A composition containing a homopolysaccharide or a derivative thereof, and a method for improving muscle satellite cells using the same

Abstract

The present invention provides compositions and methods for improving muscle satellite cell number and / or function and / or treating or preventing muscle atrophy in a subject in need of such a treatment. Such compositions include a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient. The homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units. Such methods include administering an effective dose of such a composition to a subject in need thereof.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Patent Application No. 63 / 344,158, filed on May 20, 2022, the content of which is incorporated herein by reference.

[0002] The present invention relates to compositions having pharmaceutical or functional properties. More specifically, the disclosed subject matter relates to compositions derived therefrom comprising cucurbit extracts, polysaccharides or derivatives thereof, and methods of using them, for example, as pharmaceutical compositions, functional compositions and / or supplements.

Background Art

[0003] Skeletal muscle is the largest organ of the human body and plays a very important role in maintaining the body's homeostasis and the entire metabolic process. Satellite cells (SCs), as skeletal muscle stem cells, play important roles in postnatal skeletal muscle growth, injury repair, and regeneration. Skeletal muscle satellite cells exist in a unique microenvironment (muscle sheath and basement membrane). The paired box protein Pax7 is the most important molecular marker of satellite cells and is expressed in both quiescent and activated satellite cells, while MyoD, also known as myoblast determination protein 1, is significantly upregulated only in satellite cells activated after injury and is considered a characteristic marker of activated satellite cells. In resting muscle, satellite cells are mostly quiescent but activate, proliferate, differentiate or self - renew in response to stress or injury to replenish the quiescent cell bank. Because satellite cells have a strong regenerative capacity in response to various stimuli, they are an important target for the treatment of muscle diseases.

[0004] One of the most significant effects of physical aging is the decline in skeletal muscle mass, muscle strength, and function, known as sarcopenia. After the age of 30, skeletal muscle mass decreases by approximately 3 - 8% every 10 years, and the rate of decline becomes even faster beyond the age of 60. The decrease in satellite cell number and the decline in the stem cell properties of satellite cells are important causes of age-related sarcopenia. Normally, satellite cells need to maintain a quiescent state, retain self-renewal ability, and be effectively activated to complete the regeneration process even when damaged. Aging affects the self-renewal ability of satellite cells, leading to an age-dependent decrease in the satellite cell pool and a decline in activation and / or proliferation ability, resulting in a decrease in regenerative capacity.

[0005] Previous studies have been somewhat controversial about whether satellite cell number decreases with aging. Some studies have suggested that the satellite cell numbers in young and old mice are similar. However, with the development of satellite cell labeling techniques, new studies have shown that satellite cells decrease with aging and are reduced by approximately 50% in old age. And it has been shown that improving the ability of satellite cells can restore muscle function in old mice. Therefore, the discovery of drugs that can regulate satellite cell function to treat age-related sarcopenia has attracted great attention.

[0006] During the aging process, the coordinated regulation of endogenous and exogenous signals such as p38 MAPK, JAK / STAT3, Wnt, Smad3, TGFβ, FGF2, SPRY1, etc. leads to a decrease in satellite cell number and loss of function. The development of intervention strategies targeting these important signaling pathways is also a focus of related research. For example, downregulation of JAK / STAT3 or p38 MAPK signaling through pharmacological inhibition or RNA interference (RNAi) can enhance the regenerative capacity of aged muscle stem cells. Wnt protein antagonists can restore satellite cell function in aged muscle.

[0007] By inhibiting TGFβ-p-Smad3 signaling, the function of satellite cells in aged muscle can also be restored. The mechanism of muscle atrophy due to the decrease in the number of stem cells with aging has been studied in increasing detail, and corresponding intervention strategies have been successful in animal experiments, but the safety of drugs targeting various signaling pathways needs to be considered in clinical applications.

Summary of the Invention

[0008] The present invention provides a composition and a method for using this composition to improve muscle satellite cell number and / or function and / or to treat or prevent muscle atrophy in a subject in need thereof.

[0009] In one aspect, the present invention provides a method for improving muscle satellite cell number and / or function and / or treating or preventing muscle atrophy in a subject in need thereof. According to some embodiments, the method comprises administering an effective dose of a composition comprising a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient, to a subject in need thereof. The homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units.

[0010] In some embodiments, the subject is a mammal, preferably a human subject, and may be a healthy human, an elderly adult, or an adult suffering from muscle atrophy.

[0011] The composition may be a pharmaceutical composition, a functional composition, and / or a supplement. In certain embodiments, the composition is administered orally or by forced oral administration. The composition may be in the form of a tablet or in a liquid form. For example, in certain embodiments, the composition is a pharmaceutical composition in the form of a tablet that can be orally administered. In certain embodiments, the composition may be a functional composition and may be in the form of a dry powder. The composition may also be formulated in the form of a sports drink or a snack bar.

[0012] The molecular weight of the homopolysaccharide is in the range of about 10 kDa to about 150 kDa, for example, about 10 kDa to about 100 kDa, about 10 kDa to 90 kDa, about 10 kDa to 60 kDa, or other suitable ranges. In the homopolysaccharide, the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid is in the range of 30 - 70:20 - 60:0.1 - 10:0.1 - 10.

[0013] For example, in the homopolysaccharide called LBP1C-2 used in the embodiments of the present invention, the ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid is 49.9:33.6:8.0:8.5. Its molecular weight may be a nearly specific value or in a narrow range within the range of about 10 kDa to about 150 kDa. Each polysaccharide obtained is homogeneous, and the molecular weight distribution is uniform or narrow.

[0014] In some embodiments, the homopolysaccharide described herein is the only polysaccharide in the composition.

[0015] In other embodiments, the composition further comprises other polysaccharides separated from the Lycium barbarum extract. The homopolysaccharide accounts for 15% or more of the total polysaccharide content in the composition. All polysaccharides are derived from Lycium barbarum. For example, the polysaccharides used in the present invention are Lycium barbarum polysaccharides (referred to as LBP).

[0016] In some embodiments, the composition optionally further comprises one or more of a flavonoid, a carotenoid, a polyphenol, a pigment, or any compound isolated from a goji extract.

[0017] In some embodiments, the composition comprises a chemically modified derivative of the homopolysaccharide described herein. For example, such a derivative is a pharmaceutically acceptable ester or salt thereof. The pharmaceutically acceptable ester or salt thereof is a sulfate ester derivative of a homopolysaccharide called a sulfated polysaccharide.

[0018] In some embodiments, the composition comprises a chemically modified derivative of the homopolysaccharide described herein. For example, such a derivative is a pharmaceutically acceptable ester or salt thereof. The pharmaceutically acceptable ester or salt thereof is a sulfate ester derivative of a homopolysaccharide called a sulfated polysaccharide.

[0019] The recipient 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 of the foregoing.

[0020] The composition may be administered in any suitable amount. For example, in some embodiments, the effective dose of the composition (expressed herein in terms of the amount of the homopolysaccharide) ranges from 10 mg / kg to 500 mg / kg based on the total weight of the homopolysaccharide per day / subject's daily body weight. The composition may be administered once a day, twice a day, or multiple times a day.

[0021] In another aspect, the present invention provides a composition (described herein) for improving muscle satellite cell number and / or function and / or treating or preventing muscle atrophy in a subject in need thereof. Such a composition comprises an effective dose of a homopolysaccharide and a pharmaceutically acceptable excipient. The homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units.

