Compositions comprising homogeneous polysaccharides or derivatives thereof and methods for preventing and / or

Abstract

The present invention provides compositions and methods for improving bone formation, bone mass, and / or strength, and / or treating or preventing bone loss, such as senescent bone loss, in a subject in need of such therapy. 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 mainly composed of arabinose, galactose, rhamnose and galacturonic acid as monomeric units. Such methods comprise administering to a subject in need thereof an effective dose of such a composition.

Description

[0001] Priority and cross references

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

[0003] The present invention relates to a composition having pharmaceutical or functional properties. Specifically, the disclosed subject matter relates to wolfberry extracts, compositions containing polysaccharides or derivatives thereof, and methods of using the same as, for example, pharmaceutical compositions, functional compositions and / or dietary supplements. Background Art

[0004] In a recent study on the epidemiology of osteoporosis in the European Union, the prevalence of osteoporosis was 47% in women aged 50-55 years and 80 years and older. For EU men of the same age, the prevalence was 2.5% and 16%, respectively. Bone loss is not limited to postmenopausal women, but increasing attention has also turned to osteoporosis in older men, and this number is expected to increase steadily as the population ages. Studies have shown that one-third of women or one-fifth of men over the age of 50 will experience a rapid increase in fracture risk and mortality due to bone loss. Currently, more than 200 million people suffer from osteoporosis worldwide. Osteoporosis is characterized by a decrease in bone mass and density, accompanied by a destruction of bone microstructure. Therefore, osteoporosis in the elderly has become an emerging medical and socioeconomic threat due to its high prevalence, high disability and high mortality.

[0005] Senile osteoporosis is mainly associated with decreased bone formation and decreased bone metabolism. Age-related bone loss is due to decreased bone formation and increased bone resorption. Bone morphogenetic protein (BMP) signaling plays a fundamental role in skeletal development and bone homeostasis. BMPs are members of the transforming growth factor β (TGF-β) superfamily and transmit signals by binding and assembling with type I and type II transmembrane serine / threonine receptor kinases. After the ligand-induced assembly of two type I receptors and two type II receptors, the resident active type II receptor kinase phosphorylates and activates the type I receptor, which is further phosphorylated and causes the signal to be transmitted through Smads or MAPKs, thereby activating the transcription of specific target genes involved in osteogenic differentiation and bone formation. Noggin is widely considered to be a potent inhibitor of BMPs, which can inhibit osteogenic function in vivo and in vitro. Noggin has a strong affinity for binding to BMPs and can prevent BMPs from binding to BMP receptors. BMPs play an important role in many steps of bone development. Studies have shown that BMP-2 plays a unique role in bone formation in humans after birth. As a potent osteogenic growth factor approved by the U.S. Food and Drug Administration, human recombinant bone morphogenetic protein-2 (rhBMP2) has been widely used in bone tissue engineering. Therefore, it is of great significance to discover natural products that have the mimicking effect of BMP-2 in promoting bone formation. Summary of the invention

[0006] The present invention provides a composition and the use of the composition for improving the formation, quality and / or strength of bones and / or treating or preventing bone loss in a subject in need thereof.

[0007] In one aspect, the present invention provides a method for improving bone formation, bone mass and / or bone strength. This method can be a method for treating or preventing bone loss in a subject in need, such as senile bone loss. According to some embodiments, this method comprises administering to a subject in need an effective dose of a composition comprising a homogeneous polysaccharide, the composition comprising a homogeneous polysaccharide or a derivative thereof. The homogeneous polysaccharide is mainly composed of arabinose, galactose, rhamnose and galacturonic acid as monomer units.

[0008] In some embodiments, the subject is a mammal, preferably a human subject, and can be a healthy human, or an aging adult, or an adult suffering from bone loss.

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

[0010] The molecular weight of the homogeneous polysaccharide may be 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 90 kDa, from about 10 kDa to 60 kDa, or any other suitable range. In the homogeneous polysaccharide, the molar ratio of the 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 examples of the present invention, called LBP1C-2, has a ratio of arabinose, galactose, rhamnose and galacturonic acid monomer units of 49.9:33.6:8.0:8.5. Its molecular weight may be about a specific value or a narrow range in the range of about 10 kDa to about 150 kDa. Each obtained polysaccharide is homogeneous and has 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 polysaccharide accounts for more than 15% of the total polysaccharide content in the composition. All of the polysaccharides may be derived from wolfberry. For example, the polysaccharide used in the present invention is wolfberry polysaccharide (referred to as LBP).

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

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

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

[0016] The 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 weight of homogeneous polysaccharide per day / subject's daily body weight. The composition can be administered once a day, twice a day, or more than twice a day.

[0017] In another aspect, the present invention provides a composition for improving bone formation, bone mass and / or bone strength (as described herein), which can be used to treat or prevent bone loss (such as aging-related bone loss) in a subject in need thereof. The composition comprises an effective dose of a homogeneous polysaccharide or a derivative thereof and a pharmaceutically acceptable excipient. The homogeneous polysaccharide is mainly composed of arabinose, galactose, rhamnose and galacturonic acid as monomer units.

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

[0019] 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.

[0020] The molecular weight of the homogeneous polysaccharide is 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 the 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 certain embodiments, the homogeneous polysaccharide has a ratio of arabinose, galactose, rhamnose and galacturonic acid monomer units of 49.9:33.6:8.0:8.5. Its molecular weight may be about a specific value or a narrow range in the range of about 10 kDa to about 150 kDa. Each polysaccharide obtained is homogeneous and has 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 a homogeneous polysaccharide as described herein. For example, such a derivative is a pharmaceutically acceptable ester or salt thereof. A pharmaceutically acceptable ester or salt thereof is a sulfate ester derivative of a homogeneous polysaccharide, also known as a sulfated polysaccharide.

[0024] The present invention also provides the use of a homogeneous polysaccharide as described herein or a derivative thereof in the manufacture of a medicament 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. The method may include preparing or isolating the homogeneous polysaccharide. The method may also include mixing an excipient with the homogeneous polysaccharide. The method may include chemical modification, such as sulfating the homogeneous polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The content of the present invention is best understood through the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, according to common practice, the various features in the drawings are not necessarily drawn to scale. On the contrary, the sizes of various features are arbitrarily enlarged or reduced for clarity. The same reference numerals represent the same features throughout the specification and drawings.

