBMP2), a potent osteogenic growth factor approved by the U.S. Food and Drug Administration, has been widely used in bone tissue engineering. Therefore, the discovery of natural products that mimic the bone-forming effects of BMP-2 is of great significance. Summary of the Invention

[0006] The present invention provides a composition, and uses the composition to improve the formation, quality and / or strength of bone in subjects in need, and / or to treat or prevent bone loss.

[0007] On one hand, the present invention provides a method for improving bone formation, bone mass, and / or bone strength. This method can be a treatment or prevention of bone loss in a subject in need, such as age-related bone loss. According to some embodiments, the method includes administering to the subject in need an effective dose of a composition comprising a homogeneous polysaccharide, said composition comprising a homogeneous polysaccharide or a derivative thereof. The homogeneous polysaccharide is primarily composed of arabinose, galactose, rhamnose, and galacturonic acid as monomeric units.

[0008] In some implementations, the subject is a mammal, preferably a human subject, which may be a healthy human, an aging adult, or an adult with bone loss.

[0009] The composition may be a pharmaceutical composition, a functional composition, and / or a dietary supplement. In some embodiments, the composition is administered orally or by gavage. The composition may be in tablet or liquid form. For example, in some embodiments, the composition is a pharmaceutical composition in tablet form that can be administered orally. In some embodiments, the composition may be a functional composition and is in powder form. The composition may also be formulated as a sports drink or snack bar.

[0010] The molecular weight of the homogeneous polysaccharide may be in the range of approximately 10 kDa to approximately 150 kDa, for example, from approximately 10 kDa to approximately 100 kDa, from approximately 10 kDa to 90 kDa, from approximately 10 kDa to 60 kDa, or any other suitable range. In the homogeneous polysaccharide, the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid is in the range of 30-70:20-60:0.1-10:0.1-10. For example, the homogeneous polysaccharide used in the embodiments of the present invention, referred to as LBP1C-2, has a monomer unit ratio of arabinose, galactose, rhamnose, and galacturonic acid of 49.9:33.6:8.0:8.5. Its molecular weight may be approximately a specific value or in a narrow range from approximately 10 kDa to approximately 150 kDa. Each obtained polysaccharide is homogeneous, with a uniform or narrow molecular weight distribution.

[0011] In some embodiments, the homogeneous polysaccharide described herein is the only polysaccharide in the composition.

[0012] In other embodiments, the composition further comprises additional polysaccharides isolated from wolfberry extract. The homogeneous polysaccharides constitute more than 15% of the total polysaccharide content in the composition. All polysaccharides may be derived from wolfberry. For example, the polysaccharide used in this invention is wolfberry polysaccharide (referred to as LBP).

[0013] In some embodiments, the composition may further optionally comprise one or more of flavonoids, carotenoids, polyphenols, pigments, or any compounds isolated from wolfberry extract.

[0014] In some embodiments, the composition comprises a chemically modified derivative of the homogeneous polysaccharide described herein. For example, such a derivative is a pharmaceutically acceptable ester or a salt thereof. A pharmaceutically acceptable ester or salt thereof is a sulfated derivative of the homogeneous polysaccharide, also known as a sulfated polysaccharide.

[0015] The excipient may be a solvent, co-solvent, colorant, preservative, antimicrobial agent, filler, binder, disintegrant, lubricant, surfactant, emulsifier, suspending agent, or any combination thereof.

[0016] This composition can be administered in any suitable amount. For example, in some embodiments, the effective dose of the composition (described herein as the amount of homogeneous polysaccharide) is in the range of 10 mg / kg to 500 mg / kg, based on the total daily weight of homogeneous polysaccharide / the subject's daily body weight. The composition can be administered once daily, twice daily, or more than twice daily.

[0017] On the other hand, the present invention provides a composition (as described herein) 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 subjects of need. The composition comprises an effective dose of a homogeneous polysaccharide or a derivative thereof and pharmaceutically acceptable excipients. The homogeneous polysaccharide is primarily composed of arabinose, galactose, rhamnose, and galacturonic acid as monomeric units.

[0018] As described herein, the composition is a pharmaceutical composition, a functional composition, and / or a dietary supplement. For example, in some embodiments, the composition is an oral composition and / or is available in tablet form.

[0019] The excipient may be selected from solvents, cosolvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

[0020] The homogeneous polysaccharide has a molecular weight in the range of approximately 10 kDa to approximately 150 kDa, for example, from approximately 10 kDa to approximately 100 kDa, from approximately 10 kDa to approximately 80 kDa, from approximately 10 kDa to approximately 60 kDa, or any other suitable range. In the homogeneous polysaccharide, the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid is in the range of 30-70:20-60:0.1-10:0.1-10. For example, in some embodiments, the homogeneous polysaccharide has a ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid of 49.9:33.6:8.0:8.5. Its molecular weight may be approximately a specific value or a narrow range in the range of approximately 10 kDa to approximately 150 kDa. Each resulting polysaccharide is homogeneous, with a uniform or narrow molecular weight distribution.

[0021] In some embodiments, the homogeneous polysaccharide described herein is the only polysaccharide in the composition. In other embodiments, the composition further comprises additional polysaccharides isolated from wolfberry extract, and / or, the homogeneous polysaccharide accounts for more than 15% of the total polysaccharide content in the composition.

[0022] In some embodiments, the composition may selectively include flavonoids, carotenoids, polyphenols, pigments, or any combination thereof isolated from wolfberry extract. In other embodiments, the composition does not include flavonoids, carotenoids, or polyphenols isolated from wolfberry extract.

[0023] In some embodiments, the composition comprises a chemically modified derivative of the homogeneous polysaccharide described herein. For example, such a derivative is a pharmaceutically acceptable ester or a salt thereof. A pharmaceutically acceptable ester or salt thereof is a sulfated derivative of the homogeneous polysaccharide, also known as a sulfated polysaccharide.

[0024] The present invention also provides the use of homogeneous polysaccharides or derivatives thereof as described herein in the manufacture of medicaments for treating any of the medical conditions described herein.

[0025] In another aspect, the present invention also provides a method for preparing the composition or homogeneous polysaccharide. This method may include preparing or separating the homogeneous polysaccharide. The method may also include mixing an excipient with the homogeneous polysaccharide. The method may include chemical modification, such as sulfation of the homogeneous polysaccharide. Attached Figure Description

[0026] The present invention is best understood through the following detailed description and accompanying drawings. It should be emphasized that, as is customary, the various features in the drawings are not necessarily drawn to scale. Instead, for clarity, the dimensions of the various features are arbitrarily enlarged or reduced. The same reference numerals denote the same features throughout the specification and drawings.

[0027] Figure 1 The trabecular bone structure of control mice and experimental mice treated with Lycium barbarum aqueous extract (LBE), Lycium barbarum crude polysaccharide (LBP), and Lycium barbarum homogeneous polysaccharide (LBP1C-2) are shown.

[0028] Figure 2 shows the bone mineral density (BMD) values ​​of the control group mice and the experimental group mice treated with LBE, LBP, and LBP1C-2, respectively. Figure 2A ), the ratio of trabecular bone volume to total volume (BV / TV) ( Figure 2B ), number of trabeculae (Tb.N) Figure 2C ), trabecular thickness (Tb.Th)( Figure 2D ), and trabecular separation (Tb.Sp)( Figure 2E ).