[0022] As described in this specification, the composition is a pharmaceutical composition, a functional composition, and / or a supplement. For example, in certain embodiments, the composition exists in the form of an oral composition and / or a tablet.

[0023] The recipient may be selected from a solvent, a co-solvent, a coloring agent, a preservative, an antimicrobial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination of the above.

[0024] The molecular weight of the homopolysaccharide ranges from about 10 kDa to about 150 kDa, for example, about 10 kDa to about 100 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about 60 kDa, or any other suitable range. In the homopolysaccharide, the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid ranges from 30 - 70:20 - 60:0.1 - 10:0.1 - 10.

[0025] For example, in certain embodiments, the homopolysaccharide has a ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid of 49.9:33.6:8.0:8.5. Its molecular weight may be approximately a specific value or within a narrow range of about 10 kDa to about 150 kDa. Each polysaccharide obtained is homogeneous, and the molecular weight distribution is uniform or narrow.

[0026] In some embodiments, the homopolysaccharide described in this specification is the only polysaccharide in the composition. In some other embodiments, the composition further contains other polysaccharides separated from the kukko extract, and / or the homopolysaccharide accounts for 15% or more of the total polysaccharide content in the composition.

[0027] In some embodiments, the composition may optionally contain flavonoids, carotenoids, polyphenols, pigments, or any combination thereof separated from the kukko extract. In some other embodiments, the composition does not contain flavonoids, carotenoids or polyphenols separated from the kukko extract, and the composition may contain only polysaccharides separated from the kukko extract.

[0028] In some embodiments, the composition contains a chemically modified derivative of the homopolysaccharide described herein. For example, such a derivative is a pharmaceutically acceptable ester or salt thereof. The pharmaceutically acceptable ester or salt thereof is a sulfate ester derivative of a homopolysaccharide called a sulfated polysaccharide.

[0029] The present invention also provides the use of the homopolysaccharide or its derivative described herein in the manufacture of a drug for treating any disease state described herein.

[0030] In another aspect, the present invention provides a method for preparing the composition or the homopolysaccharide. This method may include preparing or separating the homopolysaccharide. This method may further include mixing the recipient and the homopolysaccharide.

Brief Description of the Drawings

[0031] The content of the present invention is best understood by reading the following detailed description with reference to the drawings. Usually, the various features in the drawings are not necessarily drawn to scale. Conversely, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. Throughout the specification and drawings, the same reference numerals refer to the same features.

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BEST MODE FOR CARRYING OUT THE INVENTION

[0032] In the present invention, the singular forms "one", "a kind", and "the" include plural, and a reference to a specific numerical value includes at least that specific value unless the context clearly indicates otherwise. Thus, for example, a reference to "additive" is a reference to one or more such compounds and equivalents known to those skilled in the art. When a value is expressed as an approximate value using the prefix "about", it is understood that that specific value forms another embodiment. As used herein, "about X" (where X is a numerical value) preferably means including ±10% of the recited value, including the endpoint values.

[0033] For example, the phrase "about 8" preferably includes values from 7.2 to 8.8, including the endpoint values. As another example, the phrase "about 8%" preferably (but not always) means including values from 7.2% to 8.8%. If present, all ranges are inclusive and combinable. For example, if the range "1 to 5" is recited, the recited range should be interpreted to include ranges such as "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", "2 to 5", etc. Also, if alternatives are affirmatively provided, such items can be interpreted to mean that any alternative may be excluded, for example, by negative limitations in the claims.

[0034] For example, if the range "1 to 5" is recited, the recited range can be interpreted to include cases where any one of 1, 2, 3, 4, or 5 is negatively excluded. Thus, the recitation "1 to 5" can be interpreted as "1 and 3 to 5, but not including 2", or simply "but not including 2". Components, elements, attributes, or steps explicitly recited in this specification are intended to be able to be explicitly excluded in the claims, regardless of whether such components, elements, attributes, or steps are described as alternatives or are described individually.

[0035] Components, elements, attributes, or steps positively cited in this specification can be explicitly excluded in the claims, regardless of whether such components, elements, attributes, or steps are described as alternatives or are described individually.

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

[0037] As used herein, the term "drug" refers to any molecule, compound, method, and / or substance for preventing, treating, managing, and / or diagnosing a disease or condition. As used herein, the term "effective dose" refers to a dose of one therapy that is sufficient to prevent a disease or condition and the progression, recurrence, or onset of one or more of its symptoms, enhance or improve the prophylactic effect of another therapy, reduce the severity of a disease or condition, shorten the duration of a disease or condition, reduce 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.

[0038] As used herein, the phrase "pharmaceutically acceptable" means approved by a regulatory authority of the federal or state government or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopeias for use in animals, more specifically in humans.

[0039] As used herein, the term "therapeutic agent" refers to any molecule, compound, and / or substance used for treating and / or managing a disease or disorder.

[0040] As used herein, the terms "treatment method" and "treatment" may refer to any method, composition, and / or drug for preventing, treating, and / or managing a disease or condition, or one or more symptoms thereof. In certain embodiments, the terms "treatment method" and "treatment" refer to small molecule treatment methods.

[0041] As used herein, the terms "treatment", "treatment method", and "under treatment" refer to reducing or inhibiting the progression and / or duration of a disease or condition, reducing or improving the severity of a disease or condition such as cancer, and / or improving one or more symptoms as a result of administration of one or more treatment methods, in the context of subjecting a subject to a treatment method.

[0042] As used herein, the term "excipient" refers to an inert substance that functions as a carrier or medium for a drug or other active substance. Examples of suitable excipients include, but are not limited to, solvents, co-solvents, colorants, preservatives, antimicrobial agents, fillers, binders, disintegrants, lubricants, surfactants, emulsifiers, suspending agents, or any combination thereof.

[0043] Molecular weight is measured by gel permeation chromatography (GPC). GPC is an analytical technique that separates molecules within a polymer by size and provides the molecular weight or molecular weight distribution of the material. The homopolysaccharides described herein contain only one peak in GPC and have a uniform molecular weight.

[0044] Muscle stem cells, also known as satellite cells (SCs), are used for muscle repair and reconstruction, and when their function is impaired, the muscle regeneration ability decreases. There is an urgent need to find satellite cell-based treatment strategies to improve muscle function and restore age-related muscle degeneration.

[0045] The mechanism of muscle atrophy caused by the decrease in the number of stem cells with aging has been studied in increasing detail, and corresponding intervention strategies have been successful in animal experiments. However, in clinical applications, it is necessary to consider the safety of drugs targeting various signaling pathways.

[0046] Therefore, within the context of the present invention, the discovery of natural plants with the ability to improve muscle function is considered important for the development of clinical pharmaceutical intervention strategies targeting satellite cell regulation to improve aging- and age-related muscle diseases.

[0047] Gookbong has been used as a traditional Chinese medicine and has effects such as anti-fatigue and anti-aging. Modern research has verified the anti-aging effect of Gookbong in models such as cells, Drosophila, zebrafish, and mice. However, so far, little is known about the molecular mechanism and function of Gookbong, and the active ingredients are also unknown.

[0048] The inventors of the present invention used C57BL / 6 mice as a model and discovered that the aqueous extract of Gookbong significantly increased the muscle weight ratio of the anterior tibial muscle and gastrocnemius muscle of mice, decreased the fat content, and increased the average maximum running distance of mice, that is, it could increase the muscle endurance.

