A composition comprising homopolysaccharides or their derivatives, and a method for preventing and / or treating bone loss using the same.

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 Application) The present application claims the benefit of U.S. Patent Application 63 / 389,082 filed on July 14, 2022, the content of which is incorporated herein by reference.

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

[0003] 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 years and women over 80 years old. For men in the same age group in the European Union, the prevalence was 2.5% and 16% respectively. Bone loss is not limited to postmenopausal women, attention is also focused on osteoporosis in elderly men, and the number of affected people is expected to increase steadily with the aging of the population. Studies have shown that among the elderly aged over 50, one third of women and one fifth of men experience bone loss, which leads to a sharp increase in the risk of fracture and death.

[0004] Currently, more than 200 million people worldwide suffer from osteoporosis. Osteoporosis is characterized by decreased bone mass, reduced bone density, and destruction of the bone microstructure. Therefore, senile osteoporosis has become a new medical and socio-economic threat due to its high morbidity, disability rate and mortality.

[0005] Senile osteoporosis 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 maintaining bone homeostasis. BMPs are members of the transforming growth factor β (TGF-β) superfamily and transmit signals by binding to type I and type II transmembrane serine / threonine receptor kinases. Following ligand-induced assembly of two type I receptors and two type II receptors, constitutively active type II receptor kinases phosphorylate and activate the type I receptors, leading to further phosphorylation of the type I receptors and subsequent Smad or MAPK-mediated signaling, which activates 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 both in vivo and in vitro. Noggin exhibits strong binding affinity to BMPs, preventing them from binding to BMP receptors. BMPs play crucial roles in many stages of bone development. Studies have shown that BMP-2 plays a unique role in postnatal human bone formation. Human recombinant osteomorphonectomy protein 2 (rhBMP2) is widely used in bone tissue engineering as a potent bone growth factor approved by the U.S. Food and Drug Administration. Therefore, discovering natural products with a mimicry effect of BMP-2 that promotes bone formation is of paramount importance. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention provides a composition and its use in a subject requiring it to improve skeletal formation, mass, and / or strength, and / or to treat or prevent bone loss. [Means for solving the problem]

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

[0009] In some embodiments, the subject may be 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 through forced oral administration. The composition may exist in the form of a tablet or in the form of a liquid. For example, in certain embodiments, the composition is a pharmaceutical composition in the form of an orally administered tablet. In certain embodiments, the composition may be a functional composition and may exist 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 other suitable ranges. In the homopolysaccharide, the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid is in the range of 30 to 70:20 to 60:0.1 to 10:0.1 to 10.

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

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

[0014] In other embodiments, the composition further comprises other polysaccharides isolated from goji berry extract. Homopolysaccharides account for 15% or more of the total polysaccharide content in the composition. All polysaccharides are derived from goji berries. For example, the polysaccharide used in this invention is goji berry polysaccharide (referred to as LBP).

[0015] In some embodiments, the composition optionally further comprises one or more of any compounds isolated from flavonoids, carotenoids, polyphenols, pigments, or goji berry extracts.

[0016] In some embodiments, the composition comprises chemically modified derivatives of homopolysaccharides described herein. For example, such derivatives are pharmaceutically acceptable esters or salts thereof. These pharmaceutically acceptable esters or salts are sulfate ester derivatives of homopolysaccharides, known 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 thereof.

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

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

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

[0021] The excipient may be selected from solvents, co-solvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

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

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

[0024] In some embodiments, the homopolysaccharide described herein is the only polysaccharide in the composition. In some other embodiments, the composition further comprises other polysaccharides isolated from Lycium barbarum extract, and / or the homopolysaccharide accounts for 15% or more of the total polysaccharide content in the composition.

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

[0026] In some embodiments, the composition comprises chemically modified derivatives of the homopolysaccharide described herein. For example, such derivatives are pharmaceutically acceptable esters or salts thereof. Said pharmaceutically acceptable esters or salts thereof are sulfate derivatives of the homopolysaccharide, referred to as sulfated polysaccharides.

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

[0028] In another aspect, the present invention also provides a method for producing said composition or homopolysaccharide. Such a method may comprise producing or isolating the homopolysaccharide. The method may further comprise mixing the homopolysaccharide with an excipient. The method may comprise chemical modification, such as sulfation, of the homopolysaccharide. Brief Description of the Drawings