[0029] Figure 3 The maximum load force a (as assessed by the three-point bending test) is shown in the control group mice and the experimental group mice given LBE, LBP and LBP1C-2 respectively.

[0030] Figure 4 The bone formation rate is shown in the control group mice and the experimental group mice treated with LBE, LBP, and LBP1C-2, respectively. The abbreviation "BFR / BS" represents the bone formation rate per unit bone surface area.

[0031] Figure 5 The bone mineralization deposition rate (MAR) is shown in control mice and experimental mice treated with LBE, LBP, and LBP1C-2, respectively.

[0032] Figure 6 The study showed the serum osteocalcin levels in control mice and experimental mice treated with LBE, LBP, and LBP1C-2, respectively.

[0033] Figure 7 The study showed the serum levels of type I procollagen N-terminal propeptide (PINP) in control mice and experimental mice treated with LBE, LBP, and LBP1C-2, respectively.

[0034] Figure 8 This study shows the proliferation of osteoblasts in human mesenchymal stem cells (hMSCs) treated with LBE, LBP, and LBP1C-2 compared to the control group in a CFU (clonal forming unit) experiment.

[0035] Figure 9 The expression levels of osteocalcin in hMSCs treated with LBE, LBP, and LBP1C-2 and in the control group were shown.

[0036] Figure 10 The alkaline phosphatase (ALP) activity in hMSCs treated with LBE, LBP, and LBP1C-2 and in the control group was shown.

[0037] Figure 11 The expression levels of bone formation-related genes in the femur of control mice and experimental mice given LBP1C-2 were shown.

[0038] Figure 12 The expression levels of genes related to bone resorption in the femur of control mice and experimental mice given LBP1C-2 were shown.

[0039] Figure 13-15 The expression levels of bone formation-related genes Runx2, Col1α, and Bglap were shown in LBP1C-2-treated preosteoblasts and the control group after interference with BMPR1a and BMPR2 genes.

[0040] Figure 16-17The results show the direct interaction between LBP1C-2 and BMPRIA and BMPII in the control group and the experimental group given LBP1C-2, characterized by surface plasmon resonance (SPR) method.

[0041] Figure 18-19 The results show the interaction between LBP1C-2 and BMPRIA and BMPRII, determined using a fluorescence-based protein thermal shift method.

[0042] Figures 20A-20B The phosphorylation levels of Smad1 / 5 / 8 in LBP1C-2 treated hMSCs and control hMSCs were shown. Figure 20A The phosphorylation levels of Smad1 / 5 / 8, and the expression levels of Smad1 / 5 / 8 and GAPDH were shown. Figure 20B The effect of LBP1C-2 on Smad1 / 5 / 8 phosphorylation levels was shown by Western blot analysis. GAPDH was used as a loading control.

[0043] Figure 21 The results show the direct interaction between LBP1C-2 and Noggin as determined by surface plasmon resonance (SPR).

[0044] Figure 22 The bone morphogenetic protein 2 (BMP2) release levels were shown in hMSCs treated with LBP1C-2 and in control hMSCs.

[0045] Figures 23A-23B The study showed that hMSCs treated with LBP1C-2 and S-LBP1C-2 (sulfated LBP1C-2) had the early bone formation marker RUNX2 in comparison with control hMSCs. Figure 23A ) and SP7 ( Figure 23B ) level.

[0046] Figure 24A The alkaline phosphatase (ALP) staining results (day 7) in LBP1C-2 treated hMSCs, S-LBP1C-2 treated hMSCs and control hMSCs are shown.

[0047] Figure 24B The ALP enzyme activity in LBP1C-2, S-LBP1C-2 and control hMSCs was shown.

[0048] Figure 25A Alizarin Red S staining results (day 21) are shown in hMSCs treated with LBP1C-2, S-LBP1C-2 and control group hMSCs.

[0049] Figure 25BThe expression levels of osteocalcin in hMSCs treated with LBP1C-2, S-LBP1C-2 and control hMSCs are shown. Detailed Implementation

[0050] 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.

[0051] In this invention, the singular forms “an,” “a,” and “the” include plural references, and unless the context explicitly indicates otherwise, a reference to a particular numerical value includes at least that particular value. Thus, for example, a reference to “additive” is a reference to one or more such compounds and their equivalents, which are known to those skilled in the art, etc. When a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10% of the listed value, including the end values. For example, the phrase “about 8” preferably refers to a value including 7.2 to 8.8, including the end values; as another example, the phrase “about 8%” preferably (but not always) refers to a value including 7.2% to 8.8%. Where applicable, all ranges are inclusive and composable. For example, when listing the range "1 to 5", the listed range should be interpreted to include the ranges "1 to 4", "1 to 3", "1-2", "1-2 and 4-5", "1-3 and 5", "2-5", etc. Furthermore, when actively providing alternatives, such an item can be interpreted to mean that any alternative can be excluded, for example, by negative limitation in the claims. For example, when listing the range "1 to 5", the listed range can be interpreted to include cases where any one of 1, 2, 3, 4, or 5 is negatively excluded; therefore, listing "1 to 5" can be interpreted as "1 and 3-5, but excluding 2", or simply as "excluding 2". The intention is that any component, element, property, or step affirmatively listed herein may be expressly excluded in the claims, whether such component, element, property, or step is listed as an alternative or listed separately. Any component, element, property, or step actively referenced herein may be expressly excluded in the claims, whether such component, element, property, or step is listed as an alternative or cited in isolation.

[0052] The terms "subject" and "patient" as used herein are used interchangeably. The term "patient" as used herein refers to an animal, preferably a mammal such as non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys and humans), most preferably a human. In some embodiments, the subject is a non-human animal, such as a farm animal (e.g., a horse, pig, or cattle) or a pet (e.g., a dog or cat). In a particular embodiment, the subject is a human. In another embodiment, the subject is an adult.

[0053] As used herein, the term "medicine" refers to any molecule, compound, method, and / or substance used for the prevention, treatment, management, and / or diagnosis of a disease or condition. The term "effective dose" as used herein refers to a dose of a therapy sufficient to prevent the development, recurrence, or onset of a disease or condition and one or more of its symptoms; to enhance or improve the preventive effect of another therapy; to reduce the severity of a disease or condition; to shorten the duration of a disease or condition; to alleviate one or more symptoms of a disease or condition; to prevent the progression of a disease or condition; to cause degeneration of a disease or condition; and / or to enhance or improve the therapeutic effect of another therapy. The disease or condition (such as a medical condition) is related to bone loss or to bone formation, bone mass, and / or bone strength.

[0054] The phrase “pharmaceutically acceptable” as used herein means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeia for use in animals, and more specifically, in humans.

[0055] As used herein, the term "therapeutic agent" refers to any molecule, compound, and / or substance used to treat and / or manage a disease or illness.

[0056] As used herein, the terms "therapy" and "treatment" can refer to any method, composition, and / or medicine that can be used to prevent, treat, and / or manage a disease or condition, or one or more symptoms thereof. In some embodiments, the terms "therapy" and "treatment" refer to small molecule therapies.

[0057] As used herein, the terms “treatment,” “treatment method,” and “being treated” in the context of administering a therapy to a subject refer to reducing or inhibiting the progression and / or duration of a disease or condition, alleviating or improving the severity of a disease or condition, such as cancer, and / or improving one or more of its symptoms as a result of administering one or more therapies.

