Compositions comprising homogeneous polysaccharides or derivatives thereof and methods for

Abstract

The present invention provides compositions and methods for improving the number and / or function of muscle satellite cells, and / or treating or preventing muscle atrophy 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. The method comprises administering an effective dose of such a composition to a subject in need thereof.

Description

[0001] Priority and cross references

[0002] This application claims the benefit of U.S. patent application 63 / 344,158, filed on May 20, 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. More 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] Skeletal muscle is the largest organ in the human body and plays an extremely important role in maintaining body homeostasis and the entire metabolic process. Satellite cells (SCs), as skeletal muscle stem cells, play an important role in skeletal muscle growth, injury repair and regeneration after birth. Skeletal muscle satellite cells are located in a unique microenvironment (sarcolemma and basement membrane). The paired box protein Pax7 is the most important molecular marker of satellite cells, which is expressed in both resting and activated satellite cells, while MyoD, also known as myoblast-determining protein 1, is significantly upregulated only in satellite cells activated after injury and is considered to be a characteristic marker of activated satellite cells. In resting muscles, satellite cells are mostly quiescent; however, in response to stress or injury, they are activated and proliferate, and then differentiate or self-renew to replenish the resting cell pool. Since satellite cells have a strong regenerative capacity in response to various stimuli, they are an important target for the treatment of muscle diseases.

[0005] One of the most significant effects of physical aging is the loss of skeletal muscle mass, strength, and function, which is known as muscle atrophy. After the age of 30, skeletal muscle mass decreases by approximately 3-8% per decade, and the decline is faster after the age of 60. The reduction in the number of satellite cells and the reduction in the stemness of satellite cells are important causes of age-related muscle atrophy. Under normal circumstances, satellite cells need to remain quiescent and maintain the ability to self-renew, and can be effectively activated to complete the regeneration process when damaged. Aging affects the self-renewal capacity of satellite cells, resulting in an age-dependent decline in the satellite cell pool, as well as a decrease in activation and / or proliferation capacity, resulting in a decrease in regeneration capacity. Previous studies have been somewhat controversial as to whether the number of satellite cells decreases with age, with some studies showing that the number of satellite cells in young and old mice is similar. However, with the development of satellite cell labeling technology, new studies have shown that satellite cells do decline with age, with a decline of about 50% in old age. And it has been shown that improving the capacity of satellite cells can restore muscle function in old mice. Therefore, the discovery of drugs that can modulate satellite cell function to treat muscle atrophy in aging has attracted great attention.

[0006] During the aging process, the coordinated regulation of endogenous and exogenous signals, including p38 MAPK, JAK / STAT3, Wnt, Smad3, TGFβ, FGF2, SPRY1, etc., leads to a decrease in the number of satellite cells and loss of function. The development of intervention strategies targeting these key signaling pathways is also the focus of related research. For example, drug inhibition or RNA interference (RNAi)-mediated downregulation of JAK / STAT3 or p38 MAPK signals can enhance the regenerative capacity of aged muscle stem cells; Wnt protein antagonists can restore the function of satellite cells in aged muscles; inhibition of TGFβ-p-Smad3 signals can also restore the function of satellite cells in aged muscles. Although the mechanism of muscle atrophy caused by the decrease in the number of stem cells during aging has been studied in more and more detail, and the corresponding intervention strategies have been successful in animal experiments, the safety of drugs targeting various signaling pathways should be considered in clinical applications. Summary of the invention

[0007] The present invention provides a composition, and a method for using the composition to improve the number and / or function of muscle satellite cells, and / or to treat or prevent muscle atrophy in a subject in need thereof.

[0008] On the one hand, the present invention provides a method for improving the number and / or function of muscle satellite cells, and / or treating or preventing muscle atrophy in a subject in need thereof. According to some embodiments, the method comprises administering to a subject in need thereof 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. The homogeneous polysaccharide is mainly composed of arabinose, galactose, rhamnose and galacturonic acid as monomer units.

[0009] 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 muscle atrophy.

[0010] 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.

[0011] 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.

[0012] In some embodiments, the homogeneous polysaccharide described herein is the only polysaccharide in the composition.

[0013] 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).

[0014] In some embodiments, the composition further optionally comprises one or more of flavonoids, carotenoids, polyphenols, pigments or any compound isolated from wolfberry extract.

[0015] 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.

[0016] 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.

[0017] The acceptor 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 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 multiple times a day.

[0019] In another aspect, the present invention provides a composition (as described herein) for improving the number and / or function of muscle satellite cells, and / or treating or preventing muscle atrophy in a subject in need thereof. This composition comprises an effective amount of a homogeneous polysaccharide and a pharmaceutically acceptable excipient. The homogeneous polysaccharide 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 dietary supplement. For example, the composition is an oral composition and / or is in the form of a tablet in certain embodiments.

[0021] The receptor 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 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 in 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.

[0023] 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.

[0024] 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, and the composition may only include polysaccharides isolated from wolfberry extract.

[0025] 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.

[0026] 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.

[0027] 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 the receptor and the homogeneous polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 Shown are the relative mRNA levels of Pax7 in the tibialis anterior (TA) muscle of adult male mice that were or were not treated with LBE (wolfberry extract) for 4 months, n=8 or 7 per group.

[0030] Figure 2 Shown are Western blot analyses of Pax7 levels in TA muscle of 6-month-old mice with or without 4-month LBE treatment, GAPDH (uronate 3-phosphate dehydrogenase) was used as an internal control, n=4 / group.

[0031] Figure 3 The expression of Pax7 in TA muscle of 6-month-old mice was shown by IF (immunofluorescence staining). + Quantification of cells in mice that did or did not receive LBE treatment for 4 months, n = 8 or 7 per group.

[0032] Figure 4 Satellite cells (CD34 + CD31- CD45 - Sca1 - The number of cells was 4 / group.

[0033] Figure 5 Shown are the relative mRNA levels of Pax7 in TA muscle of aged male mice treated with or without LBE for 4 months, n=6 / group.