[0029] The details of this invention are best understood by referring to the drawings and reading the following detailed description. Note that, generally, various features in the drawings are not necessarily depicted to scale. Conversely, the dimensions of various features have been arbitrarily enlarged or reduced for clarity. Throughout the specification and drawings, the same reference numeral refers to the same feature. [Figure 1] The trabecular structure of mice in the control group and mice in the experimental group administered goji berry water extract (LBE), goji berry crude polysaccharide (LBP), and goji berry homopolysaccharide (LBP1C-2), respectively, is shown. [Figure 2] Figure 2A shows bone mineral density (BMD), bone volume / tissue volume ratio (BV / TV) (Figure 2B), trabecular bone number (Tb.N) (Figure 2C), trabecular bone thickness (Tb.Th) (Figure 2D), and trabecular bone space (Tb.Sp) (Figure 2E) for the control group mice and the experimental group mice administered LBE, LBP, and LBP1C-2, respectively. [Figure 3] The maximum load-bearing force a of the control group mice and the experimental group mice administered LBE, LBP, and LBP1C-2, respectively, is shown (maximum load-bearing force of the femur was evaluated using a three-point bending test). [Figure 4] The bone formation rates for the control group of mice and the experimental group of mice administered LBE, LBP, and LBP1C-2, respectively, are shown. The abbreviation "BFR / BS" represents the bone formation rate per unit bone surface. [Figure 5] The bone mineralization rate (MAR) of the control group of mice and the experimental group of mice administered LBE, LBP, and LBP1C-2, respectively, is shown. [Figure 6] The serum osteocalcin content of mice in the control group and in the experimental group mice administered LBE, LBP, and LBP1C-2, respectively, is shown. [Figure 7] The serum content of type I procollagen N-terminal propeptide (PINP) in control mice and in experimental mice administered LBE, LBP, and LBP1C-2, respectively, is shown. [Figure 8] In CFU (colony-forming unit) experiments, osteoblast proliferation is shown in human mesenchymal stem cells (hMSCs) treated with LBE, LBP, and LBP1C-2, as well as in hMSCs from the control group. [Figure 9] The expression levels of osteocalcin in hMSCs treated with LBE, LBP, and LBP1C-2, as well as in hMSCs from the control group, are shown. [Figure 10] This shows the enzymatic activity of alkaline phosphatase (ALP) in hMSCs treated with LBE, LBP, and LBP1C-2, as well as in control hMSCs. [Figure 11] This shows the expression levels of genes related to bone formation in the femur of control mice and mice administered LBP1C-2. [Figure 12] This shows the expression levels of genes related to bone resorption in the femur of control mice and mice administered LBP1C-2. [Figure 13-15] The expression levels of the osteogenic genes Runx2, Col1α, and Bglap in preosteoblasts treated with LBP1C-2 after BMPR1a and BMPR2 gene interference, as well as in the control group, are shown. [Figure 16-17] The results of the direct interaction between LBP1C-2 and BMPRIA and BMPII in the control group and the experimental group administered LBP1C-2, as represented by surface plasmon resonance (SPR), are shown. [Figure 18-19] The results of direct interactions between LBP1C-2 and BMPRIA and BMPRII, measured using fluorescence-based protein thermal shift spectroscopy, are shown. [Figures 20A-20B]Figure 20A shows the phosphorylation levels of Smad1 / 5 / 8 in hMSCs treated with LBP1C-2 and in hMSCs from the control group. Figure 20B analyzes the effect of LBP1C-2 on Smad1 / 5 / 8 phosphorylation levels, analyzed by Western blot. GAPDH is used as a loading control. [Figure 21] The results of the direct interaction between LBP1C-2 and Noggin, measured by surface plasmon resonance (SPR), are shown. [Figure 22] This shows the release levels of bone morphogenetic protein 2 (BMP2) in hMSCs treated with LBP1C-2 and in hMSCs from the control group. [Figures 23A-23B] The levels of the early bone formation markers RUNX2 (Figure 23A) and SP7 (Figure 23B) in hMSCs treated with LBP1C-2, S-LBP1C-2 (sulfated LBP1C-2), and hMSCs in the control group are shown. [Figure 24A] The results of alkaline phosphatase (ALP) staining (day 7) in hMSCs treated with LBP1C-2, S-LBP1C-2, and hMSCs in the control group are shown. [Figure 24B] This shows the ALP enzyme activity in LBP1C-2, S-LBP1C-2, and hMSCs in the control group. [Figure 25A] The results of alizarin red S staining (day 21) in hMSCs treated with LBP1C-2, S-LBP1C-2, and hMSCs in the control group are shown. [Figure 25B] The expression levels of osteocalcin in hMSCs treated with LBP1C-2, S-LBP1C-2, and control hMSCs are shown. [Modes for carrying out the invention]

[0030] The endpoints of the ranges and any values ​​disclosed herein are not limited to precise ranges or values, and should be understood to include values ​​that approximate these ranges or values. In the case of numerical ranges, one or more new numerical ranges can be obtained by combining the endpoint values ​​of each range, the endpoint values ​​of each range with individual point values, and the individual point values ​​with each other. These numerical ranges should be considered as specifically disclosed herein.

[0031] In this invention, the singular forms "one," "one type," and "the" include the plural, and references to specific numerical values ​​include at least that specific value unless the context clearly indicates otherwise. Therefore, for example, a reference to "additive" is a reference to one or more such compounds and equivalents known to those skilled in the art.

[0032] When the antecedent “about” is used to express a value as an approximation, it is understood that the particular value may form another embodiment. As used herein, “about X” (where X is a number) preferably means including ±10% of the stated value, and also includes the endpoint value. For example, the phrase “about 8” preferably includes values ​​from 7.2 to 8.8, and also includes the endpoint value.

[0033] As another example, the phrase "approximately 8%" preferably (but not always) means that the value includes values ​​between 7.2% and 8.8%. Where present, all ranges are inclusive and combinable. For example, if the range "1 to 5" is given, the given range should be interpreted to include ranges such as "1 to 4", "1 to 3", "1 to 2", "1 to 2, and 4 to 5", "1 to 3 and 5", and "2 to 5". Also, if alternatives are actively offered, such items may be interpreted to mean that any alternatives may be excluded, for example, by negative limitations in the claims.

[0034] For example, if the range "1-5" is given, it can be interpreted that the given range includes cases where 1, 2, 3, 4, or 5 is negatively excluded. Therefore, the enumeration "1-5" can be interpreted as "1 and 3-5, but 2 is not included," or simply "but 2 is not included."

[0035] Any component, element, attribute, or step expressly cited herein is intended to be expressly excluded in the claims, regardless of whether such component, element, attribute, or step is described as an alternative or separately. Any component, element, attribute, or step positively cited herein is intended to be expressly excluded in the claims, regardless of whether such component, element, attribute, or step is described as an alternative or separately.

[0036] The terms “subject” and “patient” are used interchangeably herein. As used herein, the term “patient” refers to mammals, preferably animals, including non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys and humans), and preferably humans. In some embodiments, the subject is a non-human animal, such as a livestock animal (e.g., a horse, pig, or cattle) or a pet (e.g., a dog or cat). In certain embodiments, the subject is a human. In other embodiments, the subject is an adult.