[0058] As used herein, the term "excipient" refers to an inactive substance that serves as a carrier or medium for a drug or other active substance. Examples of suitable excipients include, but are not limited to, solvents, co-solvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

[0059] As described in this article, the “monomer units” or “monomer units” in polysaccharides refer to monosaccharide syl units or single sugar molecule units or other basic chemical units. These units are understood as groups of monosaccharides, linked together by -O- bonds through condensation reactions between hydroxyl groups.

[0060] Molecular weight is measured by gel permeation chromatography (GPC). GPC is an analytical technique that separates molecules in a polymer by molecular size and provides the molecular weight or molecular weight distribution of the material. The homogeneous polysaccharide described herein contains only one peak in GPC and has a uniform molecular weight. Homogeneous polysaccharides or derivatives thereof are provided and used in this disclosure. Due to the uniformity of their molecular weight distribution (i.e., a polydispersity index of 1 or close to 1), their weighted average molecular weight (Mw), number average molecular weight (Mn), or peak molecular weight (Mp) are the same, substantially the same, or very close. Unless explicitly stated otherwise, the molecular weight values ​​and ranges described herein may be Mw or Mn. In some embodiments, the molecular weight values ​​and ranges described herein are number average molecular weights (Mn).

[0061] Goji berries, belonging to the genus *Lycium* in the Solanaceae family, are used as a traditional Chinese medicine and health food in China and other countries. They are listed as a homologous species that can be used in both medicine and food. The fruit of the goji berry has anti-aging and anti-fatigue effects. Goji berries have been used to nourish the liver and kidneys and improve eyesight. However, to date, our understanding of the molecular mechanisms of goji berry function is very limited, and its active ingredients remain unclear.

[0062] The inventors discovered the functions of goji berries in muscles. Goji berries contain various components, such as polysaccharides, flavonoids, betaine, taurine, vitamins, and fatty acids. Goji berry polysaccharides, as the main active ingredient, possess biological activities including antioxidant, antiviral, neuroprotective, acute liver injury-modulating, and immunomodulatory effects.

[0063] In a recent study, the inventors used a unique separation method to extract and isolate an aqueous extract, namely Lycium barbarum extract (LBE), and crude polysaccharide (LBP), from Lycium barbarum fruit. A homogeneous polysaccharide called LBP1C-2 (e.g., 0.02% yield in dried fruit) was purified from LBP. LBP1C-2 was found to be a pectin. However, whether LBP1C-2 has any effect on bone remodeling remains unclear.

[0064] Lycium barbarum polysaccharide samples can promote the proliferation, differentiation, and mineralization of osteoblasts. The polysaccharides are extracted from Lycium barbarum fruit and contain six monosaccharides, including galactose, glucose, rhamnose, arabinose, mannose, and xylose. In vitro experiments were conducted using human mesenchymal stem cells (hMSCs).

[0065] However, whether wolfberry supplementation can improve age-related bone loss, the main material basis, mechanism of action, and target of action have not yet been determined.

[0066] In this invention, the inventors investigated the effects of LBE, LBP, and LBP1C-2 on naturally aging mice and identified their targets and mechanisms of action. LBP1C-2, a homogeneous polysaccharide derived from Lycium barbarum extract, was found to be the main active ingredient of Lycium barbarum. LBP1C-2 can directly bind to BMPRIA and BMPRII, promoting the phosphorylation of Smads and enhancing osteogenic differentiation and mineralization. LBP1C-2 also directly binds to Noggin, inhibiting the interaction between Noggin and BMPs, thereby increasing bone formation. This study also provides a research basis for explaining the therapeutic effects of Lycium barbarum extract components and offers scientific evidence for its future use as a natural dietary or product supplement to treat age-related bone loss.

[0067] This invention provides a composition and method for improving bone formation, bone mass, and / or bone strength. This method can be used to treat or prevent bone loss in subjects in need, such as treating or preventing age-related bone loss.

[0068] According to some embodiments, such a composition comprises a homogeneous polysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient, administered to a subject in need. The homogeneous polysaccharide is primarily composed of arabinose, galactose, rhamnose, and galacturonic acid as monomeric units or molecules. This method involves administering an effective amount of the composition comprising the homogeneous polysaccharide to a subject in need.

[0069] In some implementations, the subject is a mammal, preferably a human subject, which may be a healthy human, an aging adult, or an adult with osteoporosis.

[0070] The composition may be a pharmaceutical composition, a functional composition, and / or a dietary supplement. In some embodiments, the composition is administered orally or by gavage. The composition may be in tablet or liquid form. For example, in some embodiments, the composition is an orally administered pharmaceutical composition in tablet form. In some embodiments, the homogeneous polysaccharide or a derivative thereof is a therapeutic agent. In some embodiments, the composition may be a functional composition and is in powder form. The composition may also be formulated into sports drinks or snack bars.

[0071] The homogeneous polysaccharide has a molecular weight in the range of about 10 kDa to about 150 kDa, for example, from about 10 kDa to about 100 kDa, from about 10 kDa to about 80 kDa, from about 10 kDa to about 60 kDa, or any other suitable range. In the homogeneous polysaccharide, 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. For example, in some embodiments, the molar ratio of arabinose, galactose, rhamnose, and galacturonic acid monomer units in the homogeneous polysaccharide is 49.9:33.6:8.0:8.5. Its molecular weight may be a specific value or a narrow range in the range of about 10 kDa to about 150 kDa. Such a molecular weight value may be a weight average molecular weight (Mw) or a number average molecular weight (Mn). Because polysaccharides are homogeneous, the values ​​of Mw and Mn may be very close to each other, exhibiting a very narrow molecular weight distribution. The dispersion index (PD), i.e., the ratio of Mw to Mn, may range from approximately 1 to approximately 1.3, from approximately 1 to approximately 1.2, or from approximately 1 to approximately 1.1. In some embodiments, the PD index is close to 1. The molecular weight of the polysaccharide may vary due to factors such as the growing environment and harvest season of the raw wolfberry. Therefore, the obtained homogeneous polysaccharide may have different molecular weights. However, the polysaccharide obtained in each batch is homogeneous, i.e., its molecular weight is uniform or has a narrow distribution. The molecular weight can be controlled by controlling the quality of the raw material, for example, by controlling the same growing environment and pre-harvest growing time.

[0072] In some embodiments, the homogeneous polysaccharide described herein is the only polysaccharide in the composition.

[0073] In some embodiments, the homogeneous polysaccharide described in this invention is LBP1C-2 or a derivative thereof, such as sulfated LBP1C-2.

[0074] In some embodiments, in addition to LBP1C-2, the composition further comprises additional polysaccharides isolated from wolfberry extract. The homogeneous polysaccharides constitute more than 15% of the total polysaccharides in the composition. All polysaccharides may be derived from wolfberry. For example, the polysaccharide used in this invention is wolfberry polysaccharide (referred to as LBP).

[0075] In some embodiments, the composition may further optionally comprise one or more of flavonoids, carotenoids, polyphenols, pigments, or any compounds isolated from wolfberry extract.

[0076] Lycium barbarum extract (LBE) can be obtained by water extraction of Lycium barbarum, and the extract can exist in the form of a dry powder. The extract can also be dissolved in water and then further purified or separated by fractionation purification methods. Polysaccharides can be obtained. These polysaccharides are further separated by fractionation purification and freeze-drying to obtain one or more homogeneous polysaccharides existing in the form of a dry powder.