[0049] In this study, the inventors sought to determine the efficacy of wolfberry in improving the maintenance of skeletal muscle satellite cells and further identify the active ingredients and targets. The inventors discovered that long-term use of wolfberry extract (LBE) improved satellite cell numbers and function in adult and aged mice and promoted muscle regeneration. Furthermore, the homopolysaccharide LBP1C-2 was discovered from the wolfberry extract, which is an active ingredient of LBE that can regulate satellite cell function. LBP1C-2 activates p38 signaling by interacting with FGFR1, upregulates spry1 expression, and promotes satellite cell activation and self-renewal. This is a new discovery that LBE is involved in the regulation of muscle stem cells. More importantly, the inventors have 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 adjuvant medicinal use of wolfberry.

[0050] The present invention provides compositions and methods for improving muscle satellite cell numbers and / or function and / or treating or preventing muscle atrophy in a subject in need thereof.

[0051] According to some embodiments, such a composition comprises a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient, and is injected into a subject in need thereof. The homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units. Such a method comprises administering an effective amount of the composition to a subject in need thereof.

[0052] In some embodiments, the subject is a mammal, preferably a human subject, which may be a healthy human, an elderly adult, or an adult suffering from muscle atrophy.

[0053] The composition may be a pharmaceutical composition, a functional composition, and / or a supplement. In certain embodiments, the composition is administered orally or by forced oral administration. The composition may be in the form of a tablet or in a liquid form. For example, in certain embodiments, the composition is an orally administrable pharmaceutical composition in the form of a tablet. In certain embodiments, the composition may be a functional composition and may be in the form of a dry powder. The composition may also be formulated in the form of a sports drink or a snack bar.

[0054] The molecular weight of the homopolysaccharide ranges from about 10 kDa to about 150 kDa, for example, about 10 kDa to about 100 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about 60 kDa, or any other suitable range. In the homopolysaccharide, the molar ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid ranges from 30 - 70:20 - 60:0.1 - 10:0.1 - 10. For example, in some embodiments, the homopolysaccharide has a ratio of monomer units of arabinose, galactose, rhamnose, and galacturonic acid of 49.9:33.6:8.0:8.5.

[0055] Its molecular weight may be a specific value or a narrow range within the range of about 10 kDa to about 150 kDa. Such molecular weight values may be weight-average molecular weight (Mw) or number-average molecular weight (Mn). Since the polysaccharide is homogeneous and has a very narrow and uniform molecular weight distribution, the values of Mw and Mn are considered to be close. The polydispersity (PD) index, i.e., the ratio of Mw to Mn, may range from about 1 to about 1.3, about 1 to about 1.2, and about 1 to about 1.1. In some embodiments, the PD index is close to 1. The molecular weight of the polysaccharide made from cucurbits may vary depending on factors such as the growth environment and the harvest time.

[0056] Therefore, the resulting homopolysaccharide may have different molecular weights. However, the polysaccharide obtained in each batch is homogeneous, that is, its molecular weight is either homogeneous or has a narrow distribution. The molecular weight can be controlled by controlling the quality of the raw materials, such as making the growth environment and the growth time before harvest the same.

[0057] In some embodiments, the homopolysaccharides described herein are the only polysaccharides in the composition.

[0058] In other embodiments, the composition further comprises other polysaccharides separated from the Lycium barbarum extract. The homopolysaccharides account for 15% or more of the total polysaccharides in the composition. All of the polysaccharides may be derived from Lycium barbarum. For example, the polysaccharides used in the present invention are Lycium barbarum polysaccharides (referred to as LBP).

[0059] In some embodiments, the composition optionally further comprises one or more of flavonoids, carotenoids, polyphenols, pigments, or any compounds separated from the Lycium barbarum extract.

[0060] The Lycium barbarum extract (LBE) can be obtained by extracting Lycium barbarum with water, and this extract may exist in the form of a dry powder. The extract can be dissolved in water and further purified or separated by a fractional purification method. Polysaccharides are obtained. These polysaccharides are further separated by fractional purification and lyophilization, and one or more homopolysaccharides are obtained in the form of a dry powder.

[0061] In some embodiments, the composition comprises the Lycium barbarum extract (LBE). This Lycium barbarum extract contains polysaccharides (or Lycium barbarum polysaccharides), Lycium barbarum flavonoids, carotenoids, polyphenols, and Lycium barbarum pigments. Each component may be only one type, or two or more of the same type of component may be present. For example, the composition may contain two or more polysaccharides, two or more Lycium barbarum flavonoids, two or more carotenoids, two or more polyphenols, and / or two or more Lycium barbarum pigments.

[0062] In some embodiments, the polysaccharide content in the Lycium barbarum extract (LBE) ranges from about 10.0 wt.% to about 70.0 wt.% (e.g., about 50 wt.% to about 70 wt.%) based on the total dry weight of the extract, the Lycium barbarum 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 Lycium barbarum pigments range from about 0.1 wt.% to about 8.0 wt.%.

[0063] In some embodiments, the polysaccharide is more preferably from about 50.0 wt.% to about 70.0 wt.%. The dry weight corresponds to the equivalent weight of the extract in dry powder (water-free) form. The extract may contain other trace residues. The extract in dry powder form can be mixed with water to provide an extract in the form of an aqueous solution having the selected concentrations described herein.

[0064] In some embodiments, the Lycium barbarum extract (LBE) exists in the form of a powder. In some embodiments, the Lycium barbarum extract is dissolved in a solvent such as water to provide an aqueous solution having a concentration in the range of, for example, 0.1 g / mL to 5 g / mL. In the experiments, for the LBE used, the range of the polysaccharide content in the dry powder form of the LBE is from about 50.0 wt.% to 70.0 wt.%.

[0065] The Lycium barbarum polysaccharides, or polysaccharides (LBP), exist in the form of a powder and can be further purified from the LBE in powder form. For example, the LBE can be dissolved in water and separated through a separation column. The Lycium barbarum polysaccharides (LBP) may further contain various polysaccharides that can be separated.

[0066] The homopolysaccharide LBP1C-2 is separated from LBE and / or LBP. Based on high-performance gel permeation chromatography (HPGPC) analysis, LBP1C-2 showed a single symmetric peak, indicating that it is a homopolysaccharide. According to sugar composition analysis, LBP1C-2 is composed of arabinose (Ara), galactose (Gal), rhamnose (Rha), and galacturonic acid in a ratio of 49.9:33.6:8.0:8.5. The structure of LBP1C-2 includes a main chain in which 1,2-linked α-Rhap and 1,4-linked α-GalpA are arranged alternately, 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.

[0067] Formula 1, Formula 2, and Formula 3 show the structure of LBP1C-2, presenting the same structure in three different forms.

[0068] Referring to Formulas 1-3, LBP1C-2 is composed of Ara, Gal, Rha, and GalA, and the molar ratio is 49.9:33.6:8.0:8.5. Through structural analysis, LBP1C-2 has a main chain of 1,2-α-Rha and 1,4-α-GalA, and its branches include T-α-Ara, 1,5-α-Ara, T-β-Rha, T-β-Gal, 1,3-β-Gal, 1,6-β-Gal, and 1,3,6-β-Gal. These branches are attached to the C-4 position of the backbone sugar residue of 1,2,4-α-Rha. The repeating unit of LBP1C-2 includes the structural part shown in Formula 3, and consists of 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.