[0027] Figure 1 The trabecular bone structure of the control mice and the experimental groups of mice given wolfberry water extract (LBE), wolfberry crude polysaccharide (LBP) and wolfberry homogeneous polysaccharide (LBP1C-2), respectively, is shown.

[0028] Figure 2 shows the bone mineral density (BMD) values ​​of the control group mice and the experimental groups mice given LBE, LBP and LBP1C-2, respectively ( Figure 2A ), the ratio of trabecular bone volume to total volume (BV / TV) ( Figure 2B ), trabecular number (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 (maximum load force of femur evaluated by three-point bending test) of the control mice and the experimental groups of mice administered with LBE, LBP and LBP1C-2, respectively, is shown.

[0030] Figure 4 The bone formation rates of control mice and experimental groups of mice given LBE, LBP and LBP1C-2, respectively, are shown. The abbreviation "BFR / BS" stands for bone formation rate per unit bone surface.

[0031] Figure 5 Shown are the mineralization apposition rates (MAR) of the control mice and the experimental groups of mice given LBE, LBP, and LBP1C-2, respectively.

[0032] Figure 6 The osteocalcin levels in the serum of the control mice and the experimental groups of mice administered with LBE, LBP, and LBP1C-2, respectively, are shown.

[0033] Figure 7 The contents of type I procollagen N-terminal propeptide (PINP) in the serum of the control group mice and the experimental groups mice administered with LBE, LBP and LBP1C-2, respectively, are shown.

[0034] Figure 8 The proliferation of osteoblasts in human mesenchymal stem cells (hMSCs) treated with LBE, LBP, and LBP1C-2 and in the control group of hMSCs in a CFU (colony forming unit) assay is shown.

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

[0036] Fig.10 Alkaline phosphatase (ALP) enzyme activity in hMSCs treated with LBE, LBP, LBP1C-2 and control hMSCs is shown.

[0037] Fig.11 The expression levels of genes related to bone formation in the femurs of the control group mice and the experimental group mice administered with LBP1C-2 are shown.

[0038] Fig.12 The expression levels of genes related to bone resorption in the femurs of the control group mice and the experimental group mice administered with LBP1C-2 are shown.

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

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

[0041] Figure 18-19 Shown are the results of the interaction of LBP1C-2 with BMPRIA and BMPRII determined using a fluorescence-based protein thermal shift assay.

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

[0043] Fig.21 Shown are the results of direct interaction between LBP1C-2 and Noggin determined using surface plasmon resonance (SPR).

[0044] Fig. 22 Shown are the levels of bone morphogenetic protein 2 (BMP2) release in LBP1C-2-treated hMSCs and control hMSCs.

[0045] Figures 23A-23B The expression of RUNX2 (a marker of early bone formation) in hMSCs treated with LBP1C-2 and S-LBP1C-2 (sulfated LBP1C-2) was compared with that in hMSCs of the control group. Fig.23A ) and SP7( Fig. 23B ) level.

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

[0047] Fig. 24B ALP enzyme activity in LBP1C-2, S-LBP1C-2, and control hMSCs is shown.

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

[0049] Fig.25BThe osteocalcin expression levels in LBP1C-2, S-LBP1C-2 treated hMSCs and control group hMSCs are shown. DETAILED DESCRIPTION

[0050] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0051] In the present invention, the singular forms "a", "an" and "the" include plural references, and references to a particular numerical value include at least that particular value unless the context clearly indicates otherwise. Thus, for example, a reference to an "additive" is a reference to one or more such compounds and their equivalents, which are known to those skilled in the art, and so on. When a value is expressed as an approximation by using the antecedent "about", it is understood that the particular value forms another embodiment. As used herein, "about X" (where X is a numerical value) preferably refers to ±10% of the recited value, including the end value. For example, the phrase "about 8" preferably refers to values ​​including 7.2 to 8.8, including the end value; as another example, the phrase "about 8%" preferably (but not always) refers to values ​​including 7.2% to 8.8%. Where present, all ranges are inclusive and combinable. For example, when a range of "1 to 5" is listed, the listed range should be interpreted as including the ranges "1 to 4", "1 to 3", "1-2", "1-2 and 4-5", "1-3 and 5", "2-5", etc. In addition, when alternatives are positively provided, such terms can be interpreted to mean that any alternatives can be excluded, such as by negative limitations in the claims. For example, when a range of "1 to 5" is listed, the listed range can be interpreted as including the case where any of 1, 2, 3, 4, or 5 is negatively excluded; thus, the listing "1 to 5" can be interpreted as "1 and 3-5, but not including 2", or simply as "wherein 2 is not included". It is intended that any component, element, attribute, or step that is positively listed herein can be explicitly excluded in the claims, regardless of whether such component, element, attribute, or step is listed as an alternative or it is listed in isolation. Any component, element, attribute, or step that is positively recited herein can be explicitly excluded in the claims, regardless of whether such component, element, attribute, or step is listed as an alternative or it is recited in isolation.

[0052] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the term "patient" refers to an animal, preferably a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, mice, etc.) and a primate (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 cow) or a pet (e.g., a dog or cat). In a specific embodiment, the subject is a human. In another embodiment, the subject is an adult.

[0053] As used herein, the term "drug" refers to any molecule, compound, method, and / or substance used to prevent, treat, manage, and / or diagnose a disease or condition. As used herein, the term "effective dose" refers to a dose of a therapy that is sufficient to prevent the development, recurrence, or onset of a disease or condition and one or more symptoms thereof, enhance or improve the preventive effect of another therapy, reduce the severity of a disease or condition, shorten the duration of a disease or condition, alleviate one or more symptoms of a disease or condition, prevent the progression of a disease or condition, cause regression of a disease or condition, and / or enhance or improve the therapeutic effect of another therapy. The disease or condition (e.g., medical condition) is associated with bone loss, or is associated with bone formation, bone mass, and / or bone strength.

[0054] As used herein, the phrase "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

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

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

[0057] As used herein, the terms "treat," "treatment," and "treating," 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, lessening or ameliorating the severity of a disease or condition, such as cancer, and / or ameliorating one or more symptoms thereof as a result of the administration of one or more therapies.