[0037] As used herein, the term “drug” means any molecule, compound, method and / or substance used to prevent, treat, manage and / or diagnose a disease or condition.

[0038] As used herein, the term “effective dose” means a dose of one treatment sufficient to prevent the progression, recurrence, or onset of a disease or condition and one or more of its symptoms; to enhance or improve the preventive effect of another treatment; to reduce the severity of a disease or condition; to shorten the duration of a disease or condition; to alleviate one or more of the symptoms of a disease or condition; to prevent the progression of a disease or condition; to induce regression of a disease or condition; and / or to enhance or improve the therapeutic effect of another treatment.

[0039] The aforementioned disease or condition (e.g., medical condition) is associated with bone loss or bone formation, bone mass, and / or bone strength.

[0040] As used herein, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory authority for use in animals, and more specifically for use in humans, or that it is listed in the United States Pharmacopeia, the European Pharmacopoeia, or any other recognized pharmacopoeia.

[0041] As used herein, the term “therapeutic agent” refers to any molecule, compound, and / or substance used to treat and / or manage a disease or condition.

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

[0043] As used herein, the terms “treatment,” “treatment method,” and “treatment” refer to reducing or inhibiting the progression and / or duration of a disease or condition, reducing or improving the severity of a disease or condition such as cancer, and / or improving one or more symptoms as a result of administering one or more treatments to a subject, in the context of administering a treatment to the subject.

[0044] As used herein, the term “excipient” refers to an inert substance that functions as a carrier or medium for a drug or other active substance. Suitable examples of 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.

[0045] In this specification, “monomer units” or “monomer units” in polysaccharides refer to monoglycosyl units, single sugar molecular units, or other basic chemical units. These units are understood to be monosaccharide groups 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 according to their molecular size, providing the molecular weight or molecular weight distribution of the material. The homopolysaccharides described herein have a uniform molecular weight and contain only one peak in GPC. Homopolysaccharides or derivatives thereof are provided and used in this disclosure.

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

[0048] Goji berries, belonging to the genus Lycium in the Solanaceae family, are used in traditional Chinese medicine and health foods in China and other countries. They are listed as a related species that can be used for medicinal and edible purposes. 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 unclear.

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

[0050] In recent research, the inventors extracted and separated aqueous extracts, i.e., goji berry extract (LBE) and crude polysaccharides (LBP), from goji berry fruit using a unique separation method. A homopolysaccharide called LBP1C-2 (e.g., yielded at 0.02% in dried fruit) was purified from LBP. LBP1C-2 was found to be pectin. However, it is unclear whether LBP1C-2 affects bone remodeling.

[0051] Goji berry polysaccharide samples can promote the proliferation, differentiation, and mineralization of osteoblasts. The polysaccharides, extracted from goji berry fruit, contain six monosaccharides: galactose, glucose, rhamnose, arabinose, mannose, and xylose. In vitro experiments were conducted using human mesenchymal stem cells (hMSCs).

[0052] However, the main material basis, mechanism of action, and target of action of goji berries to improve age-related bone loss have not yet been clarified.

[0053] In this invention, the inventors studied the effects of LBE, LBP, and LBP1C-2 on naturally aged mice and determined their target and mechanism of action. LBP1C-2, a homopolysaccharide obtained from goji berry extract, was found to be the main active ingredient of goji berries. LBP1C-2 directly binds to BMPRIA and BMPRII, promoting the phosphorylation of Smads, thereby promoting bone formation, differentiation, and mineralization. It can also directly bind to Noggin, inhibiting the interaction between Noggin and BMPs, and increasing bone formation. This study also provides a research basis for explaining the therapeutic effects of goji berry extract compositions and offers scientific evidence for 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. These methods can be used to treat or prevent bone loss, such as age-related bone loss, in subjects who require them.

[0055] According to some embodiments, such a composition comprises 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, which is administered to a subject in need. The homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units or molecules. Such a method involves administering an effective amount of the composition containing the homopolysaccharide to a subject in need.

[0056] In some embodiments, the subject may be 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 through forced oral administration. The composition may exist in the form of a tablet or in the form of a liquid. For example, in certain embodiments, the composition is an orally administered 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 and may exist 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 monomer units of arabinose, galactose, rhamnose, and galacturonic acid is in the range of 30 to 70:20 to 60:0.1 to 10:0.1 to 10.

[0059] For example, in some embodiments, the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid in the homopolysaccharide is 49.9:33.6:8.0:8.5. The molecular weight may be a specific value or a narrow range of about 10 kDa to about 150 kDa. Such molecular weight values ​​may be weight-average molecular weight (Mw) or number-average molecular weight (Mn). Since polysaccharides are homogeneous and have a very narrow and uniform molecular weight distribution, the Mw and Mn values ​​are considered to be close. The polydispersity (PD) index, i.e., the ratio of Mw to Mn, may be in the range of 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 the polysaccharide may vary depending on factors such as the growth environment and harvest time of the goji berry raw material. Therefore, the resulting homopolysaccharides may have different molecular weights. However, the polysaccharides obtained in each batch are homogeneous, meaning their molecular weight is either homogeneous or has a narrow distribution. The molecular weight can be controlled by controlling the quality of the raw material, such as by ensuring the same growth environment and pre-harvest growth time.

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

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

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

[0064] In some embodiments, the composition optionally further comprises one or more of any compounds isolated from flavonoids, carotenoids, polyphenols, pigments, or goji berry extract.

[0065] Goji berry extract (LBE) can be obtained by extracting goji berries with water, and this extract may exist 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 freeze-drying to obtain one or more homopolysaccharides in the form of a dry powder.