[0077] In some embodiments, the composition comprises wolfberry extract (LBE). This wolfberry extract includes polysaccharides (or wolfberry polysaccharides), wolfberry flavonoids, carotenoids, polyphenols, and wolfberry pigments. Each component may be present only once, or there may be two or more of the same type. For example, the composition may include two or more polysaccharides, two or more wolfberry flavonoids, two or more carotenoids, two or more polyphenols, and / or two or more wolfberry pigments.

[0078] In some embodiments, based on the total dry weight of the extract, the polysaccharide content in the wolfberry extract (LBE) ranges from about 10.0 wt.% to about 70.0 wt.% (e.g., from about 50 wt.% to about 70 wt.%), the wolfberry flavonoid content ranges from about 0.1 wt.% to about 5.0 wt.%, the carotenoid content ranges from about 0.1 wt.% to about 3.0 wt.%, the polyphenol content ranges from about 0.1 wt.% to about 8.0 wt.%, and the wolfberry pigment content ranges from about 0.1 wt.% to about 8.0 wt.%. In some embodiments, the polysaccharide content is more preferably from about 50.0 wt.% to about 70.0 wt.%. Dry weight is the equivalent weight of the extract corresponding to the dried powder (anhydrous) form. The extract may contain other trace residues. The extract in dried powder form can be mixed with water to provide an extract in aqueous solution form with a selected concentration as described herein.

[0079] In some embodiments, wolfberry extract (LBE) is present in powder form. In some embodiments, the wolfberry extract is dissolved in a solvent such as water to provide an aqueous solution with a concentration, for example, 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 LBE ranges from approximately 50.0 wt.% to 70.0 wt.%.

[0080] Lycium barbarum polysaccharides or polysaccharides (LBP) existing in powder form can be further purified from LBE in powder form. For example, LBE can be dissolved in water and then separated by a separation column. Lycium barbarum polysaccharides (LBP) may contain different polysaccharides, which can be further separated.

[0081] A homogeneous polysaccharide, LBP1C-2, was isolated from LBE and / or LBP. High-performance gel permeation chromatography (HPGPC) analysis revealed that LBP1C-2 exhibited a single, symmetrical peak, indicating its homogeneity. Sugar composition analysis showed that LBP1C-2 was composed of arabinose (Ara), galactose (Gal), rhamnose (Rha), and galacturonic acid in a ratio of 49.9:33.6:8.0:8.5. The structure of LBP1C-2 comprises an alternating backbone of 1,2-linked α-Rhap and 1,4-linked α-GalpA, and terminal (T)-, 1,3-, 1,6-, and 1,3,6-linked β-Galp, T-, 1,5-, and 1,3,5-linked α-Araf, and a T-linked β-Rhap branch substituted at C-4 of the 1,2,4-linked α-Rhap.

[0082] Equations 1, 2 and 3 illustrate the structure of LBP1C-2, showing the same structure in three different formats.

[0083] 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 shows that LBP1C-2 mainly consists of 1,2-α-Rha and 1,4-α-GalA as the backbone, 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. These branches are attached to the C-4 positions of the 1,2,4-α-Rha backbone sugar residues. The repeating unit of LBP1C-2 contains the structural portion shown in Formula 3 and includes a backbone (composed 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.

[0084] Formula 1,

[0085]

[0086] Equation 2,

[0087]

[0088] Formula 3,

[0089]

[0090] In Equations 1-3, n ranges from 2 to 20. The molecular weight is directly proportional to the value of n. For example, when a sample exhibits a molecular weight of approximately 13.2 kDa, n is approximately 2. When a sample exhibits a molecular weight of approximately 99.8 kDa, n is approximately 13.

[0091] The inventors of this invention have demonstrated that homogeneous polysaccharide LBP1C-2, a polysaccharide or active ingredient in LBE or LBP, has the function of improving bone loss, bone mass, and / or strength. Homogeneous polysaccharide 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.

[0092] In some embodiments, the composition comprises a chemically modified derivative of the homogeneous polysaccharide as described herein. For example, the derivative is a pharmaceutically acceptable ester or salt thereof. A pharmaceutically acceptable ester or salt thereof is a sulfated derivative of the homogeneous polysaccharide, also known as a sulfated polysaccharide. During the synthesis of the sulfated homogeneous polysaccharide, the hydroxyl groups in the homogeneous polysaccharide react with a modifying agent such as chlorosulfonic acid to form -O-SO3H groups. The modified molecular weight is the same as that of the homogeneous polysaccharide described above. The degree of sulfate substitution of the sulfated derivative of the homogeneous polysaccharide is in the range of 0.5 to 0.9, for example, in the range of 0.6 to 0.8. The degree of substitution indicates the number of substituent groups on the sugar unit. For example, a degree of substitution of 0.74 indicates that the number of sulfate substituents on each hexose or pentose unit is 0.74. Sulfuric acid substitution can bind to proteins and enhance biological activity.

[0093] The composition may be a pharmaceutical composition, a functional composition, and / or a dietary supplement. For example, the composition may be a pharmaceutical composition that can be taken orally. In embodiments of the invention, an aqueous extract of wolfberry is administered by gavage or oral administration, but is not limited thereto. Any method of administration that delivers the composition into the stomach may be suitable.

[0094] 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.

[0095] This composition can be administered in any suitable amount. For example, in some embodiments, the effective daily dose of the composition (described herein as being based on the amount of the homogeneous polysaccharide) is in the range of 10 mg / kg to 500 mg / kg, based on the total weight of the homogeneous polysaccharide / the subject's body weight. In some embodiments, the dose of Lycium barbarum extract (LBE) or LBP or LBP1C-2 is in the range of 4 mg / kg to 70 mg / kg (total daily dry weight of LBP1C-2 / the subject's body weight, such as a human's) per day, for example, 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 any other suitable range. This composition can be administered once daily, twice daily, or more than twice daily.

[0096] In some embodiments of the present invention, the dosage of Lycium barbarum extract (LBE), LBP, or LBP1C-2 is 40 mg / kg per day (total daily dry weight of LBP1C-2 / body weight of an animal such as a mouse). The total daily dose may be administered once, twice, or more than once daily. In embodiments of the present invention, mice are administered LBE, LBP, or LBP1C-2 once daily.

[0097] The dosage and administration methods described herein also apply to derivatives of the homogeneous polysaccharide LBP1C-2.

[0098] In some embodiments, these compositions can be taken with beverages, food, or related ingredients. The wolfberry water extract provided by this invention is homologous in both pharmaceuticals and food and can be 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 containing homogeneous polysaccharides. Other ingredients can be selected by those skilled in the art according to specific formulations or uses.

[0099] In some embodiments, the composition is a food or health product, including sports drinks, protein powder, snack bars, etc.

[0100] The present invention also provides the use of homogeneous polysaccharides or derivatives thereof as described herein in the manufacture of medicaments for treating any of the medical conditions described herein.

[0101] In another aspect, the present invention provides a method for preparing the composition or homogeneous polysaccharide. This method may include preparing or separating the homogeneous polysaccharide. The method may further include mixing the excipient and the homogeneous polysaccharide.

[0102] The features and effects of the present invention will be explained through embodiments and test examples. However, the following embodiments and test examples are for illustrative purposes only and do not limit the scope of the invention.