[0034] Figure 6 Shown are Western blot analyses of Pax7 levels in TA muscle of 18-month-old mice treated with or without LBE for 4 months, GAPDH was used as an internal control. n=6 / group.

[0035] Figure 7 Immunofluorescence staining of Pax7 in TA muscles of 18-month-old mice treated with or without LBE for 4 months is shown. + Quantitative analysis of cells, n=6 / group.

[0036] Figure 8 Shown are representative images of H&E staining of TA muscle sections from adult mice treated with or without LBE 10 days after injury induced by BaCl2 injection, n=6 / group.

[0037] Figures 9A-9B Shown are the relative mRNA levels of Pax7 and MyoD in the tibialis anterior muscle of adult mice treated or not with LBE at 1 day after injury, n=6 / group.

[0038] Fig.10 Figure 3 shows Pax7 expression in tibialis anterior muscle of adult mice treated with or without LBE at 30 days after injury. + MyoD - Quantitative analysis of cells, n=6 / group.

[0039] Fig.11 Representative images of H&E staining of tibialis anterior muscle sections from aged mice treated or not with LBE at 30 days after injury are shown, n=6 / group.

[0040] Fig.12 The expression of Pax7 in cultured single muscle fibers was shown. + / MyoD - ,Pax7 – / MyoD + and Pax7 + / MyoD + For the number of cells, more than 50 myofibers were analyzed.

[0041] Figures 13A-13BShown are the relative mRNA levels of MyoG and MyHC in myofiber-derived satellite cells treated with or without LBE on the first day after differentiation.

[0042] Fig.14 showed that Pax7 + Quantitative analysis of cells in mice treated or not with LBP for 4 months, n = 6 / group.

[0043] Fig.15 Pax7 in isolated primary myoblasts treated or untreated with LBP is shown + / MyoD - ,Pax7 – / MyoD + and Pax7 + / MyoD + Statistical analysis of cell numbers. More than 200 primary myoblasts were analyzed.

[0044] Figures 16A-16B Shown are the relative mRNA levels of Pax7 and MyoD in satellite cells sorted from Pax7-nGFP transgenic mice cultured with or without LBP for 18 h.

[0045] Fig.17 Shown are the relative mRNA levels of Pax7 in satellite cells sorted from Pax7-nGFP transgenic mice cultured with or without LBP for 48 h.

[0046] Figures 18A-18B Shown are the relative mRNA levels of MyoG and MyHC in differentiated C2C12 cells treated with or without LBP.

[0047] Fig.19 Shown are the relative mRNA levels of Pax7 in primary myoblasts treated with LBP and LBP1C-2 for 18 h.

[0048] Fig. 20 Pax7 in primary myoblasts treated or untreated with LBP1C-2 is shown + / MyoD - ,Pax7 – / MyoD + and Pax7 + / MyoD + For statistical analysis of cell numbers, more than 200 primary myoblasts were analyzed.

[0049] Fig.21Shown are Western blot analyses of p-FGFR1, FGFR1, p-P38, P38, and MyoD levels in muscle of adult mice treated with or without LBE 1 day after BaCl2-induced injury, GAPDH was used as a loading control.

[0050] Fig.22A and 22B Shown are the relative mRNA levels of FGFR1 and MyoD in C2C12 cells transfected with FGFR1 siRNA and treated with or without LBP1C-2.

[0051] Fig.23 Shown are Western blot analyses of FGFR1, p-P38, and P38 levels in C2C12 cells transfected with FGFR1 siRNA and treated with or without LBP1C-2, GAPDH was used as a loading control.

[0052] Fig.24 Shown are the results of the cell thermostability reaction CETSA used to evaluate the interaction of LBP1C-2 with FGFR1 in C2C12 cells.

[0053] Fig.25 Shown are the results of SPR spectroscopy used to assess the interaction of LBP1C-2 and FGFR1 in vitro.

[0054] Fig.26 Shown are the relative mRNA levels of Spry1 in the muscles of adult mice treated or not with LBE 30 days after injury, n=6 / group.

[0055] Fig. 27 Relative mRNA levels of Spry1 in C2C12 cells treated or untreated with LBP and LBP1C-2 are shown.

[0056] Figures 28A-28B Shown are the relative mRNA levels of Spry1 and Pax7 in C2C12 cells transfected with Spry1 siRNA and treated with or without LBP1C-2.

[0057] Figures 29A-29B Shown are the effects of LBP1C-2 on the relative mRNA levels of Spry1 in C2C12 cells pretreated with the FGFR1 inhibitor PD173074 or transfected with Fgfr1 siRNA.

[0058] Figures 30A-30B Shown are the relative mRNA levels of MyoD and the protein levels of p-p38 in C2C12 cells treated with LBP1C-2 and S-LBP1C-2 for 24 h.

[0059] Fig.31A Shown are the relative mRNA levels of MyoD in C2C12 cells after treatment with different doses of S-LBP1C-2 for 24 h.

[0060] Fig.31B Shown are the relative protein levels of p-p38 and MyoD in C2C12 cells after treatment with different doses of S-LBP1C-2 for 24 h.

[0061] Fig.32A Shown are the relative mRNA levels of Spry1 in C2C12 cells after treatment with LBP1C-2 and S-LBP1C-2 for 48 h.

[0062] Fig.32B Shown are the relative mRNA levels of Spry1 in C2C12 cells treated with different doses of S-LBP1C-2 for 48 h. DETAILED DESCRIPTION

[0063] 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.

[0064] 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., cattle, 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 cattle) 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. In another embodiment, the subject is a child. In yet another embodiment, the subject is a human infant.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Molecular weight is measured by gel permeation chromatography (GPC). GPC is an analytical technique that separates molecules in a polymer by size and provides a molecular weight or molecular weight distribution of a material. A homogeneous polysaccharide as described herein contains only one peak in GPC with a uniform molecular weight.

[0072] Muscle stem cells, also known as satellite cells (SCs), are responsible for repairing and rebuilding muscles, and impaired function of SCs results in a decline in muscle regeneration capacity. There is an urgent need to find satellite cell-based therapeutic strategies to improve muscle function or rescue age-related muscle degeneration.