[0066] In some embodiments, the composition comprises goji berry extract (LBE). This goji berry extract contains polysaccharides (or goji polysaccharides), goji berry flavonoids, carotenoids, polyphenols, and goji berry pigments. Each component may be present only once, or there may be two or more components of the same type. For example, the composition may contain two or more polysaccharides, two or more goji berry flavonoids, two or more carotenoids, two or more polyphenols, and / or two or more goji berry pigments.

[0067] In some embodiments, the polysaccharide content in the goji berry 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.%) based on the total dry weight of the extract, goji berry flavonoids are in the range of about 0.1 wt.% to about 5.0 wt.%, carotenoids are in the range of about 0.1 wt.% to about 3.0 wt.%, polyphenols are in the range of about 0.1 wt.% to about 8.0 wt.%, and goji berry pigment is in the range of about 0.1 wt.% to about 8.0 wt.%.

[0068] In some embodiments, the polysaccharide content is more preferably about 50.0 wt.% to about 70.0 wt.%. The dry weight is the equivalent weight corresponding to the dry powder (water-free) extract. The extract may contain other trace residues. The dry powder extract can be mixed with water to provide an extract in the form of an aqueous solution having the selected concentrations described herein.

[0069] In some embodiments, goji berry extract (LBE) exists in powder form. In some embodiments, the goji berry extract is dissolved in a solvent such as water to provide an aqueous solution having a constant concentration, for example, in the range of 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.%.

[0070] Lycium berry polysaccharides, or polysaccharides (LBPs), existing in powder form can be further purified from LBEs in powder form. For example, LBEs can be dissolved in water and separated through a separation column. Lycium berry polysaccharides (LBPs) may contain various other polysaccharides that can be further separated.

[0071] The homopolysaccharide LBP1C-2 is isolated from LBE and / or LBP. Based on high-performance gel permeation chromatography (HPGPC) analysis, LBP1C-2 showed a single symmetrical peak, indicating that it is a homopolysaccharide. Sugar composition analysis revealed that LBP1C-2 consists 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 includes a main chain consisting of alternating 1,2-linked α-Rhap and 1,4-linked α-GalpA, and branching 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] Equations 1, 2, and 3 show the structure of LBP1C-2, illustrating the same structure in three different forms.

[0074] Referring to Equation 1-3, LBP1C-2 is composed of Ara, Gal, Rha, and GalA, with a molar ratio of 49.9:33.6:8.0:8.5. Structural analysis shows that LBP1C-2 has 1,2-α-Rha and 1,4-α-GalA as its main chain, and its branches include 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, with these branches attached to the C-4 position of the skeletal sugar residue of 1,2,4-α-Rha.

[0075] The repeating unit of LBP1C-2 includes the structural part shown in Equation 3, and comprises a skeleton (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] TIFF0007920430000001.tif250170

[0077] TIFF0007920430000002.tif171170

[0078] TIFF0007920430000003.tif201170

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

[0080] The 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 who require it.

[0081] In some embodiments, the composition includes chemically modified derivatives of the homopolysaccharides described herein. For example, the derivative is a pharmaceutically acceptable ester or salt thereof. The pharmaceutically acceptable ester or salt is a sulfated ester derivative of a homopolysaccharide, called a sulfated polysaccharide. During the synthesis of the sulfated homopolysaccharide, the hydroxyl group in the homopolysaccharide reacts with a modifier such as chlorosulfonic acid to form an -O-SO3H group. The molecular weight after modification is the same as 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, for example, from 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 there are 0.74 sulfate substituents on each hexose or pentose unit. Sulfate substitution can be combined with proteins to improve their biological activity.

[0083] The composition may be a pharmaceutical composition, a functional composition, and / or a supplement. For example, the composition is an orally administered pharmaceutical composition. In embodiments of the present invention, the water extract of the goji berry is administered by forced oral administration or orally, but is not limited to these. Any form of administration of the composition into the stomach may be appropriate.

[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 appropriate amount. For example, in some embodiments, the effective daily dose of the composition (converted as the amount of homopolysaccharides as specified herein) is in the range of 10 mg / kg to 500 mg / kg, based on the total weight of homopolysaccharides / body weight of the subject. In some embodiments, the dose of goji berry extract (LBE) or LBP or LBP1C-2 is in the range of 4 mg / kg to 70 mg / kg per day (total dry weight of LBP1C-2 per day / body weight of a human or other subject), 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 other appropriate 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 goji berry 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 can be administered once or twice or more per day. In embodiments of the present invention, mice are administered LBE, LBP, or LBP1C-2 once a day.

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

[0088] In some embodiments, these compositions may be taken together with drinks, foods, or related ingredients. The goji berry water extract according to the present invention embodies the principle of "food as medicine" and can be used as a health food. There are no particular restrictions on the method of producing the food or health food. For example, it may be in the form of tablets, drinks, candies, etc. Each food formulation may contain, in addition to homopolysaccharides, other formulations used in the art. Other formulations may be selected by those skilled in the art according to a particular formulation or use.

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

[0090] The present invention also provides the use of homopolysaccharides or derivatives thereof as described herein in the manufacture of agents for treating any of the disorders described herein.

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

[0092] The features and effects of the present invention will be explained with reference to examples and test examples. However, the following examples and test examples are merely illustrative and do not limit the scope of the present invention.

[0093] Examples 1. Preparation of materials 1-1. Preparation of Goji Berry Extract The preparation process for the goji berry extract ("LBE") is as follows: The goji berries were sourced from Zhongning County, Yinchuan City, Ningxia Hui Autonomous Region, China. The dried goji berries were washed 3 to 5 times, then soaked in redistilled water (pH=7) at room temperature for 2 hours and crushed. 5 to 8 times the amount of neutral water was added to the soaked goji berry powder and mixed uniformly. The mixture was 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 filtrates were combined and evaporated under vacuum at 30-55°C to remove water and obtain a concentrate. The obtained concentrate was freeze-dried into a powder and stored in a desiccator so that it could be used at the appropriate concentration in subsequent experiments.