[0103] Example

[0104] 1. Material preparation

[0105] 1-1. Preparation of Lycium barbarum extract:

[0106] The preparation process of Lycium barbarum extract (“LBE”) is as follows: Lycium barbarum fruits were sourced from Zhongning County, Yinchuan City, Ningxia, China. Dried Lycium barbarum fruits were washed 3-5 times and soaked in double-distilled water (pH=7) for 2 hours at room temperature, followed by crushing. The soaked Lycium barbarum powder was added to 5-8 times its volume of neutral water, mixed thoroughly, and then decocted twice at boiling point for 2.0 hours and 1.5 hours respectively. The combined concentrated decoction was filtered through a fiber membrane. The combined filtrates were then evaporated under vacuum at 30-55°C to remove moisture and obtain a concentrate. The obtained concentrate was freeze-dried into a powder and stored in a desiccator for use in subsequent experiments at an appropriate concentration.

[0107] In the experiment, an exemplary wolfberry extract was used. This exemplary extract is an aqueous solution with a concentration of 0.4 g / mL (dry powder or dry weight of the extract / volume of the extract). This concentration is for illustrative purposes only. The aqueous extract can be adjusted to any suitable concentration, for example, from about 0.1 g / mL to 5 g / mL (dry weight of the example extract / volume of the extract). In some embodiments, this aqueous extract may be diluted for use. The aqueous extract may be further diluted for cell experiments.

[0108] The wolfberry extract (LBE) used in this invention mainly comprises water-soluble wolfberry polysaccharides, wolfberry flavonoids, carotenoids, polyphenols, and pigments. In exemplary LBEs, based on the total equivalent weight of the extract in dry powder form, the polysaccharide content ranges from about 50.0 wt.% to 70.0 wt.% (e.g., about 54%-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 brine. Comparisons are made using LBEs with the same composition.

[0109] 1-2. Isolation of Lycium barbarum polysaccharides and homogeneous polysaccharides: Lycium barbarum polysaccharides (“LBP”) and homogeneous polysaccharides, such as the example labeled LBP1C-2 described herein, can be isolated from LBE or Lycium barbarum fruit, for example, by the following example methods.

[0110] The method for extracting the polysaccharide includes: pulverizing the dried fruit, adding 15-30 times the amount of deionized water and mixing well, adding 3 wt.% cellulase, 1 wt.% amylase and 0.5 wt.% papain, extracting at 55-60 degrees Celsius for 1 hour, inactivating the enzymes by heating, centrifuging, concentrating the obtained filtrate, dialyzing, concentrating again, adding 5 times the amount of 95% ethanol, centrifuging to obtain a precipitate, washing the precipitate three times alternately with anhydrous ethanol and acetone, and vacuum drying to obtain crude polysaccharide (LBP). The above method also includes: dissolving the crude polysaccharide in 10-15 times its volume of water, centrifuging, collecting the supernatant, purifying it by fractionation using a diethylaminoethyl cellulose (DEAE) anion exchange column, eluting sequentially with water, 0.05M, 0.1M, and 0.2M sodium chloride, collecting the 0.2M sodium chloride eluent, concentrating, dialyzing, and freeze-drying to obtain preliminarily purified Lycium barbarum polysaccharide (LBP1C), dissolving the obtained polysaccharide LBP1C in 0.2M sodium chloride, centrifuging, collecting the supernatant, eluting it using a Sephacryl-300 (RTM: poly((acrylyl dextran)-co-N,N'-methylenebisacrylamide)) column, collecting the eluent, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharide LBP1C-2.

[0111] LBP1C-2 has the composition and structure described herein.

[0112] 1-3. Molecular weight determination of LBP1C-2:

[0113] The molecular weight and homogeneity of LBP1C-2 were measured by high-performance gel permeation chromatography (HP-GPC). Only one symmetrical peak appeared in the HP-GPC results. Referring to the known molecular weight dextran standard used in the HP-GPC measurements, the estimated weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity (PD) index were 13,181 Da, 10,750 Da, and 1.22, respectively.

[0114] 1-4. Preparation of sulfated polysaccharide derivatives of LBP1C-2 and measurement of the degree of sulfate substitution (DS)

[0115] The homogeneous polysaccharide was sulfated using the chlorosulfonic acid-pyridine method. LBP1C-2 (50 mg) was dissolved in 2.5 mL of dry formamide. 1.5 mL of a sulfation reagent, prepared from 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, followed by dialyzed against distilled water. The residue was lyophilized to obtain the sulfated derivative S-LBP1C-2. The degree of sulfation was calculated using the chlorosulfonic acid-pyridine method, using formula (1) as follows:

[0116]

[0117] Where DS is the degree of sulfate substitution and %S is the percentage of sulfur content.

[0118] Based on the sulfur content (10%), and referring to the standard curve of sulfate content detected by the chlorosulfonic acid-pyridine method, the degree of sulfate substitution (DS) of S-LBP1C-2 was calculated to be 0.74.

[0119] 2. Biological Experiments:

[0120] 2-1. Animals and Grouping

[0121] Healthy, wild-type (WT) adult male C57BL / 6J mice (2 months old) and naturally aged male C57BL / 6J mice (14 months old) were housed in an SPF barrier facility. Mice were randomly assigned to adult (n=15 per group) and aged (n=6 per group) groups. Adult and aged mice were further divided into four subgroups: control, LBE, LBP, and LBP1C-2. Control mice were administered distilled water (10 mL / kg) orally via gavage. The three extracts were administered once daily via gavage at doses of LBE (40 mg / kg), LBP (40 mg / kg), or LBP1C-2 (40 mg / kg) for 4 months. All animal experiments were conducted in accordance with the ARRIVE guidelines and the UK Animal (Scientific Procedures) Act 1986 and related guidelines.

[0122] 2-2. Cell Culture

[0123] hMSCs were purchased from ScienCell Research Laboratories. The culture medium used in this study was α-minimum basal medium (α-MEM; Gibco, 12571063), supplemented with 10% fetal bovine serum (FBS; Gibco, 16140071) and 1% penicillin-streptomycin (HyClone, SH40003-12). The differentiation culture medium 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. The cell cultures were incubated in a humid environment at 37°C with 5% CO2.

[0124] 2-3. Detection of serum bone formation markers

[0125] 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℃). The levels of osteocalcin and PINP were detected using the osteocalcin ELISA kit and the PINP ELISA kit, respectively, according to the experimental procedures of the kits. Osteocalcin and PINP are typical markers of bone formation.

[0126] 2-4. Bone analysis using micro-computed tomography (CT) scans

[0127] 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), bone volume fraction (BV / TV), 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 trabeculae analysis is 1-2 mm below the distal femoral growth plate.

[0128] 2-5. Three-point bending test

[0129] 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 distal support points and one central loading point. Biomechanical measurements were collected from load-deformation curves.

[0130] 2-6. Surface Plasmon Resonance (SPR) Analysis

[0131] The affinity of LBP1C-2 for protein binding was measured using a BIACORE T200 (GE Healthcare, Stockholm, Sweden). BMPRIA, BMPRII, and Noggin proteins were immobilized on a CM5 sensor chip via amino-coupling. For interaction measurements, different concentrations of LBP1C-2 were injected into the chip. All operations were performed in HBS-EP run buffer (pH 7.4) containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 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.

[0132] 2-7. Cellular Thermal Transfer Assay (CETSA)

[0133] CETSA experiments were performed according to the previously described protocol (Li et al., 2017). hMSCs were cultured for 96 hours in medium containing either control or 4 μM LBP1C-2. Cells were then collected. RIPA lysis buffer was added, and the cells were subjected to three freeze-thaw cycles with liquid nitrogen and centrifuged at 12,000 g for 10 minutes at 4 °C. The supernatant from the control group was divided into two fractions. One fraction was treated with ddH2O2 for 30 minutes, while the other fraction, along with both the control and 8 μM LBP1C-2 treatment groups, was treated with the 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.