[0069] TIFF2025518622000001.tif229170

[0070] TIFF2025518622000002.tif196170

[0071] TIFF2025518622000003.tif200170

[0072] In Formulas 1 to 3, the range of n is 2 to 20. The molecular weight is proportional to the value of n. For example, when the molecular weight of the sample is about 13.2 kDa, n is about 2. When the molecular weight of the sample is about 99.8 kDa, n is about 13.

[0073] The present inventors provide homopolysaccharides such as LBE or LBP polysaccharides or LBP1C-2 which is an active ingredient thereof, having an effect of improving the number and / or function of muscle satellite cells and / or treating or alleviating muscle atrophy in an individual in need thereof. Derivatives of the homopolysaccharides, esters or salts thereof, or solvates thereof have the same effect.

[0074] In some embodiments, the composition comprises a chemically modified derivative of the homopolysaccharide described herein. For example, such a derivative is a pharmaceutically acceptable ester or salt thereof. The pharmaceutically acceptable ester or salt thereof is a sulfate ester derivative of a homopolysaccharide called a sulfated polysaccharide. During the synthesis of the sulfated homopolysaccharide, the hydroxy in the homopolysaccharide reacts with a modifier such as chlorosulfonic acid to form a -O-SO3H group. The molecular weight after modification is the same as that of the above homopolysaccharide.

[0075] The degree of sulfate substitution of the sulfate ester derivative of the homopolysaccharide 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 substituents on the sugar unit. For example, a degree of substitution of 0.74 means that the number of sulfated substituents on each hexose or pentose unit is 0.74. Combining sulfate substitution with a protein can improve biological activity.

[0076] The composition may be a pharmaceutical composition, a functional composition, and / or a supplement. For example, the composition is an orally administrable pharmaceutical composition. In the examples of the present invention, the aqueous extract of Lycium barbarum is administered by forced oral administration or oral administration, but is not limited thereto. Any form of administration of the composition to the stomach may be appropriate.

[0077] The recipient may be a solvent (such as water or an aqueous solvent), a co-solvent, a coloring agent, a preservative, an antimicrobial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination thereof.

[0078] The composition may be administered in any suitable amount. For example, in some embodiments, the daily effective amount of the composition (converted herein in terms of the amount of homopolysaccharide) ranges from 10 mg / kg to 500 mg / kg based on the total weight of the homopolysaccharide / body weight of the subject. In some embodiments, the dosage of Lycium barbarum extract (LBE) or LBP or LBP1C-2 is within the range of 4 mg / kg to 70 mg / kg per day (total dry weight of LBP1C-2 per day / body weight of a subject such as a human), for example, 10 mg / kg to 70 mg / kg, 10 mg / kg to 60 mg / kg, 20 mg / kg to 70 mg / kg, 20 mg / kg to 60 mg / kg, 20 mg / kg to 50 mg / kg, or other suitable ranges. The composition can be administered once a day, twice a day, or more than twice a day.

[0079] In some embodiments of the present invention, the dosage of Lycium barbarum extract (LBE) or LBP or LBP1C-2 is 40 mg / kg per day (total dry weight of LBP1C-2 per day / body weight of an animal such as a mouse). This dosage is a total of three dosages per day.

[0080] In some embodiments, these compositions may be ingested together with drinks, foods, or related components. The kukso water extract according to the present invention is both a medicine and a food, and can be used as a health food. There are no particular restrictions on food and health food formulations. For example, it can be made into tablets, drinks, candies, etc. Each food formulation may contain, in addition to the homopolysaccharide, other compounding ingredients used in the art. Other compounding ingredients can be selected by those skilled in the art according to specific formulations or applications.

[0081] In some embodiments, the composition is a food or health product including sports drinks, protein powders, snack bars, etc.

[0082] The present invention also provides the use of the homopolysaccharide or its derivative described herein in the manufacture of a medicament for treating any medical condition described herein.

[0083] In another aspect, the present invention provides a method for preparing the composition or the homopolysaccharide. This method may include preparing the homopolysaccharide. This method may further include mixing the recipient with the homopolysaccharide.

[0084] The features and effects of the present invention will be described by way of examples and test examples. However, the following examples and test examples are merely illustrative and do not limit the scope of the present invention.

[0085] Examples 1. Preparation of Materials 1-1. Preparation of Kukso Extract The preparation process of Lycium barbarum extract (「LBE」) is as follows. The fruits of Lycium barbarum are cultivated and harvested in Zhongning County, Yinchuan City, Ningxia Hui Autonomous Region, China. After washing and pulverizing 3 to 5 times, the dried fruits were immersed in redistilled water (pH = 7) at room temperature for 2 hours. 5 to 8 times of neutral water was added to the immersed fruit powder and mixed uniformly, and it was decocted twice at the boiling point temperature, and the decoction times were 2.0 hours and 1.5 hours respectively. The combined concentrated decoction was filtered through a hollow fiber membrane. The above filtrates were combined and evaporated under vacuum at 30 to 55 °C to remove water, and a concentrate was obtained. The obtained concentrate was freeze-dried into a powder and stored in a desiccator so that it could be used at an appropriate concentration in subsequent experiments.

[0086] In the experiment, an exemplary Lycium barbarum 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 illustrative purposes only. The aqueous extract can be adjusted to any appropriate concentration in the range of, for example, about 0.1 g / ml to 5 g / ml (e.g., dry weight of the extract / volume of the extract). In certain embodiments, this aqueous extract can be diluted for use. The aqueous extract can be further diluted for cell experiments.

[0087] The Lycium barbarum extract (LBE) used in the present invention mainly contains water-soluble Lycium barbarum polysaccharides, Lycium barbarum flavonoids, carotenoids, polyphenols, and pigments. In an exemplary LBE, the polysaccharide content ranges from about 50.0 wt.% to 70.0 wt.% (e.g., about 54% - 56% or 54%) based on the total equivalent of the extract in dry powder form, the Lycium barbarum flavonoid content ranges from about 0.1 wt.% to 5.0 wt.%, the carotenoid ranges from about 0.1 wt.% to 3.0 wt.%, the polyphenol ranges from about 0.1 wt.% to 8.0 wt.%, and the Lycium barbarum pigment ranges from about 0.1 wt.% to 8.0 wt.%. LBE is in the form of a powder and is soluble in water or saline. LBE with the same composition was used for comparison.

[0088] 1 - 2. Separation of Lycium barbarum polysaccharides and homopolysaccharides Cocosaccharides (「LBP」) and homopolysaccharides such as the examples described herein labeled with LBP1C-2 can be separated from LBE or coconut fruits by the following exemplary methods.

[0089] The extraction methods of the above polysaccharides include the following. The dried fruits were pulverized, 15 - 30 times deionized water was added and mixed well, 3 wt.% cellulase, 1 wt.% amylase, and 0.5 wt.% papain were added, and extraction was carried out at 55 - 60 °C for 1 hour. After raising the temperature to inactivate the enzyme, centrifugation was performed, the obtained filtrate was concentrated, dialyzed, concentrated again, 5 times 95% ethanol was added, centrifuged to obtain a precipitate, washed 3 times alternately with absolute ethanol and acetone, and vacuum dried to obtain crude polysaccharides (LBP).