[0058] The term "excipient" as used herein 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, cosolvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

[0059] As used herein, "monomer unit" or "monomer units" in polysaccharides refers to monosaccharide units or single sugar molecule units or other basic chemical units. These units are understood to be groups of monosaccharides and are 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 a molecular weight or molecular weight distribution of a 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 the present disclosure. Due to the uniformity of its molecular weight distribution (i.e., the polydispersity index is 1 or close to 1), its weighted average molecular weight (Mw), number average molecular weight (Mn) or peak molecular weight (Mp) are the same, or approximately the same, or very close. Unless expressly stated otherwise, the molecular weight values ​​and ranges described herein can be Mw or Mn. In some embodiments, the molecular weight values ​​and ranges described herein are number average molecular weights (Mn).

[0061] Wolfberry is used as a traditional Chinese medicine and health food in China and other countries. It belongs to the genus Lycium in the Solanaceae family. Wolfberry is listed as a homologous species that can be used for medicine and food. The fruit of wolfberry has anti-aging and anti-fatigue effects. Wolfberry has been used to nourish the liver and kidneys and improve vision. However, so far, we know little about the molecular mechanism of wolfberry and its active ingredients are not clear.

[0062] The inventor discovered the function of wolfberry in muscle. Wolfberry contains a variety of ingredients, such as polysaccharides, flavonoids, betaine, taurine, vitamins, fatty acids, etc. Lycium barbarum polysaccharides, as the main active ingredient, have biological activities such as antioxidant, antiviral, neuroprotective, acute liver damage and immunomodulatory.

[0063] In recent studies, the inventors extracted and separated a water extract, namely wolfberry extract (LBE) and crude polysaccharide (LBP) from wolfberry fruit using a unique separation method. A homogeneous polysaccharide named LBP1C-2 (e.g., 0.02% yield in dry fruit) was purified from LBP. LBP1C-2 was found to be a pectin. However, whether LBP1C-2 has an effect on bone remodeling remains unclear.

[0064] Lycium barbarum polysaccharide samples can promote the proliferation, differentiation and mineralization of osteoblasts. The polysaccharide is extracted from wolfberry fruit and contains 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 bone loss caused by aging, its main material basis, mechanism of action and target of action have not yet been determined.

[0066] In the present invention, the inventors studied the effects of LBE, LBP and LBP1C-2 on naturally aged mice, and determined the targets and mechanisms of action. LBP1C-2, a homogeneous polysaccharide from wolfberry extract, was found to be the main active ingredient of wolfberry. LBP1C-2 can directly bind to BMPRIA and BMPRII, promote the phosphorylation of Smads, enhance osteogenic differentiation and mineralization, and LBP1C-2 can also directly bind to Noggin, inhibit the interaction between Noggin and BMPs, thereby increasing bone formation. This study also provides a research basis for explaining the therapeutic effects of wolfberry extract composition and provides scientific evidence for the future treatment of age-related bone loss as a natural dietary or product supplement.

[0067] The present invention provides a composition and method for improving bone formation, bone mass and / or bone strength. The method can be used to treat or prevent bone loss in a subject in need thereof, such as treating or preventing aging-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, which is administered to a subject in need thereof. The homogeneous polysaccharide is mainly composed of arabinose, galactose, rhamnose and galacturonic acid as monomer units or molecules. This method comprises administering an effective amount of a composition comprising a homogeneous polysaccharide to a subject in need thereof.

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

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

[0071] The molecular weight of the homogeneous polysaccharide is 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 the polysaccharide is uniform, the values ​​of Mw and Mn may be close to each other, with a very narrow molecular weight distribution. The polydispersity (PD) index, that is, the ratio of Mw to Mn, may be in the range of about 1 to about 1.3, from about 1 to about 1.2, and from about 1 to about 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 growth environment and harvest season of the raw wolfberry. Therefore, the uniform polysaccharide obtained may have different molecular weights. However, each batch of polysaccharides obtained is uniform, that is, its molecular weight is uniform or has a narrow distribution. The molecular weight can be controlled by controlling the quality of the raw materials, for example, controlling the same growth environment and growth time before harvest.

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

[0073] In some embodiments, the homogeneous polysaccharide of the present 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 an additional polysaccharide isolated from a wolfberry extract. The homogeneous polysaccharide accounts for more than 15% of the total amount of all polysaccharides in the composition. All polysaccharides may be derived from wolfberries. For example, the polysaccharide used in the present invention is wolfberry polysaccharide (referred to as LBP).

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

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

[0077] In some embodiments, the composition comprises a wolfberry extract (LBE). The wolfberry extract comprises polysaccharides (or wolfberry polysaccharides), wolfberry flavonoids, carotenoids, polyphenols and wolfberry pigments. Each component may have only one or two or more similar components. 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., about 50 wt.% to about 70 wt.%), the wolfberry flavonoids range from about 0.1 wt.% to about 5.0 wt.%, the carotenoids range from about 0.1 wt.% to about 3.0 wt.%, the polyphenols range from about 0.1 wt.% to about 8.0 wt.%, and the wolfberry pigments range from about 0.1 wt.% to about 8.0 wt.%. In some embodiments, the polysaccharide or polysaccharides are more preferably from about 50.0 wt.% to about 70.0 wt.%. The dry weight is the equivalent weight corresponding to the extract in the dry powder (without water). The extract may contain other trace residues. The extract in the form of a dry powder can be mixed with water to provide an extract in the form of an aqueous solution with a selected concentration as described herein.

[0079] In some embodiments, the wolfberry extract (LBE) is in powder form. In some embodiments, the wolfberry extract is dissolved in a solvent such as water to provide an aqueous solution having a certain concentration, for example, a concentration ranging from 0.1 g / mL to 5 g / mL. In the LBE used in the examples, the content of polysaccharides in the dry powder form of the LBE ranges from about 50.0 wt.% to 70.0 wt.%.

[0080] Lycium barbarum polysaccharides or polysaccharides (LBP) 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. Based on high performance gel permeation chromatography (HPGPC) analysis, LBP1C-2 showed a single and symmetrical peak, which indicated that it was a homogeneous polysaccharide. According to the sugar composition analysis, 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 included a backbone of alternating 1,2-linked α-Rhap and 1,4-linked α-GalpA, and branches of terminal (T)-, 1,3-, 1,6-, and 1,3,6-linked β-Galp, T-, 1,5-, and 1,3,5-linked α-Araf, and T-linked β-Rhap substituted at C-4 of 1,2,4-linked α-Rhap.