[0134] 2-8. Quantitative Real-Time PCR (RT-PCR) Experiment

[0135] Femoral tissue or cells were collected, 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 each treatment group rat were assessed using the 2-ΔΔCq method.

[0136] 2-9. Western blot analysis

[0137] Femoral tissue or cells were prepared for analysis and lysed using RIPA buffer. The RIPA buffer consisted of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 5 mM EDTA, and a mixture of protease inhibitors (Roche). Next, 40 mg of protein was separated from each sample by SDS-PAGE and transferred to an NC membrane. The membrane was then blocked for 1 hour in TBST solution containing 5% skim milk powder (TBST: 10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4). After blocking, the membrane was incubated overnight with primary antibody at 4°C. The main antibodies used in this study included anti-p-Smad1 / 5 / 8 (1:1000), anti-Smad1 / 5 / 8 (1:1000), and anti-GAPDH (1:2000). Subsequently, the membranes were washed with TBST and incubated for 1 hour at room temperature with a secondary antibody labeled with horseradish peroxidase (HRP) (1:2000 dilution, Zhong-shan Jin-qiao Pharmaceutical Co., Ltd., Beijing, China). The membranes were washed three times with TBST. For visualization of 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).

[0138] 2-10. ALP staining and ALP measurement

[0139] ALP staining was performed using the Vector Blue substrate kit (procedure number SK-5300, Vector Laboratories). hMSCs were stained at a concentration of 1 × 10⁻⁶ according to the protocol. 5 hMSCs 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, hMSCs were incubated with substrate working solution for 30 minutes. Throughout the process, cells were protected from light. ALP assays were performed based on the previously described procedure.

[0140] 2-11. Alizarin Red S staining

[0141] hMSCs at 1×10 5 Cells were seeded at a density of 10 cells per well in 24-well plates and cultured in growth medium for 3 days. Osteoblast differentiation was induced by culturing in differentiation medium. On day 14 after osteoblast differentiation was induced, 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.

[0142] 3. Results

[0143] In the following experiments, LBE, LBP, and LBP1C-2 solutions were prepared fresh by dissolving the powder in distilled water and administered once daily. Control group mice were administered distilled water orally via gavage, while treatment group mice were administered LBE (40 mg / kg), LBP (40 mg / kg), or LBP1C-2 (40 mg / kg) once daily via gavage for 4 months. The effective dose of LBP1C-2 (based on the amount of homogeneous polysaccharide described herein) was 40 mg / kg, calculated as the total daily weight of homogeneous polysaccharide divided by the subject's body weight. The doses of LBE and LBP (40 mg / kg) were calculated as the total dry weight of LBE or LBP divided by the subject's body weight.

[0144] 3-1. Treatment with LBE, LBP, and LBP1C-2 can increase bone mass and bone stability.

[0145] Aging is significantly associated with bone loss and deterioration of bone microstructure. Therefore, we investigated whether LBE, LBP, and LBP1C-2 affect trabecular bone structure parameters in mice using microcomputed tomography (μCT). μCT was used to quantify the morphological parameters of the femur in treated and control mice. Compared to adult and aging control groups, LBE, LBP, and LBP1C-2 treatments showed better bone morphology. Figure 1 Compared with adult and aging control groups, LBE, LBP, and LBP1C-2 treatments improved age-related bone loss because mice treated with LBE and LBP showed higher BMD (bone mineral density). Figure 2A Higher bone volume fraction (BV / TV) Figure 2B ), and a higher number of trabeculae (Tb.N) Figure 2C Thicker trabecular bone thickness (Tb.Th) Figure 2D and smaller trabecular separation (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 aging control group).

[0146] Next, we examined whether LBE, LBP, and LBP1C-2 could modulate bone biomechanical properties. The results showed that, compared with adult and aged control groups, mice treated with LBE, LBP, and LBP1C-2 exhibited a significant increase in maximum load-bearing capacity. Figure 3 ).

[0147] 3-2. Treatment with LBE, LBP, and LBP1C-2 promotes bone formation.

[0148] This study analyzed the rate of bone formation (BFR) and bone mineralization deposition rate (MAR) within the femur. BFR represents the amount of new bone formed per unit time. It is typically measured by quantifying the amount of mineralized bone matrix or osteogenic matrix (unmineralized bone) formed in a specific bone surface region. MAR specifically focuses on the mineralization rate of newly formed bone matrix. It measures the rate at which mineral crystals, primarily hydroxyapatite, are deposited onto the osteogenic matrix surface during bone formation. The BFR of mice treated with LBE, LBP, and LBP1C-2 was determined using a calcein-alizarin red fluorescence double labeling method. Figure 4 ) and bone mineralization deposition rate (MAR, Figure 5 The level of osteocalcin () was significantly increased. Compared with the control group, serum osteocalcin () was significantly increased. Figure 6 ), a bone formation marker, and P1NP (a bone formation marker, Figure 7 The levels of LBE, LBP, and LBP1C-2 were significantly increased. The effect of LBP1C-2 was even more pronounced. In summary, 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 aging control group)

[0149] 3-3. LBE, LBP, and LBP1C-2 can promote the proliferation, differentiation, and mineralization of osteoblasts.

[0150] This study aimed to investigate whether LBE, LBP, and LBP1C-2 affect the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts and to further understand the effects of these specific compounds. The proliferation, differentiation, and mineralization of osteoblasts were analyzed using hMSCs. Results showed that LBE, LBP, and LBP1C-2 significantly promoted the formation of CFU-F (fibroblast colony-forming units) colonies and promoted hMSC proliferation. Figure 8Similarly, LBE, LBP, and LBP1C-2 significantly promoted osteoblast differentiation and mineralization. Furthermore, in cells treated with LBE, LBP, and LBP1C-2, the expression levels of two osteoblast differentiation markers, Bglap, were significantly increased. Figure 9 ) and ALP enzyme activity ( Figure 10 All of these results significantly increased. All of these results indicate that LBE, LBP, and LBP1C-2 treatments enhanced the differentiation and mineralization of hMSCs into osteoblasts.

[0151] 3-4. LBP1C-2 promotes bone formation and has little effect on bone resorption.

[0152] To elucidate the mechanism of action of LBP1C-2, the expression of bone formation and bone resorption genes was analyzed by RT-PCR. In LBP1C-2-treated cells, 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, the mRNA expression of osteoclast markers in the femur, including Octamp, Oscar, Opg, and Rank1, was measured. The results showed no significant differences. Figure 12 These results indicate that LBP1C-2 promotes bone formation without affecting bone resorption.

[0153] 3-5. LBP1C-2 enhances bone formation by relying on BMPRIA and BMPRII.

[0154] Since BMPR is a key receptor for osteogenic differentiation, this study investigated whether its expression is associated with LBP1C-2-induced osteogenic differentiation. Different doses of BMPR-siRNA were used to confirm specific inhibitory effects against BMPRIA and BMPRII. Quantitative real-time PCR analysis revealed the expression levels of downstream genes Col1α, Runx2, and Bglap. Furthermore, silencing of BMPRIA and BMPRII led to Runx2 (… Figure 13 ), Col1α ( Figure 14 ) and Bglap Figure 15 The downregulation of BMPRIA and BMPRII indicates that LBP1C-2 treatment-induced osteogenic differentiation and mineralization were inhibited by the downregulation of BMPRIA and BMPRII. Therefore, the results suggest that LBP1C-2-dependent osteogenic differentiation and mineralization are dependent on BMPRIA and BMPRII.