[0090] The above method further includes the following. The crude polysaccharides were dissolved in 10 - 15 times water, centrifuged, the supernatant was collected, fractionated and purified through a diethylaminoethyl (DEAE) cellulose anion exchange column, and then eluted successively with water, 0.05 M, 0.1 M, and 0.2 M sodium chloride. The fraction eluted with 0.2 M sodium chloride was collected, concentrated, dialyzed, and freeze-dried to obtain preliminarily purified cocosaccharides (LBP1C). The obtained polysaccharide LBP1C was dissolved in 0.2 M sodium chloride, centrifuged, the supernatant was collected, elution was performed on a Sephacryl-300 (RTM: poly((propenyldextran)-co-N,N'-methylenebisacrylamide)) column, the eluted fractions were collected, concentrated, dialyzed, and freeze-dried to obtain homopolysaccharide LBP1C-2.

[0091] LBC and LBP1C-2 have the composition and structure described herein.

[0092] 1-3. Detection of the molecular weight of LBP1C-2 The molecular weight and homogeneity of LBP1C-2 were measured by high-performance gel permeation chromatography (HP-GPC). Only one symmetric peak was shown in the HP-GPC result figure. Referring to the dextran standards with known molecular weights 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.

[0093] 1-4. Preparation of sulfated polysaccharide derivatives of LBP1C-2 and measurement of degree of sulfate substitution (DS) This 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 consisting of chlorosulfonic acid and pyridine (3:1, v / v) was added under an ice bath. Then, the mixture was stirred at 40 °C for 4 hours, cooled, and neutralized with 5 M NaOH. First, the solution was dialyzed against saturated NaHCO3, and then against distilled water. The residue was 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. TIFF2025518622000004.tif11170

[0094] Here, DS is the degree of sulfate substitution, and %S is the sulfur content.

[0095] Based on the sulfur content (10%), referring to the sulfate content standard curve detected by the chlorosulfonic acid-pyridine method, the degree of sulfate group substitution (DS) of S-LBP1C-2 was calculated to be 0.74.

[0096] 2. Biological experiments 2-1. Mouse strains and animal management The LBE treatment and muscle regeneration experiments were performed using male C57BL / 6J mice purchased from Vital River. The Pax7-nGFP transgenic mice were donated by the research team of Zhu Dahai (Peking Union Medical College, Beijing, China). All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Chinese Academy of Sciences.

[0097] 2-2. LBE or LBP Treatment and Skeletal Muscle Regeneration In this study, a mouse model with five different treatments was used. Under physiological conditions, while using the same volume of normal saline as a control, adult (2 months old) and aged (14 months old) mice were intragastrically administered 2.5 g / kg of LBE daily for 4 months. Adult muscle regeneration model mice were intragastrically administered 2.5 g / kg of LBE daily starting 1 week before injury, and the administration was continued throughout the regeneration process. Aged mice started intragastric administration of LBE 1 month before injury and the administration was continued throughout the regeneration process. After inducing muscle injury by injecting 20 μL of 1.2% BaCl2 (Sigma) in phosphate-buffered saline (PBS) into the center of the TA muscle, the muscle regeneration of the mice was monitored.

[0098] 2-3. Isolation and Culture of Single Muscle Fibers Single muscle fibers were isolated by digesting mouse muscle with collagenase I (Sigma, C-0130). Each muscle sample was collected in a shaking water bath at 37°C and incubated in serum-free DMEM with 0.2% collagenase I for 60 - 90 minutes. Digestion was considered complete when the muscle became softer, slightly swollen, and single hair-like muscle fibers seemed to radiate from the ends of the muscle.

[0099] Next, the muscle was placed in a culture dish, and muscle fibers were isolated under a microscope. Single muscle fibers were placed in a 6-well plate pre-coated with Matrigel (1:3) and allowed to adhere for 3 minutes. Subsequently, 2 ml of fiber medium consisting of DMEM supplemented with 10% fetal bovine serum, 0.5% chicken embryo extract, and 1% antibiotic / antifungal agent was added. The fibers were incubated at 37 °C / 5% CO2 for 72 hours, then fixed with 4% paraformaldehyde and stained to detect Pax7 + / MyoD + The number of cells was used for statistical analysis. In each experiment, measurements were taken on 5 mice.

[0100] When culturing satellite cells derived from muscle fibers, the muscle fibers were removed when the satellite cells migrated from the muscle fibers to the culture dish. The culture medium of satellite cells was replaced with growth medium (F-10 Ham's medium supplemented with 20% FBS, 2.5 ng / mL basic fibroblast growth factor (bFGF), and 1% penicillin-streptomycin). After culturing for 1 day, immunofluorescence staining was performed after fixation. In the study evaluating the differentiation of satellite cells, the satellite cells began to differentiate after 4 days in culture and 1 day in differentiation medium.

[0101] 2 - 4. Isolation and culture of primary myoblasts Isolation of primary myoblasts from the skeletal muscle of 4-week-old mice: The muscle was minced and digested with 2 mL of digestion solution containing 0.2% collagenase (Gibco, 17101015), 2.4 U / mL dispase (Gibco, 17105041), and 2.5 mM CaCl2, and incubated at 37 °C for 30 minutes. An equal volume of 10% FBS was added to stop the digestion 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 separated cells were placed in growth medium (F-10 Ham's medium supplemented with 20% FBS, 2.5 ng / mL bFGF, and 1% penicillin-streptomycin) and cultured in an environment of 37 °C, 5% CO2 using a collagen-coated cell culture plate.

[0102] 2-5. Isolation of satellite cells by fluorescence-activated cell sorting (FACS) Sorting by flow cytometry using single muscle cell suspensions: Muscles obtained from adult or aged mice (LBE-treated or untreated) were washed with PBS, minced finely, 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.

[0103] Next, the cells were sorted using a BD FACS Aria II fluorescence-activated cell sorter. CD34 + / CD31 - CD45 - Sca1 - The cells were defined as satellite cells. Satellite cells derived from the skeletal muscle of Pax7-nGFP reporter mice were also fluorescence sorted in the same manner, but fluorescence antibody staining was not performed.

[0104] 2-6. Culture and transfection of C2C12 cells C2C12 cells (female mouse myoblast cell line) were cultured in DMEM (Gibco) supplemented with 4.5 g / L glucose, 10% fetal bovine serum, and 1% antibiotic / antifungal agent, and cultured in an atmosphere of 37 °C and 5% CO2. When the cells reached a density of 70 - 80%, they were cultured for 3 days in differentiation medium (DMEM containing 2% horse serum) in the presence or absence of LBP. For gene knockdown, siRNA was transiently transfected into C2C12 myoblasts using Lipofectamine 2000 (Invitrogen). Mock siRNA was used as a negative control.

[0105] 2-7. Histological analysis of muscle and H&E staining The tibialis anterior (TA) muscles were harvested 1, 3, 10, and 30 days after BaCl2-induced injury. These muscles were fixed overnight in PBS containing 4% paraformaldehyde (Sigma) and then embedded in paraffin for subsequent H&E staining. The percentage of fibers with a central core within the damaged area (representing regenerating fibers) was calculated, and the cross-sectional area (CSA) of muscle fibers was measured using ImageJ software.