[0073] Although the mechanism of muscle atrophy caused by the decrease in stem cell numbers with aging has been studied in increasing detail, and corresponding intervention strategies have been successful in animal experiments, the safety of drugs targeting various signaling pathways should be considered in clinical applications.

[0074] Therefore, it is contemplated in the present invention that the discovery of natural plants with the ability to improve muscle function is crucial for the development of clinical drug intervention strategies targeting satellite cell regulation to improve aging and age-related muscle diseases.

[0075] Wolfberry can be used as a Chinese herbal medicine with anti-fatigue and anti-aging effects. Modern research has verified the anti-aging effects of wolfberry in models such as cells, fruit flies, zebrafish, and mice. However, so far, we know very little about the molecular mechanism of wolfberry, and its active ingredients are not clear.

[0076] The inventors used C57BL / 6 mice as a model and found that wolfberry water extract can significantly increase the muscle weight ratio of the tibialis anterior and gastrocnemius muscles of mice, reduce fat content, and increase the average maximum running distance of mice, that is, increase muscle endurance.

[0077] In this study, the inventors attempted to determine the efficacy of wolfberry in improving the maintenance of skeletal muscle satellite cells and further identify active ingredients and targets. The inventors found that long-term use of wolfberry extract (LBE) can improve the number and function of satellite cells in adult and elderly mice and enhance muscle regeneration. In addition, a homogeneous polysaccharide, LBP1C-2, was discovered from wolfberry extract, which is the active ingredient in LBE that can regulate satellite cell function. LBP1C-2 promotes satellite cell activation and self-renewal by interacting with FGFR1 to activate p38 signaling and upregulate spry1 expression. This is a new discovery that LBE is involved in regulating muscle stem cells. More importantly, the inventors identified for the first time the active ingredients and targets of LBE that exert this effect. This study provides a new scientific explanation for the function of wolfberry and also lays the foundation for the medicinal or auxiliary medicinal use of wolfberry.

[0078] The present invention provides a composition and method for improving the number and / or function of muscle satellite cells, and / or treating or preventing muscle atrophy in a subject in need thereof.

[0079] 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 introduced into a subject in need thereof. The homogeneous polysaccharide is mainly composed of arabinose, galactose, rhamnose and galacturonic acid as monomer units. This method comprises introducing an effective amount of the composition into a subject in need thereof.

[0080] 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 muscle atrophy.

[0081] 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 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.

[0082] 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 ratio of arabinose, galactose, rhamnose and galacturonic acid monomer units of 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). The values ​​of Mw and Mn may be close to each other because the polysaccharide is uniform and has 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. Because the molecular weight of the polysaccharide using wolfberry as a raw material may vary due to factors such as the growth environment and the harvest season. So 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 material, for example, by having the same growth environment and growth time before harvest.

[0083] In some embodiments, the homogeneous polysaccharide described herein is the only polysaccharide in the composition.

[0084] In other embodiments, the composition further comprises additional polysaccharides isolated from wolfberry extract. Homogeneous polysaccharides account for more than 15% of all polysaccharides in the composition. All polysaccharides may be derived from wolfberry. For example, the polysaccharide used in the present invention is wolfberry polysaccharide (referred to as LBP).

[0085] In some embodiments, the composition further optionally comprises one or more of flavonoids, carotenoids, polyphenols, pigments or any compound isolated from wolfberry extract.

[0086] Lycium barbarum extract (LBE) can be obtained by extracting wolfberry with water, and the extract can be 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 uniform polysaccharides, which are in the form of dry powder.

[0087] 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.

[0088] 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.

[0089] In some embodiments, the wolfberry extract (LBE) is in powder form. In some embodiments, the wolfberry extract is dissolved in a solvent such as water to provide an aqueous solution having a concentration, for example, ranging from 0.1 g / mL to 5 g / mL. In the experiment, the LBE used had a polysaccharide content in the dry powder form of the LBE ranging from about 50.0 wt.% to 70.0 wt.%.

[0090] 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 passing through a separation column. Lycium barbarum polysaccharides (LBP) may contain different polysaccharides, which can be further separated.

[0091] 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.

[0092] Formula 1, Formula 2, and Formula 3 show the structure of LBP1C-2, and they show the same structure in three different formats.

[0093] 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.

[0094] Formula 1,

[0095]

[0096] Formula 2,

[0097]

[0098] Formula 3,

[0099]

[0100] 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.

[0101] The inventors of the present invention provide a homogeneous polysaccharide, such as LBP1C-2, which is a polysaccharide or active ingredient in LBE or LBP, having the effect of improving the number and / or function of muscle satellite cells, and / or treating or preventing muscle atrophy in a subject in need thereof. The derivatives of the homogeneous polysaccharide, its ester or salt, or its solvate also have the same effect.

[0102] 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. Pharmaceutically acceptable esters or salts thereof are sulfate ester derivatives of homogeneous polysaccharides, also known as sulfated polysaccharides. In the process of synthesizing sulfated homogeneous polysaccharides, hydroxyl groups in the homogeneous polysaccharide react with a modifier such as chlorosulfonic acid to form -O-SO3H groups. The molecular weight after modification is the same as that of the above-mentioned homogeneous polysaccharide. The degree of sulfate substitution of the sulfate ester 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 sulfated substituents on each hexose or pentose unit is 0.74. Sulfate substitution can bind to proteins and increase biological activity.

[0103] 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.

[0104] The receptor can be a solvent (such as water or an aqueous-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.

[0105] The composition can be administered in any appropriate amount. For example, in some embodiments, the daily 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 / 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, from 10 mg / kg to 70 mg / kg, from 10 mg / kg to 60 mg / kg, from 20 mg / kg to 70 mg / kg, from 20 mg / kg to 60 mg / kg, from 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.

[0106] 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). This dosage is the total amount of three administrations per day.

[0107] 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 food or health food preparations. For example, it can be made into tablets, beverages, candies, etc. In addition to containing homologous 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.