[0082] Formula 1, Formula 2, and Formula 3 show the structure of LBP1C-2, and they show the same structure in three different formats.

[0083] Referring to Formula 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 is mainly composed of 1,2-α-Rha and 1,4-α-GalA as the main chain, and the 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, which are attached to the C-4 position of the 1,2,4-α-Rha backbone sugar residue. The repeating unit of LBP1C-2 contains the structural part shown in Formula 3, and contains 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] Formula 2,

[0087]

[0088] Formula 3,

[0089]

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

[0091] The inventors of the present invention have demonstrated that the homogeneous polysaccharide LBP1C-2 is a polysaccharide or active ingredient in LBE or LBP, which has the function of improving bone loss, bone mass and / or strength. The homogeneous polysaccharide LBP1C-2 is an active ingredient that can be used to treat or prevent bone loss, such as aging-related bone loss, in a subject in need thereof.

[0092] In some embodiments, the composition comprises a chemically modified derivative of a homogeneous polysaccharide as described herein. For example, the derivative is a pharmaceutically acceptable ester or salt thereof. Its pharmaceutically acceptable ester or salt is a sulfate derivative of a homogeneous polysaccharide, also known as a sulfated polysaccharide. In the process of synthesizing sulfated homogeneous polysaccharides, hydroxyl groups in the homogeneous polysaccharide react 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 above-mentioned homogeneous polysaccharide. The degree of sulfate substitution of the sulfate 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 substituted 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. Sulfate substitution can bind to proteins and increase biological activity.

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

[0094] The excipient can be a solvent (such as water or a water-based 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] The composition can be administered in any appropriate amount. For example, in some embodiments, the daily effective dose of the composition (described herein in terms of 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 / body weight of the subject. In some embodiments, the dose of wolfberry extract (LBE) or LBP or LBP1C-2 is in the range of 4 mg / kg to 70 mg / kg (total daily dry weight of LBP1C-2 / body weight of the subject such as a person) 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 appropriate range. The composition can be administered once a day, twice a day, or more than twice a day.

[0096] In some embodiments of the present invention, the dosage of wolfberry extract (LBE) or 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 dosage may be administered once or twice or more per day. In an embodiment of the present invention, mice were administered LBE, LBP or LBP1C-2 once per day.

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

[0098] In some embodiments, these compositions can be taken together with beverages, food or related ingredients. The wolfberry water extract provided by the present invention is homologous in medicine and food and can be used as a health food. There are no particular restrictions on the preparation method of food or health food. For example, it can be made into tablets, beverages, candies, etc. In addition to containing uniform polysaccharides, each food preparation can also include other ingredients used in the art. Other ingredients can be selected by those skilled in the art according to a specific formula or use.

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

[0100] The present invention also provides the use of a homogeneous polysaccharide as described herein or a derivative thereof in the manufacture of a medicament 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. The method may include preparing or isolating the homogeneous polysaccharide. The method may further include mixing an excipient and the homogeneous polysaccharide.

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

[0103] Example

[0104] 1. Material Preparation

[0105] 1-1. Preparation of wolfberry extract:

[0106] The preparation process of wolfberry extract ("LBE") is as follows: wolfberry fruits are collected from Zhongning County, Yinchuan, Ningxia, China. The dried wolfberry fruits are washed 3-5 times, soaked in double distilled water (pH=7) at room temperature for 2 hours, and then crushed. The soaked wolfberry powder is added with 5-8 times of neutral water, mixed evenly and decocted twice at boiling temperature, with the decoction time being 2.0 hours and 1.5 hours respectively. The combined concentrated decoction is filtered through a fiber membrane. After the above filtrates are combined, they are evaporated under vacuum at 30-55°C to remove water and obtain a concentrate. The obtained concentrate is freeze-dried into a powder and stored in a desiccator for subsequent experiments at an appropriate concentration.

[0107] In the experiment, an exemplary wolfberry extract was used. This exemplary extract is an aqueous extract 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 illustration only. The water extract can be adjusted to any appropriate concentration, for example, ranging from about 0.1 g / mL to 5 g / mL (dry weight of the exemplary extract / volume of the extract). In certain embodiments, this water extract may be diluted for use. The water extract may be further diluted for cell experiments.

[0108] The wolfberry extract (LBE) used in the present invention mainly includes water-soluble wolfberry polysaccharides, wolfberry flavonoids, carotenoids, polyphenols and pigments. In an exemplary LBE, based on the total equivalent weight of the extract in the form of dry powder, the content of polysaccharides ranges from about 50.0wt.% to 70.0wt.% (e.g., about 54%-56% or 54%), the range of wolfberry flavonoids ranges from about 0.1wt.% to 5.0wt.%, the range of carotenoids ranges from about 0.1wt.% to 3.0wt.%, the range of polyphenols ranges from about 0.1wt.% to 8.0wt.%, and the range of wolfberry pigments ranges from about 0.1wt.% to 8.0wt.%. LBE is in powder form and can be dissolved in water or salt water. LBE with the same composition is used for comparison.

[0109] 1-2. Separation 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 separated from LBE or wolfberry fruit, for example, by the following exemplary method.

[0110] The polysaccharide extraction method comprises: crushing the dried fruit, adding 15-30 times of deionized water to mix, adding 3wt.% cellulase, 1wt.% amylase and 0.5wt.% papain, extracting at 55-60 degrees Celsius for 1 hour, heating to inactivate the enzymes, centrifuging, concentrating the obtained filtrate, dialyzing, concentrating again, adding 5 times of 95% ethanol, centrifuging to obtain a precipitate, washing the precipitate with anhydrous ethanol and acetone alternately for 3 times, and vacuum drying to obtain crude polysaccharide (LBP). The above method also includes: adding 10-15 times water to dissolve the crude polysaccharide, centrifuging, collecting the supernatant, grading and purifying through a diethylaminoethylcellulose (DEAE) anion exchange column, eluting with water, 0.05M, 0.1M, and 0.2M sodium chloride in sequence, collecting the 0.2M sodium chloride elution component, concentrating, dialyzing, and freeze-drying to obtain a preliminarily purified wolfberry polysaccharide (LBP1C), dissolving the obtained polysaccharide LBP1C in 0.2M sodium chloride, centrifuging, collecting the supernatant, eluting through a Sephacryl-300 (RTM: poly((acryl glucan)-co-N,N'-methylenebisacrylamide)) column, collecting the elution components, concentrating, dialyzing, and freeze-drying to obtain a uniform polysaccharide LBP1C-2.