[0106] 2-7. Immunofluorescence (IF) staining For Pax7 immunostaining, fresh sections were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with methanol (-20 °C) for 6 minutes, and then the antigens were repaired by treating the sections with a 100 mM sodium citrate process (98 °C) for 5 minutes. After washing with PBS, the sections were blocked with a PBS solution containing 4% bovine serum albumin (BSA, Jackson) for 2 hours and incubated with the Pax7 antibody (1:20) overnight at 4 °C. After washing with PBS, the sections were incubated with biotinylated goat anti-mouse IgG1 (1:1000) and Cy3-labeled streptavidin (1:2500). The nuclei were stained with Hoechst 33258 (Beyotime).

[0107] For MyoD immunostaining, fresh sections were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.5% Triton X-100 / PBS (PBST) for 10 minutes, and then blocked with 4% BSA at room temperature for 2 hours. Immunostaining was performed using the MyoD antibody (1:50) and incubated overnight at 4 °C. After washing with PBS, the sections were incubated with an FITC-conjugated goat anti-rabbit IgG antibody. The fluorescence signals were observed using a confocal laser scanning microscope (LSM750) (Carl Zeiss).

[0108] 2-8. Western blot analysis 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 an NC membrane. The membrane was blocked in TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20, pH 7.4) containing 5% non-fat milk powder for 1 hour and incubated overnight at 4 °C with the primary antibody.

[0109] 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 with TBST, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Zhongshan Golden Bridge, Beijing, China) at a dilution ratio of 1:2000 for 1 hour at room temperature and then washed three times with TBST.

[0110] Each membrane was placed in ECL solution (Thermo Scientific, Waltham, MA, USA), and signals were detected using a Molecular Imager ChemiDoc XRS+ (BioRad).

[0111] 2-9. Quantitative reverse transcription polymerase chain reaction analysis Skeletal muscle tissues or cells were collected, and total RNA was extracted using INVITROGENTM TRIzol (trademark) reagent according to the manufacturer's (Invitrogen) instructions. TRIzol (trademark) reagent is an acid-guanine-phenol-based reagent for extracting RNA (as well as DNA and proteins) from various biological samples. Next, the RNA samples were reverse transcribed using M-MuLV reverse transcriptase (Promega, Madison, Wisconsin, USA), and the mRNA levels of related genes were measured by qRT-PCR using a 7500 Real-Time PCR System (Applied Biosystems, California, USA). The samples were heated at 95 °C for 2 minutes, followed by 40 cycles of amplification (94 °C for 1 minute, 58 °C for 1 minute, 72 °C for 1 minute), and then a final extension at 72 °C for 10 minutes.

[0112] 2-10. Cellular Thermal Shift Assay (CETSA) C2C12 cells were collected 24 hours after LBP1C-2 treatment, washed with PBS (using PBS as a control), and the cell suspension of each group was divided into eight aliquots. One experimental sample and one control sample were paired and heated from 34.0 °C to 76.0 °C for 5 minutes. Next, the cell suspension was repeatedly frozen and thawed in liquid nitrogen three times. The cell lysate was centrifuged at 12,000 rpm at 4 °C for 10 minutes and boiled for 15 minutes. The soluble supernatant was used for Western blot analysis.

[0113] 2-11. Surface Plasmon Resonance (SPR) Analysis The measurement of the interaction was performed by surface plasmon resonance (SPR) spectroscopy. The SPR measurement was carried out using a Biacore 3000 biosensor from Biacore AB (Uppsala, Sweden). Using 100 mM PBS (pH 7.4) as the medium, the binding affinity between FGFR1 and LBP1C-2 was studied at 25 °C.

[0114] By standard methods, FGFR1 was immobilized via amine coupling onto the flow channels of a research-grade CM5 chip (Biacore AB, Sweden). Samples were injected into the CM5 chip at a flow rate of 50 μL / min at seven different concentrations (ranging from 0.25 μM to 16 μM), with the ratio of protein to LBP1C-2 fixed at 1:1. After injection of each sample (150 seconds), PBS (flow rate: 50 μL / min) was passed through the chip, and the dissociation of the complex was monitored for 150 seconds. Subsequently, the chip surface was regenerated by injecting 50 μL of 2 M NaCl in 10 mM sodium acetate (pH 4.5). The control channel was used by flowing PBS buffer at the same flow rate. Data were analyzed kinetically using BIA evaluation software (v3.0).

[0115] 2-12. Quantitative and Statistical Analysis Results are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Statistical analysis was performed using the GraphPad Prism software package (GraphPad Software, La Jolla, CA, USA). The statistical significance of the difference between two means was calculated using a two-sided Student's t-test. When comparing three or more groups, one-way analysis of variance was performed. When there were two factors influencing the experiment, two-way analysis of variance was used to analyze statistical significance. In 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.

[0116] 3. Results 3-1. LBE increases the number of satellite cells in young mice. For 2-month-old male C57BL / 6J mice, the effect of Lycium barbarum extract (LBE) on skeletal muscle satellite cells (satellite cells) was evaluated. These mice were gavaged with a dose of 2.5 g / kg of LBE for 4 months (saline as control). Pax7 is a marker of satellite cells, and both the RNA (Figure 1) and protein (Figure 2) levels were upregulated by LBE.

[0117] To further determine whether the upregulation of Pax7 is due solely to the upregulation of gene transcription or to an increase in the satellite cell population, in the present invention, immunofluorescent staining of Pax7 was performed on cryosections of the tibialis anterior muscle. The results showed that LBE increased the number of Pax7-positive cells in the tibialis anterior muscle (Figure 3). Furthermore, muscle satellite cells of the control group and the LBE-treated group were separated by flow cytometry. The cell surface markers CD34 + CD31 - CD45 - Sca1 - The number of satellite cells sorted by was significantly increased by LBE (Figure 4). Therefore, long-term treatment with LBE increased the number of satellite cells under normal physiological conditions.

[0118] 3-2. LBE reverses the degeneration of muscle satellite cells in the elderly. The decrease in the satellite cell bank is an important cause of muscle dysfunction in the elderly. To evaluate the effect of LBE on muscle satellite cells in the elderly, mice at 14 months of age were fed a diet containing LBE for 4 months. Pax7 mRNA (Figure 5) and protein (Figure 6), and Pax7 + cells were increased by LBE (Figure 7). This indicates that LBE can reverse the age-dependent decrease in the number of satellite cells.

[0119] 3-3. LBE promotes muscle regeneration in BaCl2-induced muscle injury. To investigate whether LBE also affects satellite cell function, in the present invention, a BaCl2-induced muscle regeneration model in C57 mice was established. One week after intragastric administration of LBE, BaCl2 was intramuscularly injected into the tibialis anterior muscle of the mice, and samples were collected and analyzed on days 1, 3, 10, and 30 after injury. On day 10 after injury, regenerated muscle fibers characterized by central cores were larger in the LBE group than in the control group, indicating that the muscle regeneration process was accelerated after LBE treatment (Figure 8).

[0120] Next, in order to evaluate the effect of LBE on the activation and proliferation of satellite cells during regeneration, in the present invention, the levels of Pax7 and MyoD were analyzed. Pax7 was expressed in quiescent satellite cells and activated satellite cells, while MyoD was expressed in activated satellite cells during muscle regeneration. One day after BaCl2 injection, the mRNA levels of Pax7 and MyoD were higher in the LBE group (Figs. 9A - 9B), indicating that LBE promoted the activation of satellite cells at the initial stage of muscle regeneration.