[0094] In the experiment, an exemplary goji berry extract was used. This exemplary extract is an aqueous solution of the extract at a concentration of 0.4 g / mL (dry powder of extract or dry weight / volume of extract). This concentration is for illustrative purposes only. The aqueous extract can be adjusted to any suitable concentration in the range of, for example, about 0.1 g / mL to 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 goji berry extract (LBE) used in this invention mainly contains water-soluble goji berry polysaccharides, goji berry flavonoids, carotenoids, polyphenols, and pigments. In an exemplary LBE, the polysaccharide content ranges from about 50.0 wt.% to 70.0 wt.% (e.g., about 54% to 56% or 54%) based on the total equivalent weight of the extract in dry powder form, the goji berry 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 goji berry pigment ranges from about 0.1 wt.% to 8.0 wt.%. The LBE is in powder form and is soluble in water or brine. LBE of the same composition was used for comparison.

[0096] 1-2. Separation of Lycium berry polysaccharides and homopolysaccharides Lycium polysaccharides ("LBP") and homopolysaccharides such as those labeled LBP1C-2 as described herein can be isolated from LBE or Lycium berries, for example, by the following exemplary methods.

[0097] The extraction method for the polysaccharides described above may include the following: Dried fruit is crushed, 15 to 30 times its volume of deionized water is added and mixed well, 3 wt.% cellulase, 1 wt.% amylase, and 0.5 wt.% papain are added, and the mixture is extracted at 55 to 60°C for 1 hour. After increasing the temperature to inactivate the enzymes, the mixture is centrifuged, the resulting filtrate is concentrated, dialyzed, concentrated again, 5 times its volume of 95% ethanol is added, and the mixture is centrifuged to obtain a precipitate. The precipitate is washed three times alternately with anhydrous ethanol and acetone, and then vacuum dried to obtain crude polysaccharides (LBP).

[0098] The above method further includes the following: Crude polysaccharides are dissolved in 10 to 15 times their volume of water, centrifuged, the supernatant is collected, fractionally purified through a diethylaminoethylcellulose (DEAE) anion exchange column, and then eluted sequentially with water, 0.05 M, 0.1 M, and 0.2 M sodium chloride. The fraction eluted with 0.2 M sodium chloride is collected, concentrated, dialyzed, and freeze-dried to obtain pre-purified goji berry polysaccharide (LBP1C). The obtained polysaccharide LBP1C is dissolved in 0.2 M sodium chloride, centrifuged, the supernatant is collected, and eluted by Sephacryl-300 (RTM: poly((propenyldextran)-co-N,N'-methylenebisacrylamide)) column elution. The eluted fraction is collected, concentrated, dialyzed, and freeze-dried to obtain 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 result figure showed only one symmetrical peak. Referring to dextran standards of known molecular weights used in 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 sulfurized according to 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 consisting of chlorosulfonic acid and pyridine (3:1, v / v) was added under ice bath. The resulting mixture was then stirred at 40°C for 4 hours, cooled, and neutralized with 5 M NaOH. First, the solution was dialyzed against saturated NaHCO3, and then against distilled water. The residue was freeze-dried to obtain the sulfated derivative S-LBP1C-2. The degree of sulfation was calculated according to the chlorosulfonic acid-pyridine method using formula (1) as follows. TIFF0007920430000004.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 referring to the standard curve for sulfate content detected by the chlorosulfonic acid-pyridine method.

[0104] 2. Biological experiments 2-1. Animals and Grouping 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. The mice were randomly divided into adult groups (15 mice each) and aged groups (6 mice each). The adult and aged groups were further divided into four groups: control, LBE, LBP, and LBP1C-2. The control group mice were force-fed distilled water (10 mL / kg) and then force-fed the three extracts 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 1986 UK Animal (Scientific Procedures) Act and related guidelines.

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

[0106] The culture medium used for differentiation 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 two days. The cell cultures were incubated at 37°C in a humid atmosphere of 5% CO2.

[0107] 2-3. Detection of serum bone formation markers After treating mice with drugs, they were anesthetized, and blood samples were collected from the fundus of the mouse eyes. Serum was obtained by centrifugation (2000 × g, 20 min, 4°C). Osteocalcin and PINP content were detected using an osteocalcin ELISA kit and a PINP ELISA kit, respectively, according to the experimental steps of each kit. Osteocalcin and PINP are typical bone formation markers.

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

[0109] In short, a right femoral sample was scanned in vitro and in vitro under the following conditions: effective pixel size of 8.89 μm, voltage of 60 kV, current of 220 μA, exposure time of 1500 ms, 1° exposure per scan, and 360 exposures in total. The entire image dataset contained 1536 tomographic images, with an effective pixel size of 8.89 μm in all three axes. Three-dimensional (3D) visualizations were reconstructed using the 2D images, and parameter analysis was performed using Inveon Research Workplace (Siemens). The region of interest (ROI) for femoral trabecular analysis was 1–2 mm below the distal femoral growth plate.

[0110] 2-5.3-point bending test The three-point bending test was performed using a bone biomechanical testing apparatus (Instron 4302, Instron, Norwood, Massachusetts) for small animal bone strength testing. The bone strength of fresh femurs from each group of mice was detected using the three-point bending test immediately after harvesting. The three-point bending test used two end support points and one central load point. Biomechanical measurement data was collected from load-deformation curves.

[0111] 2-6. Surface Plasmon Resonance (SPR) Analysis The binding affinity of LBP1C-2 to proteins was measured using the 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, LBP1C-2 at various concentrations was injected into the tip. All operations 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 to a 1:1 binding model using Biacore T200 Evaluation Software version 1.0.