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

[0112] 1-3. Molecular weight detection 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 result graph. With reference to the known molecular weight dextran standards used in the HP-GPC measurement, 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.

[0114] 1-4. Preparation of sulfated polysaccharide derivatives of LBP1C-2 and measurement of sulfate substitution degree (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 made of chlorosulfonic acid and pyridine (3:1, v / v) was added under an ice bath. The resulting mixture was then stirred at 40°C for 4 hours, cooled and neutralized with 5 M NaOH. The solution was first dialyzed against saturated NaHCO3 and then against distilled water. The retentate was lyophilized 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:

[0116]

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

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

[0119] 2. Biological experiments:

[0120] 2-1. Animals and groups

[0121] Healthy wild-type (WT) adult C57BL / 6J mice (male, 2 months old) and naturally aged male C57BL / 6J mice aged 14 months were housed in an SPF barrier facility. The mice were randomly divided into: adult group (15 in each group) and aged group (6 in each group). The adult and aged mice were divided into 4 groups: control group, LBE group, LBP group, and LBP1C-2 group. The control group mice were orally gavaged with distilled water (10 mL / kg) by oral gavage, and the three extracts were gavaged once a day at a dose of LBE (40 mg / kg), LBP (40 mg / kg), or LBP1C-2 (40 mg / kg) for 4 months. All animal experiments were in accordance with the ARRIVE guidelines and the UK Animals (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-minimal essential medium (α-MEM; Gibco, 12571063) supplemented with 10% fetal bovine serum (FBS; Gibco, 16140071) and 1% penicillin-streptomycin (HyClone, SH40003-12). The culture medium used for differentiation culture was α-MEM supplemented with 10% FBS, L-ascorbic acid (50 μg / ml; Sigma, A8960-5G), 0.1 μM dexamethasone (Sigma, D8893-1MG), 10 mM β-glycerophosphate (Sigma, G9422-10G) and 1% penicillin-streptomycin (HyClone, SH40003-12). The culture medium was changed every 2 days. The cell cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C.

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

[0125] After the mice were treated with drugs, they were anesthetized, blood samples were collected from the fundus of the mice, and serum was obtained by centrifugation (2000×g, 20 minutes, 4°C). The contents of osteocalcin and PINP were detected using an osteocalcin ELISA kit and a PINP ELISA kit according to the experimental procedures of the kits, respectively. Osteocalcin and PINP are typical bone formation markers.

[0126] 2-4. Bone analysis by micro-computed tomography

[0127] CT scanning was performed using a micro-computed tomography (micro-CT) imaging system (Inveon MM system, Siemens, Munich, Germany), and trabecular morphology was analyzed according to the manufacturer's protocol. Trabecular parameters measured included bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Briefly, right femoral samples were scanned in vivo and ex vivo under the conditions of an effective pixel value of 8.89 μm, a voltage of 60 kV, a current of 220 μA, an exposure time of 1500 ms, and 360 exposures per 1° exposure during the entire scanning process. The entire image contained 1536 tomograms, and the effective pixel value in the three axial directions was 8.89 μm. The two-dimensional images were reconstructed into three-dimensional (3D) visualization 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.

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

[0129] Three-point bending tests were performed using a small animal bone strength tester (Instron 4302, Instron, Norwood, Massachusetts) bone biomechanical testing equipment. Fresh femoral bone strength was tested using a three-point bending test immediately after sampling from each group of mice. The three-point bending test used two end support points and one central loading point. Biomechanical measurement data were collected from load-deformation curves.

[0130] 2-6. Surface plasmon resonance (SPR) analysis

[0131] The affinity of LBP1C-2 binding to proteins was measured using BIACORE T200 (GE Healthcare, Stockholm, Sweden). BMPRIA, BMPRII and Noggin proteins were immobilized on a CM5 sensor chip by amino coupling. For the measurement of interaction, different concentrations of LBP1C-2 were injected into the chip. All operations were performed in HBS-EP running buffer (pH 7.4), which contained 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA and 0.005% surfactant P20. The kinetic parameters were determined by fitting the data using a 1:1 binding model using Biacore T200 Evaluation Software version 1.0.

[0132] 2-7. Cellular thermal shift assay (CETSA)

[0133] CETSA experiments were performed according to a previously described protocol (Li et al., 2017). hMSCs were cultured in medium containing control or 4 μM LBP1C-2 for 96 h. The cells were then collected. RIPA lysis buffer was added, freeze-thawed three times with liquid nitrogen, and centrifuged at 12,000 g for 10 min at 4 °C. The supernatant of the control group was divided into two parts. One part of the control group was treated with ddH2O2 for 30 min, and the other part of the control group and the 8 μM LBP1C-2 treatment group were treated with the control solution for 30 min. Soluble proteins were collected by centrifugation at 12,000 g for 15 min 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 described previously. Changes in mRNA expression in rats in each treatment group were evaluated by 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 composition of RIPA buffer included 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). Next, 40 mg of protein was separated in each sample by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to an NC membrane. Subsequently, the membrane was blocked for 1 hour in a TBST solution containing 5% skim milk powder (TBST: 10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4). After the blocking step, the membrane was incubated with the primary antibody overnight at 4°C. The primary antibodies used in this article include anti-p-Smad1 / 5 / 8 (1:1000), anti-Smad1 / 5 / 8 (1:1000) and anti-GAPDH (1:2000). Subsequently, the membrane was washed with TBST and incubated with a secondary antibody labeled with horseradish peroxidase (HRP) (1:2000 dilution, Zhong-shan Jin-qiao Company, Beijing, China) for 1 hour at room temperature. The membrane was washed three times with TBST. In order to visualize the protein signal, each membrane was treated with ECL solution (Thermo Scientific, Waltham, USA) and the signal was detected using a molecular imager ChemiDoc XRS+ (Bio-Rad).