[0121] Since the inventors discovered that LBE increases the number of stem cells under physiological conditions, they were more interested in whether LBE affects the self-renewal ability of satellite cells. On the 30th day after injury, the number of + MyoD - Pax7 cells (Fig. 10) was higher in the LBE group, indicating that more satellite cells exited the cell cycle and returned to the quiescent state. Therefore, LBE promotes muscle regeneration by promoting the activation and self-renewal of satellite cells.

[0122] Furthermore, the BaCl2-induced muscle injury experiment was also conducted in 18-month-old aged mice administered LBE. On the 30th day after injury, HE staining showed that there were still many regenerated muscle fibers with central cores in the muscle, indicating that the muscle regeneration process in aged mice was slower than that in young mice. However, the cross-sectional area of regenerated muscle fibers in the LBE group was larger than that in the control group (Fig. 11), also indicating that LBE promoted muscle regeneration in aged mice. Overall, LBE improved muscle regeneration in aged mice by restoring the dysfunction of satellite cells in the muscle of aged mice.

[0123] 3 - 4. LBE shows a tendency to drive proliferating satellite cells into a quiescent state rather than differentiating. Differentiation of satellite cells is important for providing newly formed muscle fibers, and self-renewal of satellite cells is also necessary to replenish the satellite cell bank. Defects in self-renewal ability lead to a decrease in the number of satellite cells, resulting in depletion of the satellite cell bank and a decline in muscle regeneration ability. Upregulation of Pax7 inhibits myogenesis and cell cycle progression of satellite cells. LBE inhibits Pax7 + MyoD - To evaluate whether LBE also regulates the re-entry of satellite cells into a quiescent state in in vitro culture to increase the proportion of the cell population, single muscle fibers were isolated from the muscles of 4-week-old mice, cultured, and treated with LBE for 72 hours or left untreated.

[0124] The results of immunostaining showed typical clusters of satellite cells during proliferation (Pax7 + / MyoD + ), differentiation (Pax7 - / MyoD + ), and self-renewal (Pax7 + / MyoD - ). A higher proportion of Pax7 + / MyoD - cells were found in satellite cells derived from muscle fibers treated with LBE (Figure 12). After 4 days of culture, muscle fiber-derived satellite cells were switched to differentiation medium. On day 1 after differentiation, LBE inhibited the final muscle differentiation of satellite cells as shown by MyHC staining and the transcriptional levels of myoG and MyHC (Figures 13A - 13B). In short, in vivo and in vitro data clearly show that LBE makes satellite cells more likely to self-renew rather than differentiate.

[0125] 3 - 5. LBP is a major component of LBE and plays a role in maintaining the satellite cell bank. Since LBE plays a very important role in maintaining the muscle satellite cell bank, it is important to explore the main components of its function in order to understand the molecular mechanism. Since the main component of LBE is water-soluble kukko polysaccharide (LBP), in the present invention, the function of LBP on muscle satellite cells was investigated. Similar to the function of LBE, LBP also increased the pax7 + cells of the anterior tibial muscle under physiological conditions (Figure 14). In isolated primary myoblasts, the ratio of Pax7 + / MyoD - cells increased after LBP treatment (Figure 15).

[0126] Furthermore, in the present invention, Pax7-nGFP transgenic mice and GFP + satellite cells isolated by FACS were used. After 18 hours of culture, LBP increased the expression of Pax7 and myoD in these satellite cells, indicating the promotion of satellite cell activation (Figures 16A - 16B). Also, after 48 hours of culture, the Pax7 level of satellite cells was also higher than that of the control group, indicating the presence of more self-renewing satellite cells (Figure 17). In the C2C12 cell line, LBP inhibited cell differentiation as shown by the decrease in MyHC mRNA and protein (Figures 18A - 18B). Overall, LBP is the main component of LBE and plays a role in promoting the activation of satellite cells and maintaining the satellite cell bank.

[0127] 3 - 6. LBP1C - 2 is a homopolysaccharide and an active ingredient of LBP that can regulate the function of satellite cells. To further identify effective single components, in the present invention, the effectiveness of several components or homopolysaccharides separated from LBP was evaluated. The homopolysaccharide LBP1C - 2 increased the pax7 transcription level in primary myoblasts after 18 hours of culture (Figure 19). In isolated primary myoblasts, the increase in Pax7 + / MyoD - cells and Pax7 - MyoD +The role of LBP1C-2 in promoting satellite cell self-renewal and inhibiting differentiation was further confirmed by cell depletion (Figure 20). Therefore, LBP1C-2 is an important factor for LBP regulation of satellite cell function in vitro.

[0128] 3-7. LBP1C-2 promotes satellite cell activation by binding to FGFR1 and promoting p38 phosphorylation.

[0129] Activation of satellite cells after muscle injury is a transient but important step in muscle regeneration. The earliest marker of activated satellite cells is phosphorylated p38MAPK, followed by MyoD. The inventors examined the expression of phosphorylated p38 in the muscles of young mice 1 day after BaCl2 injury and found that LBE significantly promoted the phosphorylation of p38 and correspondingly increased the MyoD level (Figure 21).

[0130] Activation of fibroblast growth factor receptor (FGFR1) immediately after muscle injury is one of the major pathways promoting p38 phosphorylation. FGFR1-mediated activation of p38 promotes the escape of satellite cells from the quiescent state and is required for the initiation of the cell cycle. Phosphorylation of FGFR1 was also significantly upregulated (Figure 21).

[0131] Therefore, to determine whether FGFR1 is involved in LBP1C-2-mediated satellite cell activation, the function of LBP1C-2 in C2C12 cells transfected with FGFR1 siRNA was analyzed (Figure 22A). The results showed that FGFR1 deficiency reversed the upregulation of MyoD expression (Figure 22B) and the increase in p38 phosphorylation (Figure 23) induced by LBP1C-2, indicating that LBP1C-2 promotes satellite cell activation via FGFR1-mediated p38 phosphorylation.

[0132] Next, the question is whether LBP1C-2 may directly bind to the membrane receptor FGFR1 and activate the FGFR1-p38 signaling pathway.

[0133] First, the inventors studied the binding characteristics of LBP1C-2 and FGFR1 by cell thermal shift assay (CETSA). The results showed that in the presence of LBP1C-2, the stability of the FGFR1 protein was slightly enhanced, while the stability of the control protein β-actin remained unchanged (Figure 24). Next, the binding affinity of LBP1C-2 for FGFR1 was quantified using surface plasmon resonance (SPR) spectroscopy. As shown in Figure 25, the binding affinity of LBP1C-2 for FGFR1 was 26.9 μM (KD), indicating weak binding of LBP1C-2 to FGFR1.

[0134] This weak binding is also logical and explains why long-term administration of LBE under normal physiological conditions slightly activates satellite cell proliferation and self-renewal without damaging them, thereby increasing the number of satellite cells.

[0135] 3-8. LBP1C-2 promotes satellite cell self-renewal by upregulating spry1. In this study, it was found that LBE increased the expression of spry1 in muscle on the 30th day after injury (Figure 26). Spry1 is an inhibitor of receptor tyrosine kinase signaling essential for maintaining a quiescent satellite cell bank.