[0108] In some embodiments, the composition is a food or health product, including sports drinks, protein powders, snack bars, etc.

[0109] 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.

[0110] In another aspect, the present invention provides a method for preparing the composition or homogeneous polysaccharide. The method may include preparing the homogeneous polysaccharide. The method may further include mixing the receptor and the homogeneous polysaccharide.

[0111] 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.

[0112] Example

[0113] 1. Material Preparation

[0114] 1-1. Preparation of wolfberry extract:

[0115] The preparation process of wolfberry extract ("LBE") is as follows: wolfberry berries are grown and harvested in Zhongning County, Yinchuan, Ningxia, China. The dried berries are soaked in double distilled water (pH=7) at room temperature for 2 hours, after being washed 3-5 times, and then crushed. The soaked berry powder is added to 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 hollow fiber membrane. The above filtrates are combined and 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 appropriate concentrations.

[0116] In the experiment, an exemplary wolfberry extract was used. This exemplary extract is an aqueous extract solution with a concentration of 1 g / ml (dry powder or dry weight of the extract / volume of the extract). This concentration is for illustration only. The aqueous 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 aqueous extract may be diluted for use. The aqueous extract may be further diluted for cell experiments.

[0117] 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.

[0118] 1-2. Separation of Lycium barbarum polysaccharides and homogeneous polysaccharides:

[0119] Lycium barbarum polysaccharides ("LBP") and homogeneous polysaccharides, such as the example labeled LBP1C-2 described herein, can be isolated from LBE or wolfberry berries by the following exemplary methods.

[0120] 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, re-concentrating, 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 a crude polysaccharide (LBP); the method further comprises: dissolving the crude polysaccharide with 10-15 times of water, centrifuging, collecting the supernatant, and filtering the filtrate with diethylaminoethyl (D The purified protein was graded and purified using an EAE cellulose anion exchange column, eluted with water, 0.05 M, 0.1 M, and 0.2 M sodium chloride in sequence, and the 0.2 M sodium chloride elution fraction was collected, concentrated, dialyzed, and freeze-dried to obtain the preliminary purified wolfberry polysaccharide (LBP1C). The obtained polysaccharide LBP1C was dissolved in 0.2 M sodium chloride, centrifuged, and the supernatant was collected and eluted through a Sephacryl-300 (RTM: poly((acryl glucan)-co-N,N'-methylenebisacrylamide)) column, and the eluted fractions were collected, concentrated, dialyzed, and freeze-dried to obtain a uniform polysaccharide LBP1C-2.

[0121] LBC and LBP1C-2 have the composition and structure described herein.

[0122] 1-3. Molecular weight detection of LBP1C-2:

[0123] 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.

[0124] 1-4. Preparation of sulfated polysaccharide derivatives of LBP1C-2 and measurement of sulfate substitution degree (DS)

[0125] The polysaccharide was sulfated according to the chlorosulfonic acid-pyridine method. LBP1C-2 (50 mg) was dissolved in 2.5 mL of dry formamide. 1.5 mL of a sulfation reagent made of chlorosulfonic acid and pyridine (3:1, v / v) was added under an ice bath. The mixture was then stirred at 40°C for 4 hours, cooled and neutralized with 5M 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 equation (1) as follows:

[0126]

[0127] Where DS is the degree of sulfate substitution and %S is the percentage of sulfur content.

[0128] 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.

[0129] 2. Biological experiments:

[0130] 2-1. Mouse strains and animal management

[0131] LBE treatment and muscle regeneration experiments were performed using male C57BL / 6J mice purchased from Vital River. Pax7-nGFP transgenic mice were gifted by Zhu Dahai's group (Peking Union Medical College, China). All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Chinese Academy of Sciences Animal Standing Committee.

[0132] 2-2. LBE or LBP treatment and skeletal muscle regeneration

[0133] Five differently treated mouse models were used in this study. Adult (2 months old) and aged (14 months old) mice were treated with 2.5 g / kg LBE daily by intragastric perfusion for 4 months under physiological conditions, while an equal volume of saline was used as a control. Adult muscle regeneration model mice were given 2.5 g / kg LBE daily by intragastric perfusion starting one week before injury and continued throughout regeneration; aged mice were given LBE by intragastric perfusion starting one month before injury and continued throughout regeneration. Muscle regeneration was monitored in mice after induction of muscle injury by injection of 20 μL of 1.2% BaCl2 (Sigma) in phosphate buffered saline (PBS) into the mid-TA muscle.

[0134] 2-3. Isolation and culture of single muscle fibers

[0135] Single myofibers were isolated from mouse muscles by digestion using collagenase I (Sigma, C-0130). Each muscle sample was collected and incubated in serum-free DMEM medium with 0.2% collagenase I for 60-90 minutes in a shaking water bath at 37°C. Digestion was considered complete when the muscle appeared less firm and slightly swollen, with single myofibers like hair spreading out from the edges of the muscle. The muscles were then placed in a culture dish and the myofibers were isolated under a microscope. Single myofibers were placed in a six-well plate pre-coated with Matrigel (1:3) and allowed to attach for 3 minutes. Subsequently, 2 ml of fiber culture medium consisting of DMEM supplemented with 10% horse serum, 0.5% chicken embryo extract and 1% antibiotic / antimycotic was added. The fibers were cultured at 37°C / 5% CO2 for 72 hours and then fixed with 4% paraformaldehyde and stained for the detection of Pax7 and MyoD. Pax7 was determined by counting at least 30 single myofibers per mouse. + / MyoD + Cell numbers were used for statistical analysis. Five mice were assayed in each experiment.

[0136] When culturing satellite cells from myofibers, myofibers were removed as satellite cells migrated from the myofibers into the culture dish. The culture medium for satellite cells was replaced with growth medium (F-10 Ham's medium supplemented with 20% FBS, 2.5ng / mL basic fibroblast growth factor (bFGF) and 1% penicillin-streptomycin). After 1 day of culture, immunofluorescence staining was performed after fixation. In studies evaluating satellite cell differentiation, satellite cells were differentiated after 4 days of culture and 1 day of culture in differentiation medium.