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

[0139] ALP staining was performed using the Vector Blue Substrate Kit (Procedure No. SK-5300, Vector Laboratories). 5 hMSCs were seeded into 24-well plates at a density of 100 cells / well and cultured in growth medium for 3 days. On day 7, hMSCs were incubated with substrate working solution for 30 min. During the whole process, cells were protected from light. ALP assay was performed based on the previously described procedure.

[0140] 2-11. Alizarin Red S staining

[0141] hMSCs were collected at 1×10 5 The cells were seeded into 24-well plates at a density of 100 cells per well and cultured in growth medium for 3 days. Osteoblast differentiation was induced by culturing in differentiation medium. On day 14 after induction of osteoblast differentiation, 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.

[0142] 3. Results

[0143] In the following experiment, LBE, LBP and LBP1C-2 solutions were prepared by dissolving the powder in distilled water and administered once a day. The control group mice were given distilled water orally by gavage, while the treatment groups were gavaged once a day at a dose of LBE (40 mg / kg), LBP (40 mg / kg) or LBP1C-2 (40 mg / kg) for 4 months. The effective dose of LBP1C-2 (based on the amount of homogeneous polysaccharide described herein) was 40 mg / Kg, calculated based on the total weight of homogeneous polysaccharide per day / body weight of the subject. The dose of LBE and LBP (40 mg / kg) was the total dry weight of LBE or LBP / body weight of the subject.

[0144] 3-1. LBE, LBP and LBP1C-2 treatment increases bone mass and bone stability

[0145] Aging is significantly associated with decreased bone mass, which is associated with deterioration of bone microstructure. Therefore, we investigated whether LBE, LBP, and LBP1C-2 affect trabecular structural parameters in mice by microcomputed tomography (μCT). Using μCT, bone histomorphometric parameters of femurs of treated and control mice were quantified. LBE, LBP, and LBP1C-2 treatments showed improved bone histomorphometric ( Figure 1 ). LBE, LBP, and LBP1C-2 treatment improved aging-induced bone loss, as LBE- and LBP-treated mice showed higher BMD (bone mineral density) compared with adult and aging controls ( Figure 2A ), higher bone volume fraction (BV / TV) ( Figure 2B ), higher trabecular number (Tb.N) ( Figure 2C ), thicker trabecular thickness (Tb.Th) ( Figure 2D ) and smaller trabecular separation (Tb.Sp)( Figure 2E). *p<0.05, **p<0.01 (compared with adult control group); #p<0.05, ##p<0.01 (compared with aging control group).

[0146] Next, we examined whether LBE, LBP, and LBP1C-2 could modulate the biomechanical properties of bone. The results showed that the maximum load force of mice treated with LBE, LBP, and LBP1C-2 was significantly increased compared with that of adult and aged controls ( Figure 3 ).

[0147] 3-2. LBE, LBP and LBP1C-2 treatment promote bone formation

[0148] The bone formation rate and bone mineralization apposition rate in the femur were analyzed. The bone formation rate (BFR) represents the amount of new bone formed per unit time. It is usually measured by quantifying the amount of mineralized bone matrix or osteogenic matrix (unmineralized bone) formed within a specific bone surface area. The bone mineralization apposition rate (MAR) focuses specifically on the mineralization rate of newly formed bone matrix. It measures the rate at which mineral crystals, mainly hydroxyapatite, are deposited onto the surface of osteogenic matrix during bone formation. The bone formation rate (BFR, Figure 4 ) and bone mineralization apposition rate (MAR, Figure 5 ) was significantly increased compared with the control group. Figure 6 ), a bone formation marker, and P1NP (a bone formation marker, Figure 7 ) levels were significantly increased. The effect of LBP1C-2 was even more significant. Taken together, these results suggest 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 with adult controls); #p<0.05, ##p<0.01 (compared with aging controls)

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

[0150] To test whether LBE, LBP and LBP1C-2 affect the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts and to further understand the above specific compounds. The proliferation, differentiation and mineralization of osteoblasts were analyzed using hMSCs. The results showed that LBE, LBP and LBP1C-2 significantly promoted the formation of CFU-F (fibroblast colony forming unit) colonies and promoted the proliferation of hMSCs ( Figure 8Similarly, LBE, LBP, and LBP1C-2 significantly promoted osteoblast differentiation and mineralization. In addition, the expression levels of two osteoblast differentiation markers, Bglap ( Fig. 9 ) and ALP enzyme activity ( Fig.10 All these results indicate that LBE, LBP and LBP1C-2 treatment 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. The mRNA expression of osteogenic differentiation markers, such as Col1α, Runx2, Bglap, and Opn, was significantly upregulated in LBP1C-2-treated cells ( Fig.11 To further confirm the effect of LBP1C-2 on bone resorption, the mRNA expression of osteoclast markers in the femur, including Ocstamp, Oscar, Opg, and Rankl, was measured, and the results showed no significant difference ( Fig.12 ). These results suggest that LBP1C-2 promotes bone formation and does not affect bone resorption.

[0153] 3-5. LBP1C-2 enhances bone formation in a BMPRIA- and BMPRII-dependent manner

[0154] Since BMPR is a key receptor for osteogenic differentiation, we investigated whether its expression is associated with LBP1C-2-induced osteogenic differentiation. Different doses of BMPR-siRNA were used to confirm the specific inhibitory effect against BMPRIA and BMPRII. Quantitative real-time PCR analysis showed the expression levels of downstream genes Col1α, Runx2, and Bglap. In addition, silencing of BMPRIA and BMPRII resulted in the downregulation of Runx2 ( Fig.13 )、Col1α( Fig.14 ) and Bglap( Fig.15 ), indicating that osteogenic differentiation and mineralization induced by LBP1C-2 treatment were inhibited by downregulation of BMPRIA and BMPRII. Therefore, the results indicate that LBP1C-2 depends on BMPRIA and BMPRII for osteogenic differentiation and mineralization.