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

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

[0115] 2-8. Quantitative Real-Time PCR (RT-PCR) Experiment 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 the 7500 real-time PCR system (Applied Biosystems) as described above. Changes in mRNA expression in rats of each treatment group were evaluated by the 2-ΔΔCq method.

[0116] 2-9. Western blot analysis 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 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. Subsequently, the membrane was blocked for 1 hour with a TBST solution containing 5% skim milk powder (TBST: 10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4).

[0118] Following the blocking step, the membranes were incubated overnight at 4°C with the primary antibody. The primary antibodies used herein 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 with horseradish peroxidase (HRP)-labeled secondary antibody (1:2000 dilution, Zhong-shan Jin-qiao, Beijing, China) at room temperature for 1 hour. The membranes were washed three times with TBST. To visualize the protein signal, each membrane was treated with ECL solution (Thermo Scientific, Waltham, USA), and the signal was detected using a ChemiDoc XRS+ molecular imaging instrument (Bio-Rad).

[0119] 2-10. ALP staining and ALP assay ALP staining was monitored using the Vector Blue substrate kit (program number SK-5300, Vector Laboratories). hMSCs were placed in a 24-well plate at a rate of 1 × 10⁶ per well, according to the protocol. 5 The cells were inoculated at the specified density 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 kept out of light. The ALP assay was performed based on the previously described steps.

[0120] 2-11. Alizarin Red S staining hMSCs were placed in a 24-well plate, 1 x 10⁶ hMSCs per well. 5 The cells were inoculated at the specified density 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 powder in distilled water at the time of use and administered once daily. Control mice received forced oral administration of distilled water, while treatment mice received forced oral administration of LBE (40 mg / kg), LBP (40 mg / kg), or LBP1C-2 (40 mg / kg) 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 total weight of homopolysaccharide / subject body weight / day. The doses of LBE and LBP (40 mg / kg) were calculated as total dry weight of LBE or LBP / subject body weight.

[0122] 3-1. Increased bone mass and improved stability through LBE, LBP, and LBP1C-2 treatments. Aging is strongly associated with bone loss and deterioration of bone microstructure. Therefore, we investigated whether LBE, LBP, and LBP1C-2 affect trabecular structure parameters in mice using microcomputed tomography (μCT). Morphological parameters of the femur of treated and control mice were quantified using μCT. LBE, LBP, and LBP1C-2 treatment improved bone tissue morphology compared to adult and aged control groups (Figure 1).

[0123] Compared to adult and aged control groups, LBE, LBP, and LBP1C-2 treatments improved age-related bone loss. This was because mice treated with LBE and LBP showed higher BMD (bone mineral density) (Figure 2A), higher bone volume ratio (BV / TV) (Figure 2B), higher trabecular bone number (Tb.N) (Figure 2C), thicker trabecular bone thickness (Tb.Th) (Figure 2D), and smaller trabecular bone space (Tb.Sp) (Figure 2E). *p<0.05, **p<0.01 (compared to adult control group); #p<0.05, ##p<0.01 (compared to aged control group).

[0124] Next, we investigated whether LBE, LBP, and LBP1C-2 could modulate the biomechanical properties of bone. The experimental results showed that the maximum load capacity 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 through LBE, LBP, and LBP1C-2 treatments The rates of bone formation and mineralization of the femur in vivo were analyzed. Bone formation rate (BFR) indicates the amount of new bone formed per unit time. This is typically measured by quantifying the amount of mineralized bone matrix or osteogenic matrix (unmineralized bone) formed within a specific bone surface region. Mineralization rate (MAR) focuses specifically on the mineralization rate of newly formed bone matrix.

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

[0127] The effect of LBP1C-2 was even more pronounced. As described 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 elderly control group)

[0128] 3-3. Promotion of osteoblast proliferation, differentiation, and mineralization by LBE, LBP, and LBP1C-2 We investigated whether LBE, LBP, and LBP1C-2 affect the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts, and gained a deeper understanding of these specific compounds. We used hMSCs to analyze osteoblast proliferation, differentiation, and mineralization. The results showed that LBE, LBP, and LBP1C-2 significantly promoted CFU-F (fibroblast colony-forming unit) colony formation and promoted hMSC proliferation (Figure 8).

[0129] Similarly, LBE, LBP, and LBP1C-2 significantly promoted osteoblast differentiation and mineralization. Furthermore, cells treated with LBE, LBP, and LBP1C-2 showed significant improvements in both osteoblast differentiation markers: Bglap expression levels (Figure 9) and ALP enzyme activity (Figure 10). All of these results indicate that LBE, LBP, and LBP1C-2 treatment promoted the differentiation and mineralization of hMSCs into osteoblasts.

[0130] 3-4. LBP1C-2 promotes bone formation and has little effect on bone resorption. To elucidate the mechanism of action of LBP1C-2, the expression of osteogenic and osteoresorbing genes was analyzed by RT-PCR. Cells treated with LBP1C-2 showed significant upregulation of mRNA expression of osteogenic differentiation markers such as Col1α, Runx2, Bglap, and Opn (Figure 11). To further confirm the effect of LBP1C-2 on bone resorption, mRNA expression of osteoclast markers including Ocstamp, Oscar, Opg, and Rankl was measured in the femur, but no significant difference was observed (Figure 12).

[0131] These results indicate that LBP1C-2 promotes bone formation and does not affect bone resorption.

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

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

[0134] 3-6. Direct binding of LBP1C-2 to BMPRIA and BMPRII, and their massive activation. To investigate whether LBP1C-2 directly interacts with BMPRIA, we used SPR to measure their interactions. The results showed that these interactions were dose-dependent. The equilibrium dissociation constant (Kd) for LBP1C-2 and BMPRIA was approximately 3.6 μmol / L (Figure 16), and the Kd for 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. Upon reaching a plateau, LBP1C-2 dose-dependently increased the melting temperature (Tm) of BMPRIA (Figure 18) and BMPRII (Figure 19) by more than 4°C. The BMP / Smad signaling pathway is a crucial pathway that regulates the differentiation of BMSCs into osteoblasts.