[0155] 3-6. LBP1C-2 can directly bind to BMPRIA and BMPRII and activate them in large quantities.

[0156] To investigate whether LBP1C-2 directly interacts with BMPR, their interaction was measured using SPR. The results showed that these interactions are dose-dependent. The equilibrium dissociation constant (Kd) of LBP1C-2 with BMPR is approximately 3.6 μmol / L. Figure 16 ), BMPRII's Kd is 5.6 μmol / L ( Figure 17 The direct interaction between LBP1C-2 and BMPR was further confirmed by fluorescence-based protein thermal transfer experiments; before reaching the plateau phase, LBP1C-2 dose-dependently transferred BMPR (…). Figure 18 ) and BMPRII ( Figure 19 The melting temperature (Tm) of hMSCs was increased by more than 4°C. The BMP / Smads signaling pathway is a key pathway regulating the differentiation of BMSCs into osteoblasts. To verify whether LBP1C-2 in hMSCs regulates osteogenic differentiation of hMSCs through the BMP / Smads signaling pathway, the phosphorylation levels of Smad1 / 5 / 8 in hMSCs were measured 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 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. Therefore, LBP1C-2 upregulates bone formation by binding to BMPRIA and BMPRII and modulating the BMP / Smads signaling pathway.

[0157] 3-7. LBP1C-2 blocks the interaction between Noggin and BMP.

[0158] LBP1C-2 can directly activate BMPRIA and BMPRII, and the activation of BMPRIA and BMPRII ultimately promotes the phosphorylation of downstream effectors Smad1 / 5 / 8, thereby enhancing osteogenic differentiation and mineralization. Noggin is known for its inhibitory effect on the BMP signaling pathway. Since the interaction between noggin and BMP inhibits osteogenic differentiation, the inventors investigated whether LBP1C-2 activates this signaling pathway by binding to noggin. Figure 21As shown, the effect of LBP1C-2 treatment on Noggin was examined, and the interaction between LBP1C-2 and Noggin was determined by SPR analysis. The results showed that LBP1C-2 had a strong binding affinity to Noggin, with an equilibrium dissociation constant (Kd) of 0.313 μM. Furthermore, to investigate whether this binding inhibited the interaction between Noggin and BMP2, the concentration of BMP2 in the supernatant of LBP1C-2-treated hMSCs was measured. The results indicated that a certain amount of BMP2 was detected in the cell supernatant under Noggin conditions, and a large amount of BMP2 was detected under LBP1C-2 conditions. The upregulation of BMP-2 levels induced by LBP1C-2 was also inhibited by Noggin. These data suggest that LBP1C-2 interacts with Noggin and inhibits the binding of Noggin to BMP2. Figure 22 ).

[0159] Age-related bone loss occurs in both men and women, and osteoporosis-related fractures are a leading cause of morbidity and mortality in the elderly. In this study, the inventors demonstrated for the first time the protective effect of wolfberry on bone in a natural aging model. The LBE, LBD, and LBP1C-2 supplements described in this paper successfully improved age-induced osteoporosis in mice by improving overall bone microstructure. Furthermore, LBP1C-2 was identified as the primary active substance. Mechanistic studies showed that LBE, LBD, and LBP1C-2 regulate bone formation, rather than bone resorption. As a major component of wolfberry, LBP1C-2 can directly bind to BMPRIA and BMPRII, promoting the phosphorylation of downstream effectors Smads, thereby enhancing osteogenic differentiation and mineralization. On the other hand, LBP1C-2 can also directly bind to Noggin and inhibit the interaction between Noggin and BMP. This binding further upregulates the BMP / Smads signaling pathway, increasing bone formation. These results indicate that the compositions described herein have similar bone-forming potential to BMPs and can be used as dietary supplements or pharmaceutical compositions to treat or prevent age-related bone loss.

[0160] Although the experimental period is long, natural aging animal models are currently the most suitable animal models for simulating age-related bone loss, and the inventors have a clear advantage in their research on wolfberry in natural aging mouse models. The natural supplementation method of LBE, LBP, and LBP1C-2 described in this invention has successfully improved age-related bone loss in adult and aging mice.

[0161] BMPR silencing led to the downregulation of bone formation markers Col1α1, Runx2, and Bglap in LBP1C-2-treated preosteoblasts. Furthermore, SPR, protein thermal translocation assays, and CETSA were used to verify whether LBP1C-2 directly interacts with BMPR. The inventors found that LBP1C-2 can directly bind to BMPRIA and BMPRII, and the phosphorylation levels of downstream effectors Smad1 / 5 / 8 are significantly increased. On the other hand, the inventors first discovered that LBP1C-2 not only binds to BMPRIA and BMPRII, but also binds more strongly to Noggin, and this binding blocks the interaction between Noggin and BMP. These two interactions result in LBP1C-2 promoting osteogenic differentiation, indicating that LBP1C-2 mimics the ligand of BMPR and regulates osteogenic differentiation through the BMP / Smads signaling pathway.

[0162] Currently, there are no specific drugs for treating age-related osteoporosis. Conventional anti-osteoporosis drugs have limitations, and their long-term efficacy remains unclear. Clinically, treatment options for osteoporosis mainly fall into several strategies: anti-resorption drugs, such as denosumab, odanacatib, and saracatinib, are commonly used to treat osteoporosis, but inhibiting bone resorption can also interfere with bone remodeling and inhibit bone formation. These osteoporosis-specific drugs often cause serious side effects, such as osteonecrosis of the jaw and atypical fractures. Teriparatide, a recombinant parathyroid hormone, is currently the main bone formation promoting drug for treating osteoporosis, administered via daily subcutaneous injection at appropriate doses. Against this backdrop, natural products, due to their fewer side effects, may offer a better alternative or method for treating osteoporosis. Goji berries, as a traditional Chinese medicine or health food, are used to treat age-related diseases. In this invention, it was demonstrated that the homogeneous polysaccharide LBP1C-2 extracted from wolfberry significantly increased bone mass and bone strength in adult and aging mice after long-term treatment (administration to mice for 4 months), without any adverse reactions. Therefore, LBP1C-2 may be a potentially effective and safe compound suitable for the development of anti-osteoporosis drugs under both pathological and non-pathological conditions.