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

[0156] To test whether LBP1C-2 directly interacts with BMPR, SPR was used to measure the interaction. The results showed that these interactions were dose-dependent. The equilibrium dissociation constant (Kd) of LBP1C-2 and BMPRIA was approximately 3.6 μmol / L ( Fig.16 ), the Kd of BMPRII is 5.6 μmol / L ( Fig.17 The direct interaction between LBP1C-2 and BMPR was further confirmed by fluorescence-based protein heat shift assay; before reaching the plateau phase, LBP1C-2 dose-dependently increased BMPRIA ( Fig.18 ) and BMPRII( Fig.19 ) increased by more than 4°C. The BMP / Smads signaling pathway is a key pathway for regulating the differentiation of BMSCs into osteoblasts. In order to verify whether LBP1C-2 in hMSCs regulates the osteogenic differentiation of hMSCs through the BMP / Smads signaling pathway, the phosphorylation levels of Smad1 / 5 / 8 were measured after hMSCs differentiated from osteoblasts for 14 days. 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 suggest 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 regulating 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 well known for its role in inhibiting the BMP signaling pathway. Since the interaction between Noggin and BMP inhibits osteogenic differentiation, the inventors studied whether LBP1C-2 activates this signaling pathway by binding to Noggin. Fig.21As shown, the effect of LBP1C-2 on Noggin was detected after treatment, and the interaction between LBP1C-2 and Noggin was determined by SPR analysis. The results showed that LBP1C-2 had a strong binding affinity with Noggin, with an equilibrium dissociation constant (Kd) of 0.313 μM. In addition, whether this binding inhibited the interaction between Noggin and BMP2, the concentration of BMP2 in the supernatant of hMSCs treated with LBP1C-2 was measured. The results showed that under Noggin conditions, a certain amount of BMP2 was detected in the cell supernatant, and under LBP1C-2 conditions, a large amount of BMP2 was detected in the cell supernatant. The upregulation of BMP-2 levels induced by LBP1C-2 was also inhibited by Noggin. These data indicate that LBP1C-2 interacts with Noggin and inhibits the binding of Noggin to BMP2 ( Fig. 22 ).

[0159] Age-related bone loss occurs equally in men and women, and osteoporosis-induced fractures are the 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 bones in a natural aging model. As described herein, LBE, LBD, and LBP1C-2 supplements successfully improved age-induced osteoporosis in mice by improving overall bone microstructure. In addition, it was determined that the main active substance basis was LBP1C-2. Mechanistic studies have shown 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, promote the phosphorylation of downstream effectors Smads, and thus enhance 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 combination further upregulates the BMP / Smads signaling pathway and increases bone formation. These results indicate that the compositions described herein have the functional potential of promoting bone formation similar to BMPs and can be used as dietary supplements or pharmaceutical compositions to treat or prevent aging-related bone loss.

[0160] Although the experimental period is long, the natural aging animal model is currently the most suitable animal model for simulating aging-related bone loss. The inventors' research on wolfberry in the natural aging mouse model has obvious advantages. The natural supplementation method of LBE, LBP and LBP1C-2 described in the present invention successfully improved aging-related bone loss in adult mice and aging mice.

[0161] BMPR silencing resulted in downregulation of bone formation markers Col1α1, Runx2, and Bglap in LBP1C-2-treated preosteoblasts. In addition, SPR, protein thermal shift assay, and CETSA were used to verify whether LBP1C-2 directly interacted with BMPR. The inventors found that LBP1C-2 could directly bind to BMPRIA and BMPRII, and the phosphorylation level of downstream effectors Smad1 / 5 / 8 was significantly increased. On the other hand, the inventors first found that LBP1C-2 not only bound to BMPRIA and BMPRII, but also had a stronger binding to Noggin, and this binding blocked the interaction between Noggin and BMP. These two interactions led to LBP1C-2 promoting osteogenic differentiation, indicating that LBP1C-2 mimicked the ligands of BMPR and regulated osteogenic differentiation through the BMP / Smads signaling pathway.

[0162] There is currently no drug specifically for the treatment of aging-related osteoporosis. Conventional anti-osteoporosis drugs have certain limitations, and the effects of long-term use are still unclear. In clinical practice, the treatment options for osteoporosis are mainly divided into the following strategies. Anti-bone resorption drugs, such as Denosumab, Odanacatib, Saracatinib, etc., are usually used to treat osteoporosis, but inhibiting bone resorption will also interfere with bone remodeling and inhibit bone formation. These drugs for osteoporosis often have 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 the treatment of osteoporosis and is used at appropriate doses by daily subcutaneous injection. Based on this background, natural products may provide better alternatives or methods for the treatment of osteoporosis due to their small side effects. Lycium barbarum, as a traditional Chinese medicine or health food, is used to treat aging-related diseases. In the present invention, it is demonstrated that the homogeneous polysaccharide LBP1C-2 extracted from wolfberry significantly increases the bone mass and bone strength of adult and aged mice after long-term treatment (4 months of administration to mice), and no adverse reactions are found. Therefore, LBP1C-2 may be a potential effective and safe compound suitable for the development of anti-osteoporosis drugs under pathological and non-pathological conditions.

[0163] In conclusion, the studies in the present invention demonstrate that the homogeneous polysaccharide LBP1C-2 from wolfberry can be used to treat or prevent aging-related bone loss. Osteoporosis has become an emerging medical and socioeconomic threat due to its high morbidity, high disability rate and high mortality rate. As a traditional Chinese medicine and also an edible food, wolfberry has many health benefits. However, it is still unclear whether wolfberry can improve aging-related bone loss and what its main active substances are. In the present invention, the results of the study show the role of three wolfberry extracts: wolfberry water extract (LBE), wolfberry polysaccharide (LBP, isolated from LBE) and the homogeneous polysaccharide LBP1C-2 (isolated from LBP) in treating or preventing bone loss. Supplementation of 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). In addition, from a mechanistic point of view, the main active ingredient LBP1C-2 directly binds to BMP receptors BMPRIA and BMPRII, and the activation of BMPRIA and BMPRII leads to the phosphorylation of downstream effectors Smads, thereby enhancing osteogenic differentiation and mineralization. On the other hand, Noggin, as a potent inhibitor of BMP, LBP1C-2 also directly binds to Noggin and inhibits the interaction between Noggin and BMP. This binding further upregulates the BMP / Smads signaling pathway, thereby promoting bone formation. In summary, the above results show that wolfberry extract can prevent aging-induced bone loss, and the main polysaccharide component LBP1C-2 improves aging-induced 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 foods to intervene in 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 is referred to as S-LBP1C-2.