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

[0137] These data demonstrate 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 upregulated osteogenicity by binding to BMPRIA and BMPRII and modulating the BMP / Smad signaling pathway.

[0138] 3-7. Blocking of the interaction between Noggin and BMP by LBP1C-2 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 promoting osteogenic differentiation and mineralization. Noggin is known to play a role in inhibiting the BMP signaling pathway. Since the interaction between Noggin and 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 for Noggin and an equilibrium dissociation constant (Kd) of 0.313 μM. Furthermore, to 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, while a large amount of BMP2 was detected under LBP1C-2 conditions. 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 Noggin's binding to BMP2 (Figure 22).

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

[0142] Mechanistic studies have revealed that LBE, LBD, and LBP1C-2 regulate bone formation rather than bone resorption. LBP1C-2, the main component of goji berries, can directly bind to BMPRIA and BMPRII, promoting the phosphorylation of the downstream effector Smad and accelerating the differentiation and mineralization of bone formation. On the other hand, LBP1C-2 can also directly bind to Noggin, inhibiting the interaction between Noggin and BMP. 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 cycle is relatively long, the natural aging animal model is currently the most suitable animal model for simulating age-related bone loss, and the inventors' study of goji berries in a 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 aged mice.

[0145] BMPR silencing resulted in downregulation of the osteogenic markers Col1α1, Runx2, and Bglap in preosteoblasts treated with LBP1C-2. Furthermore, we investigated whether LBP1C-2 directly interacts with BMPR using SPR, protein thermal shift assays, and CETSA.

[0146] The inventors discovered that LBP1C-2 directly binds to BMPRIA and BMPRII, significantly enhancing the phosphorylation levels of downstream effectors Smad1 / 5 / 8. Furthermore, the inventors were the first to discover that LBP1C-2 not only binds to BMPRIA and BMPRII, but also binds more strongly to Noggin, and that this binding blocks the interaction between Noggin and BMP. These two interactions demonstrate that LBP1C-2 promotes osteogenic differentiation, mimicking the ligand of BMPR and regulating osteogenic differentiation via the BMP / Smad signaling pathway.

[0147] There are currently no drugs specifically formulated to treat senile osteoporosis, and conventional osteoporosis medications have certain limitations, with their long-term effects unclear. Clinically, treatment options for osteoporosis can be broadly divided into the following strategies. Bone resorption inhibitors such as denosumab, odanacatib, and saracatinib are commonly used to treat osteoporosis, but inhibiting bone resorption can also hinder bone remodeling, potentially impeding bone formation. These osteoporosis medications often cause serious side effects such as osteonecrosis of the jaw and atypical fractures.

[0148] Teriparatide is a recombinant parathyroid hormone and is currently the leading drug for promoting bone formation in the treatment of osteoporosis, administered daily by subcutaneous injection at appropriate doses. Given this background, natural products may offer a superior alternative or treatment for osteoporosis due to their fewer side effects. Goji berries have been used in traditional Chinese medicine and health foods to treat age-related diseases.

[0149] In this invention, it was demonstrated that LBP1C-2, a homopolysaccharide extracted from goji berries, significantly increased bone mass and bone strength in adult and aged mice after long-term administration (administered 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 in both pathological and non-pathological conditions.

[0150] As described above, research on the present invention demonstrates that LBP1C-2, a homopolysaccharide derived from goji berries, can be used to treat or prevent age-related bone loss. Osteoporosis is a new medical and socioeconomic threat due to its high morbidity, disability rate, and mortality rate. Goji berries, a traditional Chinese medicine and food, have many health benefits. However, it is unclear whether goji berries can improve age-related bone loss and what their main active ingredients are.

[0151] In this invention, research has shown that extracts from three types of goji berries, namely goji berry water extract (LBE), goji berry 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 bone strength in adult and aged mice. LBE, LBP, and LBP1C-2 also promoted the proliferation, differentiation, and mineralization of osteoblasts in hMSCs and treated mice (in vivo). Furthermore, from a mechanistic perspective, 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 the downstream effector Smad, promoting bone formation, 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, promoting bone formation. As described above, the results indicate that goji berry extract can prevent age-related bone loss, and that the main polysaccharide component LBP1C-2 improves age-related bone loss via the BMP / Smad signaling pathways BMPRIA, BMPRII, and Noggin. This study also provides a basis for further research on LBE, LBP, and LBP1C-2 as functional foods for intervention in senile osteoporosis.

[0154] 3-8. Results of the sulfated derivative of LBP1C-2 (S-LBP1C-2) The sulfated derivative of LBP1C-2 is 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 considered to be the main regulator of osteoblast differentiation.

[0155] RUNX2 is essential for the differentiation of mesenchymal stem cells into osteoblasts and subsequent osteoblast maturation. RUNX2 regulates the expression of several genes involved in osteoblast differentiation, extracellular matrix formation, and mineralization. SP7, on the other hand, functions downstream of RUNX2 and is thought to be 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 the major bone formation markers RUNX2 and SP7. qPCR detection 7 days after treatment with hMSCs showed that S-LBP1C-2 increased the mRNA levels of RUNX2 and SP7 (Figures 23A and 23B). These experimental results demonstrate that S-LBP1C-2 has the ability to promote bone formation and differentiation.

[0157] Furthermore, at equivalent concentrations of 4 μmol, LBP1C-2 upregulated RUNX2 by 2.13 times, while S-LBP1C-2 upregulated RUNX2 by 3.01 times. Similarly, LBP1C-2 upregulated SP7 by 1.68 times, while S-LBP1C-2 upregulated SP7 by 2.01 times. 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 evaluating osteoblast differentiation and maturation.