[0163] In summary, the research in this invention demonstrates that the homogeneous polysaccharide LBP1C-2 from Lycium barbarum can be used to treat or prevent age-related bone loss. Osteoporosis has become an emerging medical and socioeconomic threat due to its high incidence, disability rate, and mortality rate. Lycium barbarum, as a traditional Chinese medicine and also an edible food, has many health benefits. However, whether Lycium barbarum can improve age-related bone loss and what its main active substances are remains unclear. In this invention, the results indicate the role of three Lycium barbarum extracts: Lycium barbarum aqueous extract (LBE), Lycium barbarum polysaccharide (LBP, isolated from LBE), and homogeneous polysaccharide LBP1C-2 (isolated from LBP) in treating or preventing bone loss. Supplementation with LBE, LBP, and LBP1C-2 significantly increased bone mass and bone strength in adult and aging mice. LBE, LBP, and LBP1C-2 also promoted the proliferation, differentiation, and mineralization of hMSCs and osteoblasts in treated mice (in vivo). Furthermore, mechanistically, the main active ingredient, LBP1C-2, directly binds to BMP receptors BMPRIA and BMPRII. Activation of BMPRIA and BMPRII leads to phosphorylation of downstream effectors Smads, thereby enhancing osteogenic differentiation and mineralization. On the other hand, LBP1C-2 also directly binds to Noggin, a potent inhibitor of BMP, and inhibits the interaction between Noggin and BMP. This binding further upregulates the BMP / Smads signaling pathway, thereby promoting bone formation. In summary, these results indicate that Lycium barbarum extract can prevent age-related bone loss, and the main polysaccharide component, LBP1C-2, improves age-related bone loss through BMPRIA, BMPRII, and Noggin in the BMP / Smads signaling pathway. This study also provides a basis for further research on LBE, LBP, and LBP1C-2 as functional food interventions for age-related osteoporosis.

[0164] 3-8. Results of the sulfated derivative of LBP1C-2 (S-LBP1C-2)

[0165] The sulfated derivative of LBP1C-2 was prepared according to the above method, and this derivative is called S-LBP1C-2.

[0166] To investigate the role of sulfated LBP1C-2 (S-LBP1C-2) in promoting osteoblast differentiation and mineralization, human mesenchymal stem cells (hMSCs) were used to examine 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 key roles in bone formation and osteoblast differentiation. RUNX2 is considered a major regulator of osteoblast differentiation. It is crucial for the differentiation of mesenchymal stem cells into osteoblasts and the subsequent maturation of osteoblasts. RUNX2 regulates the expression of multiple genes related to osteoblast differentiation, extracellular matrix formation, and mineralization. On the other hand, SP7 acts downstream of RUNX2 and is considered a marker of mature osteoblasts. It is a transcription factor mainly expressed in osteoblasts and is responsible for osteoblast differentiation and function.

[0167] First, the levels of the major osteogenic markers RUNX2 and SP7 were detected. qPCR analysis after 7 days of hMSC treatment showed that S-LBP1C-2 increased the mRNA levels of RUNX2 and SP7. Figure 23A and 23B Experimental results showed that S-LBP1C-2 has the ability to promote osteogenic differentiation. Furthermore, at an equivalent concentration of 4 μmol, LBP1C-2 upregulated RUNX2 by 2.13-fold, while S-LBP1C-2 upregulated RUNX2 by 3.01-fold. Similarly, LBP1C-2 upregulated SP7 by 1.68-fold, while S-LBP1C-2 upregulated SP7 by 2.01-fold. Therefore, S-LBP1C-2 is more effective than LBP1C-2.

[0168] BALP staining, also known as alkaline phosphatase staining, is a technique used to assess the 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 allows for the observation and quantification of ALP activity, providing an indirect method for assessing osteoblast differentiation and maturation.

[0169] Subsequently, we evaluated the effect of S-LBP1C-2 on ALP staining ( Figure 24A ) and ALP activity ( Figure 24BThe effects of S-LBP1C-2 on ALP secretion and activity were observed. S-LBP1C-2 promoted ALP secretion and activity more than 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. It is commonly used to assess the extent of mineralized matrix deposition by osteoblasts during bone formation. Furthermore, the effects of S-LBP1C-2 on Alizarin Red S staining (…) were investigated. Figure 25A ) and the expression level of osteocalcin BGLAP in hMSCs ( Figure 25B Alizarin Red S staining results indicated that S-LBP1C-2 promotes osteogenic mineralization. BGLAP, also known as osteocalcin, is a protein that plays an important role in bone formation and mineralization. It is mainly synthesized and secreted by osteoblasts, the cells responsible for bone formation. BGLAP is considered a marker of osteoblast activity.

[0170] Furthermore, after 21 days of hMSC treatment, S-LBP1C-2 increased the mRNA level of BGLAP, the gene encoding osteocalcin. Similar to previous findings, at an equivalent concentration of 4 μmol, LBP1C-2 upregulated BGLAP by 2.21-fold, while S-LBP1C-2 upregulated BGLAP by 2.51-fold, indicating that S-LBP1C-2 had a superior effect on BGLAP expression compared to LBP1C-2.

[0171] 4. Experimental samples and control samples:

[0172] Four homogeneous polysaccharides were isolated from Lycium barbarum extract. Of these four homogeneous polysaccharides, only LBP1C-2 exhibited the function described herein. The sugar composition and structure of three of the four homogeneous polysaccharides, including LBP1A1-1, LBP1B-S-2, and LBP1C-2, were analyzed.

[0173] 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 shows that LBP1A1-1 is mainly composed of 1,4-α-Glc, 1,3-β-Gal, and 1,6-β-Gal. Its branches mainly 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 1,3-β-Gal residue and the C-3 position of the 1,6-β-Gal residue in the main chain sugar.

[0174] Equation 4, schematic structure of LBP1A1-1:

[0175]

[0176] 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 shows that LBP1B-S-2 is mainly composed of 1,3-β-Gal and 1,6-β-Gal, with branches primarily including 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 1,3-β-Gal main chain sugar residue and the C-3 position of the 1,6-β-Gal main chain.

[0177] Equation 5, schematic structure of LBP1B-S-2:

[0178]

[0179] Of the four homogeneous polysaccharides with different structures, only LBP1C-2 exhibits 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 of need, and for treating or preventing bone loss, such as age-related bone loss.

[0180] While the invention has been described through exemplary embodiments, it is not limited thereto. Rather, the appended claims should be interpreted broadly to include other variations and embodiments that may be made by those skilled in the art.

Claims

1. Use of a homogeneous polysaccharide and / or a sulfated homogeneous polysaccharide in the preparation of a pharmaceutical composition for treating or preventing bone loss and / or improving bone formation, bone mass or strength in a subject in need thereof; wherein the homogeneous polysaccharide consists of arabinose, galactose, rhamnose and galacturonic acid as monomer units, and has a structure as shown in Formula 1, Formula 1, wherein n is 2-20.

2. The use according to claim 1, wherein, The subject is a mammal.

3. The use according to claim 1, wherein, The subject is a human subject, and / or the bone loss is age-related bone loss.

4. The use according to claim 1, wherein, The composition is for oral administration.

5. The use according to claim 1, wherein, The homogeneous polysaccharide has a molecular weight ranging from 10 kDa to 150 kDa.

6. Use according to claim 5, wherein, The homogeneous polysaccharide has a molar ratio of arabinose, galactose, rhamnose and galacturonic acid monomer units of 49.9:33.6:8.0:8.

5.

7. The use according to claim 1, wherein, The composition further comprises additional polysaccharides isolated from a wolfberry extract; and / or, the homogeneous polysaccharide accounts for more than 15% of all polysaccharides in the composition.

8. The use according to claim 1, wherein, The composition comprises an excipient; The excipient is selected from a solvent, a co-solvent, a colorant, a preservative, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination thereof.

9. The use according to claim 1, wherein, The composition further comprises one or more of a flavonoid, a carotenoid, a polyphenol, a pigment, or any compound isolated from a wolfberry extract.

10. The use according to claim 1, wherein, The homogeneous polysaccharide is the only polysaccharide in the composition.

11. The use according to claim 1, wherein, The sulfated homogeneous polysaccharide has a sulfate substitution degree of 0.5 to 0.9.

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