[0166] To investigate the ability 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 a key role in bone formation and osteoblast differentiation. RUNX2 is considered to be a master regulator of osteoblast differentiation. It is essential 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 the differentiation and function of osteoblasts.

[0167] First, the levels of major osteogenic markers RUNX2 and SP7 were detected. The results of qPCR assay after 7 days of hMSCs treatment showed that S-LBP1C-2 increased the mRNA levels of RUNX2 and SP7 ( Fig.23A and 23B ). The experimental results showed that S-LBP1C-2 has the ability to promote osteogenic differentiation. In addition, at an equivalent concentration 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 is better than that of LBP1C-2.

[0168] BALP staining, also known as alkaline phosphatase staining, is a technique used to evaluate the activity of the alkaline phosphatase (ALP) enzyme during osteoblast differentiation. ALP is the predominant enzyme expressed in osteoblasts and is involved in various aspects of bone formation. During osteoblast differentiation, the expression and activity of ALP increases. ALP staining provides an indirect method for evaluating osteoblast differentiation and maturation by observing and quantifying the activity of ALP.

[0169] Subsequently, we evaluated the effect of S-LBP1C-2 on ALP staining ( Fig.24A ) and ALP activity ( Fig. 24B). It was observed that S-LBP1C-2 enhanced ALP secretion and ALP activity. The promotion of ALP secretion and activity by S-LBP1C-2 exceeded that by LBP1C-2, indicating that S-LBP1C-2 has a stronger ability to promote osteoblast differentiation. Alizarin Red S staining is a widely used technique for detecting and quantifying calcium mineralization in bone and mineralized tissues. It is often used to assess the degree of mineralized matrix deposition by osteoblasts during bone formation. In addition, the effects of S-LBP1C-2 on Alizarin Red S staining ( Fig.25A ) and the expression level of osteocalcin BGLAP in hMSCs ( Fig.25B ). Alizarin Red S staining results showed that S-LBP1C-2 promoted 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, which are the cells responsible for bone formation. BGLAP is considered a marker of osteoblast activity.

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

[0171] 4. Experimental samples and control samples:

[0172] Four homogeneous polysaccharides were isolated from wolfberry extract. Among these four homogeneous polysaccharides, only LBP1C-2 showed the functions described in this article. Among the four homogeneous polysaccharides, the sugar composition and structure of three 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) 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 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 main chain sugar 1,3-β-Gal residue and the C-3 position of 1,6-β-Gal.

[0174] Formula 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, with 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, and its branches mainly include 1,4-β-GlcA, T-β-Rha, T-β-Gal, T-α-Ara, T-β-Ara, 1,5-α-Ara and part of 1,6-β-Gal. These branches are connected to the C-6 position of the main chain sugar residue 1,3-β-Gal and the C-3 position of 1,6-β-Gal.

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

[0178]

[0179] Among the four homogeneous polysaccharides with different structures, only LBP1C-2 showed the most powerful function and activity. In some embodiments, LBP1C-2, its derivatives such as sulfate derivatives, and compositions comprising LBP1C-2 or its derivatives are preferred compositions for improving bone formation, bone mass and / or bone strength in subjects in need thereof, and for treating or preventing bone loss, such as aging-related bone loss.

[0180] Although the present invention has been described by way of 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. A method for treating or preventing bone loss and / or improving bone formation, bone mass or strength in a subject in need thereof, comprising: administering an effective dose of a composition comprising 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 to a subject in need thereof, Among them, homogeneous polysaccharides are mainly composed of arabinose, galactose, rhamnose and galacturonic acid as monomer units.

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

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

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

5. The method according to claim 1, wherein: The molecular weight of the homogeneous polysaccharide or its derivatives ranges from about 10 kDa to about 150 kDa, and / or the molar ratio of arabinose, galactose, rhamnose and galacturonic acid monomer units in the homogeneous polysaccharide ranges from 30-70:20-60:0.1-10:0.1-10.

6. The method according to claim 1, wherein: 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.

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

8. The method according to claim 1, wherein: The composition further comprises one or more of flavonoids, carotenoids, polyphenols, pigments or any compound isolated from wolfberry extract.

9. The method according to claim 1, wherein: The homogeneous polysaccharide is the only polysaccharide in the composition.

10. The method according to claim 1, wherein: The pharmaceutically acceptable esters or salts are sulfate derivatives of the homogeneous polysaccharide.

11. The method according to claim 1, wherein: The excipient is selected from solvents, co-solvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents or any combination thereof.

12. The method according to claim 1, wherein: The effective daily amount of the composition is 10 mg / kg to 500 mg / kg based on the total weight of the homogeneous polysaccharide or a pharmaceutically acceptable ester or salt thereof per body weight of the subject.

13. A composition for improving bone formation, bone mass or strength, and / or treating or preventing bone loss in a subject in need thereof, comprising: An effective amount of 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, Among them, homogeneous polysaccharides are mainly composed of arabinose, galactose, rhamnose and galacturonic acid as monomer units.

14. The composition according to claim 13, wherein The pharmaceutically acceptable ester or salt is a sulfate derivative of the homogeneous polysaccharide.

15. The composition according to claim 14, wherein The degree of substitution of sulfate groups of the sulfate ester derivatives of the homogeneous polysaccharide is in the range of 0.5 to 0.

9.

16. The composition according to claim 13, wherein The composition is a pharmaceutical composition, a functional composition and / or a dietary supplement.

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

18. The composition according to claim 13, wherein The excipient is selected from solvents, co-solvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents or any combination thereof.

19. The composition according to claim 13, wherein The molecular weight of the homogeneous polysaccharide or its derivatives ranges from about 10 kDa to about 150 kDa, The molar ratio of arabinose, galactose, rhamnose and galacturonic acid monomer units is in the range of 30-70:20-60:0.1-10:0.1-10.

20. The composition according to claim 13, wherein 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.

21. The composition according to claim 13, wherein The homogeneous polysaccharide is the only polysaccharide in the composition.

22. The composition according to claim 13, wherein The composition is a medicine composition for treating human aging bone loss.

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