[0137] 2-4. Isolation and culture of primary myoblasts

[0138] Primary myoblasts were isolated from skeletal muscle of 4-week-old mice: the muscle was minced and digested in 2 mL of digestion solution containing 0.2% collagenase (Gibco, 17101015), 2.4 U / mL dispase (Gibco, 17105041) and 2.5 mM CaCl2, and then incubated at 37°C for 30 minutes. The digestion was stopped by adding an equal volume of 10% FBS in PBS, and then the supernatant was filtered through a 40 μm nylon mesh to remove particulate matter, centrifuged at 350 g for 10 minutes, and the isolated cells were cultured in growth medium (F-10 Ham's medium supplemented with 20% FBS, 2.5 ng / mL bFGF and 1% penicillin-streptomycin) using collagen-coated cell culture plates at 37°C in a 5% CO2 environment.

[0139] 2-5. Isolation of satellite cells by fluorescence activated cell sorting (FACS)

[0140] Flow cytometry sorting was performed using muscle single cell suspensions. Muscles obtained from adult or aged mice (LBE treated or untreated) were washed with PBS, minced, and digested as described above. After centrifugation, the cells were washed twice with PBS and then suspended in PBS. These mononuclear cells were stained with CD34-FITC (1:500), CD31-PE (1:1000), CD45-APC (1:1000), and Sca1-PE-CY7 (1:1000) at 4°C for 30 minutes. Then, the cells were sorted using a BD FACS Aria II fluorescence-activated cell sorter. Definition of CD34 + / CD31 - CD45 - Sca1 - The cells are satellite cells. Satellite cells from skeletal muscle of Pax7-nGFP reporter mice were fluorescently sorted in a similar manner but without fluorescent antibody staining.

[0141] 2-6. C2C12 cell culture and transfection

[0142] C2C12 cells (a female mouse myoblast cell line) were cultured in DMEM (Gibco) medium supplemented with 4.5 g / L glucose, 10% fetal bovine serum and 1% antibiotic / antimycotic and cultured at 37°C in a 5% CO2 atmosphere. When they reached 70-80% density, the cells were cultured in differentiation medium (DMEM containing 2% horse serum) and cultured for 3 days with or without LBP. For gene knockout, C2C12 myoblasts were transiently transfected with siRNA using Lipofectamine2000 (Invitrogen). Mock siRNA was used as a negative control.

[0143] 2-7. Muscle Histochemical Analysis and H&E Staining

[0144] Tibialis anterior muscles (TA) were collected at 1, 3, 10, and 30 days after BaCl2-induced injury. These muscles were fixed overnight in PBS containing 4% paraformaldehyde (Sigma) and embedded in paraffin for subsequent H&E staining. The proportion of fibers with central nuclei (representing regenerating fibers) in the damaged area was calculated, and the cross-sectional areas (CSAs) of myofibers were measured using Image J software.

[0145] 2-7. Immunofluorescence (IF) staining

[0146] For Pax7 immunostaining, fresh sections were fixed in 4% paraformaldehyde for 20 minutes, permeabilized with methanol (-20°C) for 6 minutes, and then the sections were subjected to antigen retrieval using 100 mM sodium citrate (98°C) for 5 minutes. After washing with PBS, the sections were blocked with PBS solution containing 4% bovine serum albumin (BSA, Jackson) for 2 hours and then incubated with Pax7 antibody (1:20) at 4°C overnight. After washing with PBS, the sections were incubated with biotin-labeled goat anti-mouse IgG1 (1:1000) and Cy3-labeled streptavidin (1:2500). Nuclei were stained with Hoechst33258 (Beyotime).

[0147] For immunostaining of MyoD, fresh sections were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 / PBS (PBST) for 10 min, followed by blocking with 4% BSA for 2 h at room temperature. Immunostaining was performed using MyoD antibody (1:50) and incubated overnight at 4°C. After washing with PBS, sections were incubated with FITC-conjugated goat anti-rabbit IgG antibody. Fluorescence signals were observed using a confocal laser scanning microscope (LSM750) (Carl Zeiss).

[0148] 2-8. Western Blot Analysis

[0149] Muscle tissue or cells were lysed in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 5 mM EDTA and protease inhibitor cocktail solution (Roche). For each sample, 40 mg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to NC membrane. The membrane was blocked in TBST (10 mM Tris; 150 mM NaCl; and 0.1% Tween-20; pH 7.4) containing 5% skim milk powder for 1 hour and incubated with primary antibody overnight at 4°C. The primary antibodies included anti-Pax7 (1:500), anti-MyoD (1:1000), anti-MyHC (1:500), anti-p-FGFR1 (1:1000), anti-FGFR1 (1:1000), anti-p-p38 (1:1000), anti-p38 (1:1000), and anti-GAPDH (1:2000). After washing in TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshan Jinqiao Company, Beijing, China) at room temperature for 1 hour at a dilution ratio of 1:2000, and then washed three times with TBST. Each membrane was placed in ECL solution (Thermo Scientific, Waltham, MA, USA), and the signals were detected using Molecular Imager ChemiDoc XRS+ (BioRad).

[0150] 2-9. Quantitative reverse transcription polymerase chain reaction analysis

[0151] Collect muscle tissue or cells and use INVITROGENTM TRIzol TM Total RNA was extracted using TRIzol PCR according to the manufacturer's instructions (Invitrogen). TM The reagent is an acid-guanine-phenol-based reagent used to extract RNA (as well as DNA and protein) from various biological samples. RNA samples were then reverse transcribed using M-MuLV reverse transcriptase (Promega, Madison, Wisconsin, USA), and mRNA levels of relevant genes were measured by qRT–PCR using the 7500 Real-Time PCR System (Applied Biosystems, California, USA). Samples were heated to 95°C for 2 min and amplified for 40 cycles (1 min at 94°C, 1 min at 58°C, and 1 min at 72°C), followed by a final extension at 72°C for 10 min.