[0159] Next, the effects of S-LBP1C-2 on ALP staining (Figure 24A) and ALP activity (Figure 24B) were evaluated. S-LBP1C-2 was observed to enhance ALP secretion and ALP activity. The enhancement of ALP secretion and activity by S-LBP1C-2 surpassed 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 assess the degree of mineralization matrix deposition during osteoblast formation. Furthermore, the expression levels of S-LBP1C-2 (Figure 25A) and osteocalcin BGLAP in hMSCs (Figure 25B) were detected. The results of the alizarin red staining indicated that S-LBP1C-2 promotes osteoblast formation and mineralization. BGLAP, also known as osteocalcin, is a protein that plays a crucial role in osteoblast formation and mineralization. It is primarily synthesized and secreted 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, 21 days after treatment of hMSCs. Similar to previous findings, at equivalent concentrations of 4 μmol, LBP1C-2 was observed to upregulate BGLAP 2.21 times, while S-LBP1C-2 upregulated BGLAP 2.51 times, indicating that S-LBP1C-2 has a superior effect on BGLAP expression compared to LBP1C-2.

[0162] 4. Experimental samples and control samples Four homopolysaccharides were isolated from goji berry extract. Of these four homopolysaccharides, only LBP1C-2 exhibits the function 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 Equation 4, LBP1A1-1 is composed of rhamnose (Rha), arabinose (Ara), glucose (Glc), and galactose (Gal), with 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 branching mainly includes terminal (T)-β-Rha, T-β-Gal, T-α-Ara, T-β-Ara, and 1,5-α-Ara. These branchings are connected to the C-6 position of the main chain sugar 1,3-β-Gal residue and the C-3 position of 1,6-β-Gal.

[0164] TIFF0007920430000005.tif220170

[0165] Referring to Equation 5, LBP1B-S-2 is composed of Rha, GlcA (glucuronic acid), Gal, and Ara, with their molar ratios being 3.13:3.95:39.37:53.55. Structural analysis reveals that LBP1B-S-2 is primarily composed of 1,3-β-Gal and 1,6-β-Gal, with its branching mainly 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 branchings are connected to the C-6 position of the main chain sugar residue 1,3-β-Gal and the C-3 position of 1,6-β-Gal.

[0166] TIFF0007920430000006.tif199170

[0167] Of the four homopolysaccharides with different structures, only LBP1C-2 exhibited the most potent function and activity. In some embodiments, LBP1C-2, its derivatives such as sulfate derivatives, and compositions containing LBP1C-2 or its derivatives are preferred compositions for improving bone formation, bone mass, and / or bone strength in subjects requiring it, and for treating or preventing bone loss such as senile osteoporosis.

[0168] Although the present invention has been described above through exemplary embodiments, the present invention is not limited thereto. Rather, the appended claims should be interpreted broadly to include other modifications and embodiments that can be made by those skilled in the art.

Claims

1. A method for treating or preventing bone loss and / or improving bone formation, bone mass or bone strength in subjects who require this, The method involves administering an effective dose of a composition comprising a homopolysaccharide, a pharmaceutically acceptable ester or salt thereof, a pharmaceutically acceptable solvate thereof, or any combination thereof, with a pharmaceutically acceptable excipient to a subject (excluding humans) in need thereof. The aforementioned homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units. The method wherein the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid in the homopolysaccharide is 49.9:33.6:8.0:8.

5.

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

3. The method according to claim 1, wherein the bone loss in the subject is age-related bone loss.

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

5. The method according to claim 1, wherein the molecular weight of the homopolysaccharide or its derivative is in the range of about 10 kDa to about 150 kDa.

6. The method according to claim 1, wherein the composition further comprises other polysaccharides isolated from goji berry extract, and / or homopolysaccharides account for 15% or more of the total polysaccharides in the composition.

7. The method according to claim 1, further comprising one or more of any compounds isolated from flavonoids, carotenoids, polyphenols, pigments, or goji berry extract.

8. The method according to claim 1, wherein the homopolysaccharide is the only polysaccharide in the composition.

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

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

11. The method according to claim 1, wherein the effective daily dose of the composition is 10 mg / kg to 500 mg / kg based on the total weight of homopolysaccharides or pharmaceutically acceptable esters or salts / body weight of the subject.

12. A composition for improving bone formation, bone mass, or bone strength, and / or treating or preventing bone loss, in subjects who require it, A homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, in an effective amount, and a pharmaceutically acceptable excipient, The aforementioned homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units. A composition in which the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid in the homopolysaccharide is 49.9:33.6:8.0:8.

5.

13. The aforementioned pharmaceutically acceptable ester or salt is a pharmaceutically acceptable ester, The composition according to claim 12, wherein the pharmaceutically acceptable ester is a sulfate ester derivative of the homopolysaccharide.

14. The composition according to claim 13, wherein the degree of sulfate group substitution of the sulfate ester derivative of the homopolysaccharide is in the range of 0.5 to 0.

9.

15. The composition according to claim 12, wherein the composition is a pharmaceutical composition, a functional composition and / or a supplement.

16. The composition according to claim 12, wherein the composition is an oral composition and / or exists in the form of a tablet.

17. The composition according to claim 12, wherein the excipient is selected from a solvent, co-solvent, colorant, preservative, antimicrobial agent, filler, binder, disintegrant, lubricant, surfactant, emulsifier, suspending agent, or any combination thereof.

18. The composition according to claim 12, wherein the molecular weight of the homopolysaccharide or its derivative is in the range of about 10 kDa to about 150 kDa.

19. The composition according to claim 12, wherein the homopolysaccharide is the only polysaccharide in the composition.

20. The composition according to claim 12, which is a pharmaceutical composition for treating age-related bone loss in humans.

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