[0136] Furthermore, in C2C12 myoblasts, LBP and LBP1C-2 also upregulated the expression of Spry1, and the effect of LBP1C-2 was stronger at the same treatment concentration (Figure 27). To verify that Spry1 is actually involved in the function of LBP1C-2, spry1 was knocked down in C2C12 cells by siRNA transfection (Figure 28A). The data showed that spry1 deficiency significantly inhibited the upregulation of Pax7 (Figure 28B), suggesting that spry1 plays an important role in enhancing LBP1C-2-mediated satellite cell self-renewal.

[0137] Results of 3-9.LBP1C-2 Sulfate Derivative (S-LBP1X-2) Inhibitors of FGFR1 reversed the upregulation of Spry1 by LBP1C-2 in sorted satellite cells (Figure 29A). Consistent with this result, blocking of FGFR1 by siRNA in C2C12 cells also inhibited the upregulation of Spry1 induced by LBP1C-2 (Figure 29B). These results indicate that the upregulation of Spry1 induced by LBP1C-2 is also dependent on FGFR1, and the upregulation of Spry1 may inhibit the p38 signaling pathway through negative feedback control, thereby preventing the overactivation of satellite cells.

[0138] To verify whether sulfated LBP1C-2 (i.e., S-LBP1C-2) has the function of maintaining the skeletal muscle satellite cell bank, in the present invention, C2C12 myoblasts were used to evaluate the effect of S-LBP1C-2 on the activation of satellite cells and the expression of important markers of self-renewal.

[0139] First, the levels of phosphorylated p-p38 MAPK and MyoD, which are the most initially activated satellite cell markers in C2C12 cells treated with S-LBP1C-2 for 24 hours, were measured by Western blot and qPCR, respectively. S-LBP1C-2 increased the mRNA level of MyoD and the protein level of phosphorylated p-p38 MAPK (Figures 30A, 30B).

[0140] This indicates that S-LBP1C-2 also has the function of promoting the activation of satellite cells. At the same concentration of 200 μg / mL, the effect of S-LBP1C-2 is slightly better than that of LBP1C-2. Next, when C2C12 cells were treated with S-LBP1C-2 at various concentrations (100, 200, 400 μg / mL), it was observed that the upregulation of MyoD and p-p38 induced by S-LBP1C-2 was dose-dependent (Figures 31A, 31B).

[0141] Next, based on the discovery that LBP1C-2 promotes satellite cell self-renewal by upregulating Spry1, in the present invention, the effect of S-LBP1C-2 on Spry1 expression was further evaluated by qPCR. S-LBP1C-2 can also upregulate Spry1 expression in a dose-dependent manner in C2C12 cells, suggesting that S-LBP1C-2 may also promote satellite cell self-renewal (Figure 32A). As shown in Figure 32B, the results were compared with those using LBP1C-2.

[0142] In short, the present invention has for the first time demonstrated that long-term use of the Eucommia extract can improve the number and function of satellite cells in adult and aged mice and promote muscle regeneration. Furthermore, LBP1C-2 discovered from LBE is an active ingredient that regulates the function of satellite cells. LBP1C-2 promotes the activation and self-renewal of satellite cells by activating the FGFR1-p38 signaling pathway and upregulating Spry1 expression. This study provides a new perspective for scientifically explaining the traditional effects of Eucommia and also provides a theoretical basis for the medicinal or adjuvant medicinal use of Eucommia. It was also shown that further preparation of a sulfate derivative of the homopolysaccharide has a similar effect.

[0143] 4. Experimental Samples and Control Samples Four homopolysaccharides were isolated from the Eucommia extract. Among these four homopolysaccharides, only LBP1C-2 exhibits the functions described herein. The sugar composition and structure of three homopolysaccharides including LBP1A1-1, LBP1B-S-2, and LBP1C-2 among the four homopolysaccharides were analyzed.

[0144] Referring to Formula 4, LBP1A1-1 is composed of rhamnose (Rha), arabinose (Ara), glucose (Glc), and galactose (Gal), and the molar ratios thereof are 1.2:47.8:1.4:49.8. Structural analysis has shown 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 glycosyl 1,3-β-Gal residue and the C-3 position of 1,6-β-Gal.

[0145] TIFF2025518622000005.tif219170

[0146] Referring to Formula 5, LBP1B-S-2 is composed of Rha, GlcA (glucuronic acid), Gal, and Ara, and the molar ratios thereof are 3.13:3.95:39.37:53.55. Structural analysis has shown 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 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.

[0147] TIFF2025518622000006.tif198170

[0148] Among the four different homopolysaccharides in terms of structure, only LBP1C-2 showed the target function and activity. LBP1C-2, its derivatives such as sulfate derivatives, and compositions containing LBP1C-2 or its derivatives are preferred compositions for improving the number and / or function of muscle satellite cells and / or for treating or preventing muscle atrophy.

[0149] Although the present invention has been described through the above exemplary embodiments, the present invention is not limited thereto. Rather, the appended claims should be broadly construed to include other variations and embodiments that can be made by those skilled in the art.

Claims

1. A method for improving the number and / or function of satellite cells and / or treating or preventing muscle atrophy, comprising administering to a subject in need thereof an effective dose of a composition comprising a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient, wherein the homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units, method.

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

3. wherein the subject is a human subject, The method according to claim 1.

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

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

5. The method according to claim 1.

7. The method according to claim 1, wherein the molecular weight range of the homopolysaccharide is about 10 kDa to about 150 kDa. The method according to claim 1.

8. The method according to claim 1, wherein the composition further comprises other polysaccharides separated from the kukko extract and / or the homopolysaccharide accounts for 15% or more of the total polysaccharides in the composition.

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

10. The homopolysaccharide is the only polysaccharide in the composition. The method according to claim 1.

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

12. The recipient is selected from a solvent, a co-solvent, a coloring agent, a preservative, an antimicrobial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination thereof. The method according to claim 1.

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

14. A composition for improving muscle satellite cell number and / or function and / or treating or preventing muscle atrophy in an individual in need thereof, comprising an effective amount of a homopolysaccharide, or a pharmaceutically acceptable ester or salt thereof, or a pharmaceutically acceptable solvate thereof, or any combination thereof, and a pharmaceutically acceptable excipient. The homopolysaccharide is mainly composed of arabinose, galactose, rhamnose, and galacturonic acid as monomer units. Composition.

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

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

17. The pharmaceutically acceptable ester or salt is a sulfate ester derivative of the homopolysaccharide. The composition according to claim 14.

18. The degree of sulfate group substitution of the sulfate ester derivative of the homopolysaccharide is in the range of 0.5 to 0.

9. The composition according to claim 17.

19. The recipient is selected from a solvent, a co-solvent, a colorant, a preservative, an antimicrobial agent, a filler, a binder, a disintegrant, a lubricant, a surfactant, an emulsifier, a suspending agent, or any combination thereof. The composition according to claim 14.

20. The molecular weight range of the homopolysaccharide is from about 10 kDa to about 150 kDa. The composition according to claim 14.

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

22. The molar ratio of the monomer units of arabinose, galactose, rhamnose, and galacturonic acid in the homopolysaccharide is 49.9:33.6:8.0:8.

5. The composition according to claim 14.

23. The composition further comprises other polysaccharides separated from the kukko extract and / or the homopolysaccharide accounts for 15% or more of the total polysaccharides in the composition. The composition according to claim 14.

24. The homopolysaccharide is the only polysaccharide in the composition. The composition according to claim 14.

25. The composition does not contain flavonoids, carotenoids, or polyphenols separated from kukko extract, The composition according to claim 14.

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