[0152] 2-10. Cellular thermal shift assay (CETSA)

[0153] C2C12 cells were collected and washed with PBS (PBS was used as a control) after 24 hours of LBP1C-2 treatment, and then the cell suspension of each group was divided into eight equal parts. One experimental sample and one control sample formed a pair and were heated from 34.0°C to 76.0°C for 5 minutes. Then, the cell suspension was frozen and thawed three times using liquid nitrogen. The cell lysate was centrifuged at 12000 rpm for 10 minutes at 4°C and boiled for 15 minutes. The soluble supernatant was used for protein immunoblot analysis.

[0154] 2-11. Surface plasmon resonance (SPR) analysis

[0155] The interaction assay was performed by surface plasmon resonance (SPR) spectroscopy. SPR measurements were performed using a Biacore 3000 biosensor from Biacore AB (Uppsala, Sweden). The binding affinity between FGFR1 and LBP1C-2 was investigated at 25 °C in 100 mM PBS (pH 7.4) as the medium. FGFR1 was immobilized on the flow channel of a research-grade CM5 chip (Biacore AB, Sweden) by amine coupling by standard methods. Samples were injected into the CM5 chip at a flow rate of 50 μL / min at 7 different concentrations (ranging from 0.25 μM to 16 μM) with a fixed ratio of protein to LBP1C-2 of 1:1. After each sample injection (150 s), PBS (flow rate: 50 μL / min) was flowed through the chip and the dissociation of the complex was monitored for 150 s. The chip surface was then regenerated by injecting 50 μL of 2 M NaCl in 10 mM sodium acetate (pH 4.5). A control channel was used by passing PBS buffer at the same flow rate. Data were kinetically analyzed using BIA Evaluation software (v 3.0).

[0156] 2-12. Quantitative and statistical analysis

[0157] Results are presented as mean ± standard error (SEM) of at least 3 independent experiments. Statistical analysis was performed using GraphPad Prism software package (GraphPad Software, La Jolla, CA, USA). The statistical significance of the difference between the two mean values ​​was calculated using a two-tailed Student's t-test. When three or more groups were compared, a one-way ANOVA was performed. When the experiment had two influencing factors, a two-way ANOVA was used to analyze statistical significance. For all analyses, p<0.05 was considered statistically significant, and the significance levels were as follows: ***, p<0.001; **, p<0.01; *, p<0.05.

[0158] 3. Results

[0159] 3-1. LBE increases the number of satellite cells in young mice.

[0160] The effects of wolfberry extract (LBE) on skeletal muscle satellite cells (SSCs) were evaluated in 2-month-old male C57BL / 6J mice, which were treated with LBE at a dose of 2.5 g / kg daily by oral gavage (with saline as control) for 4 months. Pax7 is a marker of satellite cells and is expressed in RNA ( Figure 1 ) and protein levels ( Figure 2 ) were upregulated by LBE. In order to further determine whether the upregulation of Pax7 was solely due to the upregulation of gene transcription or the increase in satellite cell population, the present invention performed immunofluorescence staining of Pax7 on frozen sections of tibialis anterior muscle. The results showed that LBE increased the number of Pax7-positive cells in tibialis anterior muscle ( Figure 3 ). In addition, satellite cells in the muscles of the control and LBE-treated groups were isolated by flow cytometry. + CD31 - CD45 - Sca1 - The number of satellite cells sorted was significantly increased by LBE ( Figure 4 ). Therefore, long-term treatment with LBE increases the number of satellite cells under normal physiological conditions.

[0161] 3-2. LBE reverses the decline of aged muscle satellite cells.

[0162] The reduction of satellite cell pool is an important cause of muscle dysfunction in old age. To evaluate the effect of LBE on satellite cells in old muscle, 14-month-old mice were fed a diet containing LBE for 4 months. Pax7 mRNA ( Figure 5 ) and protein ( Figure 6 ), and Pax7 + The cells were increased by LBE ( Figure 7 ), indicating that LBE can reverse the age-dependent decline in satellite cell number.

[0163] 3-3. LBE accelerates muscle regeneration in BaCl2-induced muscle damage.

[0164] To investigate whether LBE also affects satellite cell function, a BaCl2-induced muscle regeneration model was established in C57 mice. One week after LBE was administered by gavage, BaCl2 was injected intramuscularly into the tibialis anterior muscles of mice and harvested and analyzed on days 1, 3, 10, and 30 after injury. On day 10 after injury, regenerating muscle fibers characterized by central nuclei were larger in the LBE group than in the control group, indicating that the muscle regeneration process was accelerated after LBE treatment ( Figure 8 ). Next, to evaluate the effect of LBE on satellite cell activation and proliferation during regeneration, the present invention analyzed the levels of Pax7 and MyoD. Pax7 is expressed in both resting and activated satellite cells, while MyoD is expressed in satellite cells activated during muscle regeneration. On day 1 after BaCl2 injection, the mRNA levels of Pax7 and MyoD were higher in the LBE group ( Figures 9A-9B ), indicating that LBE promotes the activation of satellite cells in the early stage of muscle regeneration. Since the inventors found that LBE increased the number of stem cells under physiological conditions, the inventors were more interested in whether LBE had an effect on the self-renewal ability of satellite cells. On the 30th day after injury, Pax7 + MyoD - cell( Fig.10 ) were higher in the LBE group, indicating that more satellite cells exited the cell cycle and returned to a quiescent state. Therefore, LBE accelerated muscle regeneration by promoting the activation and self-renewal of satellite cells.

[0165] In addition, a BaCl2-induced muscle injury experiment was performed in 18-month-old LBE-fed aged mice. At 30 days after injury, HE staining showed that there were still many regenerating myofibers with central nuclei in the muscles, indicating that the muscle regeneration process in aged mice was slower than that in young mice. However, the cross-sectional area of ​​the regenerating myofibers in the LBE group was larger than that in the control group ( Fig.11 ), indicating that LBE also promoted muscle regeneration in aged mice. In general, LBE improved muscle regeneration in aged mice by restoring the dysfunction of aged muscle satellite cells.

[0166] 3-4. LBE tends to drive proliferating satellite cells into a quiescent state rather than differentiation.

[0167] Satellite cell differentiation is essential for supplying newly formed myofibers, while satellite cell self-renewal is also required to replenish the satellite cell pool. Defects in self-renewal capacity lead to a reduction in satellite cell numbers, which results in depletion of the satellite cell pool and reduced muscle regeneration capacity. Upregulation of Pax7 inhibits myogenesis and cell cycle progression in satellite cells. LBE increases Pax7 during muscle regeneration in vivo + MyoD -This prompted us to assess whether LBE also regulates satellite cells reentering a quiescent state in vitro. Single myofibers were isolated from muscles of 4-week-old mice, cultured, and treated with or without LBE for 72 h.

[0168] Immunostaining results showed that the proliferating (Pax7 + / MyoD + ), differentiation (Pax7 – / MyoD + ) and self-renewal (Pax7 + / MyoD – A higher proportion of Pax7 was found in satellite cells derived from myofibers treated with LBE. + / MyoD – cell( Fig.12 After 4 days of culture, myofiber-derived satellite cells were switched to differentiation medium. On day 1 after differentiation, LBE inhibited terminal muscle differentiation of satellite cells, as shown by MyHC staining and the transcriptional levels of myoG and MyHC ( Figures 13A-13B Overall, our in vivo and in vitro data convincingly demonstrate that LBE renders satellite cells more likely to self-renew rather than differentiate.

[0169] 3-5. LBP is the main component of LBE and plays a role in maintaining the satellite cell pool.

[0170] Since LBE plays such an important role in maintaining the muscle satellite cell pool, it is important to explore the main components of its function to understand the molecular mechanism. The main component of LBE is water-soluble Lycium barbarum polysaccharide (LBP), therefore, the present invention explores the function of LBP on muscle satellite cells. Consistent with the function of LBE, LBP also increased pax7 in the tibialis anterior muscle under physiological conditions. + cell( Fig.14 ). In isolated primary myoblasts, Pax7 + / MyoD – The proportion of cells increased after LBP treatment ( Fig.15 ).

[0171] In addition, the present invention uses Pax7-nGFP transgenic mice and GFP isolated by FACS. + Satellite cells. After 18 hours of culture, LBP increased the expression of Pax7 and myoD in these satellite cells, indicating that it promoted the activation of satellite cells ( Figures 16A-16B ). And after 48 hours of culture, the level of Pax7 in satellite cells was also higher than that in the control group, indicating that there were more self-renewing satellite cells ( Fig.17In the C2C12 cell line, LBP inhibited cell differentiation, as shown by the reduction of MyHC mRNA and protein ( Figures 18A-18B ). In summary, LBP is a major component of LBE, which plays a role in promoting satellite cell activation and maintaining the satellite cell pool.

[0172] 3-6.LBP1C-2 is a homogeneous polysaccharide and the active component of LBP that can regulate the function of satellite cells.

[0173] To further identify the effective single component, the present invention evaluated the efficacy of some components or homogeneous polysaccharides isolated from LBP. A homogeneous polysaccharide, LBP1C-2, increased the transcription level of pax7 in primary myoblasts after 18 hours of culture ( Fig.19 ). In isolated primary myoblasts, increased Pax7 + / MyoD – Cells and reduced Pax7 - MyoD + Cells further confirmed the role of LBP1C-2 in promoting satellite cell self-renewal and inhibiting differentiation ( Fig. 20 ). Therefore, LBP1C-2 is an important component of LBP regulation of satellite cell function in vitro.

[0174] 3-7. LBP1C-2 promotes satellite cell activation by binding to FGFR1 and promoting p38 phosphorylation.

[0175] Satellite cell activation after muscle injury is a transient but critical step in muscle regeneration. The earliest marker of activated satellite cells is phosphorylated p38 MAPK, followed by MyoD. The inventors examined the expression of phosphorylated p38 in young mouse muscle one day after BaCl2 injury and found that LBE significantly promoted the phosphorylation of p38 and correspondingly increased the level of MyoD ( Fig.21 ). Activation of the fibroblast growth factor receptor (FGFR1) shortly after muscle injury is one of the major pathways that promotes p38 phosphorylation. FGFR1-mediated p38 activation promotes the exit of satellite cells from the quiescent state and is essential for entry into the cell cycle. Phosphorylation of FGFR1 is also significantly upregulated ( Fig.21 Therefore, to determine whether FGFR1 is involved in LBP1C-2-mediated satellite cell activation, the function of LBP1C-2 was analyzed in C2C12 cells transfected with FGFR1 siRNA ( Fig.22A The results showed that FGFR1 deficiency reversed the upregulation of MyoD expression caused by LBP1C-2 ( Fig. 22B ) and increased p38 phosphorylation ( Fig.23), indicating that LBP1C-2 promoted satellite cell activation through FGFR1-mediated p38 phosphorylation.

[0176] Then, a question is whether LBP1C-2 may directly bind to the membrane receptor FGFR1 to activate the FGFR1-p38 signaling pathway. First, the inventors studied the binding properties of LBP1C-2 to FGFR1 by cellular thermal shift assay (CETSA). The results showed that in the presence of LBP1C-2, the stability of FGFR1 protein was slightly enhanced, while the stability of the control protein β-actin did not change ( Fig.24 ). The binding affinity of LBP1C-2 to FGFR1 was then quantified using surface plasmon resonance (SPR) spectroscopy. Fig.25 As shown, the binding affinity of LBP1C-2 to FGFR1 is 26.9 μM (KD), which indicates that the binding between LBP1C-2 and FGFR1 is weak. This weak binding is also logical, which explains why long-term administration of LBE under normal physiological conditions can slightly activate the proliferation and self-renewal of satellite cells without damage, thereby increasing the number of satellite cells.

[0177] 3-8.LBP1C-2 promotes satellite cell self-renewal by upregulating SPRY1.

[0178] The study found that LBE increased the expression of SPRY1 in muscles 30 days after injury ( Fig.26 ). Spry1 is a receptor tyrosine kinase signaling inhibitor that is essential for maintaining the quiescent satellite cell pool. In addition, in C2C12 myoblasts, LBP and LBP1C-2 also upregulated the expression of Spry1, and at the same treatment concentration, the effect of LBP1C-2 was stronger ( Fig. 27 To verify that Spry1 is actually involved in the function of LBP1C-2, we knocked down Spry1 in C2C12 cells by siRNA transfection ( Fig.28A ). The data showed that SPRY1 deficiency significantly inhibited the upregulation of Pax7 ( Fig.28B ), suggesting that SPRY1 plays a key role in LBP1C-2-mediated enhancement of satellite cell self-renewal.

[0179] 3-9. Results of the sulfuric acid derivative of LBP1C-2 (S-LBP1X-2)

[0180] FGFR1 inhibitor reversed the upregulation of Spry1 by LBP1C-2 in sorted satellite cells ( Fig.29A ). Consistent with this result, blocking FGFR1 by siRNA in C2C12 cells also inhibited LBP1C-2-induced upregulation of Spry1 ( Fig.29B These results suggest that LBP1C-2-induced Spry1 upregulation is also dependent on FGFR1 and that Spry1 upregulation may inhibit the p38 signaling pathway through negative feedback regulation, thereby preventing satellite cell overactivation.

[0181] In order to verify whether sulfated LBP1C-2 (ie, S-LBP1C-2) has the function of maintaining the skeletal muscle satellite cell pool, the present invention uses C2C12 myoblasts to evaluate the effect of S-LBP1C-2 on the expression of important markers of satellite cell activation and self-renewal.

[0182] First, the levels of phosphorylated p-p38 MAPK and MyoD, the earliest activated satellite cell markers, were determined by western blot and qPCR, respectively, in C2C12 cells treated with S-LBP1C-2 for 24 h. S-LBP1C-2 increased the mRNA level of MyoD and the protein level of phosphorylated p-p38 MAPK ( Fig. 30A , 30B). This suggests that S-LBP1C-2 also has the function of promoting satellite cell activation. At the same concentration of 200 μg / mL, the effect of S-LBP1C-2 was slightly better than that of LBP1C-2. Then, C2C12 cells were treated with different concentrations of S-LBP1C-2 (100, 200, 400 μg / mL), and it was observed that the upregulation of MyoD and p-p38 induced by S-LBP1C-2 was dose-dependent ( Fig.31A , 31B).

[0183] Secondly, based on the finding that LBP1C-2 promotes satellite cell self-renewal by upregulating Spry1, the present invention further evaluated the effect of S-LBP1C-2 on Spry1 expression by qPCR. S-LBP1C-2 also upregulated Spry1 expression in C2C12 cells in a dose-dependent manner, suggesting that it may also promote satellite cell self-renewal ( Fig.32A ). The results were compared with those using LBP1C-2, as shown in Fig.32B shown.

[0184] In summary, the present invention demonstrates for the first time that long-term use of wolfberry extract can improve satellite cell number and function and enhance muscle regeneration in adult and elderly mice. In addition, LBP1C-2 found from LBE is an active ingredient that regulates satellite cell function. LBP1C-2 promotes satellite cell activation and self-renewal by activating the FGFR1-p38 signaling pathway and upregulating Spry1 expression. This study provides a new perspective for the scientific explanation of the traditional efficacy of wolfberry and a theoretical basis for the medicinal or auxiliary medicinal use of wolfberry. Sulfate derivatives of the homogeneous polysaccharide were further prepared to show that they also have the same effect.

[0185] 4. Experimental samples and control samples:

[0186] Four homogeneous polysaccharides were isolated from wolfberry extract. Among these four homogeneous polysaccharides, only LBP1C-2 showed the functions described here. 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.

[0187] 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.

[0188] Formula 4, schematic structure of LBP1A1-1:

[0189]

[0190] 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.

[0191] Formula 5, schematic structure of LBP1B-S-2:

[0192]

[0193] Among the four homogeneous polysaccharides with different structures, only LBP1C-2 showed the desired functions and activities. LBP1C-2, its derivatives such as sulfate derivatives, and compositions containing LBP1C-2 or its derivatives are the preferred compositions for improving the number and / or function of muscle satellite cells and / or treating or preventing muscle atrophy.

[0194] 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 improving the number and / or function of muscle satellite cells, and / or treating or preventing muscle atrophy, 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.

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

5. The method according to claim 1, wherein: The molar ratio of arabinose, galactose, rhamnose and galacturonic acid monomer units in the homogeneous polysaccharide is in the range of 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 of the homogeneous polysaccharide is 49.9:33.6:8.0:8.

5.

7. The method according to claim 1, wherein: The molecular weight of the homogeneous polysaccharide ranges from about 10 kDa to about 150 kDa.

8. 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.

9. 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.

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

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

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

13. 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.

14. A composition for improving the number and / or function of muscle satellite cells, and / or treating or preventing muscle atrophy 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.

15. The composition according to claim 14, wherein The composition is a pharmaceutical composition, a functional composition and / or a dietary supplement.

16. The composition according to claim 14, wherein The composition is an oral composition and / or is in the form of a tablet.

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

18. The composition according to claim 17, 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.

19. The composition according to claim 14, wherein The receptor is selected from solvents, co-solvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents or any combination thereof.

20. The composition according to claim 14, wherein The molecular weight of the homogeneous polysaccharide ranges from about 10 kDa to about 150 kDa.

21. The composition according to claim 14, wherein The molar ratio of arabinose, galactose, rhamnose and galacturonic acid monomer units in the homogeneous polysaccharide is in the range of 30-70:20-60:0.1-10:0.1-10.

22. The composition according to claim 14, 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.

23. The composition according to claim 14, 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.

24. The composition according to claim 14, wherein The homogeneous polysaccharide is the only polysaccharide in the composition.

25. The composition according to claim 14, wherein The composition does not include flavonoids, carotenoids or polyphenols isolated from wolfberry extract.

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