Use of flavonoid derivative in preventing and treating sarcopenia

Abstract

Use of a flavonoid derivative of formula I or a salt thereof in preparing a medicament for preventing and / or treating sarcopenia. The chemical structural formula of formula I is as follows, wherein R1 and R2 are monosaccharides or oligosaccharides composed of glucose (glc) and / or rhamnose (rha) combined in different amounts and manners.

Description

Use of flavonoid derivatives for prevention and treatment of sarcopenia TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to use of flavonoid derivatives for prevention and treatment of sarcopenia. BACKGROUND

[0002] Sarcopenia is a common disease in the elderly, mainly characterized by chronic and progressive decrease in muscle mass and quantity with age, leading to loss of muscle strength [Dent E, Morley JE, Cruz-Jentoft AJ, et al. International Clinical Practice Guidelines for Sarcopenia (ICFSR): Screening, Diagnosis and Management. J Nutr Health Aging. 2018; 22(10): 1148-1161.]. Sarcopenia is associated with adverse outcomes, including increased risk of physical disability, reduced ability to perform activities of daily living, and increased risk of fractures, which imposes a significant economic and social burden [Cruz-Jentoft AJ, Sayer AA. Sarcopenia. Lancet. 2019 Jun 29; 393(10191): 2636-2646.]. Studies have shown that human muscle reaches its peak at the age of 40, and then gradually declines in muscle mass.The 2016 Asian Sarcopenia Management Guidelines estimated the prevalence of sarcopenia in Chinese middle-aged and elderly people aged 60 to 70 to be about 4.1% to 11.5%, and the prevalence of sarcopenia in men and women aged 70 and over was 13.2% and 4.8%, respectively. The incidence in people over 80 years old increased significantly, with the highest incidence reaching 50% [Cruz-Jentoft AJ, Bahat G, Bauer J, Boirie Y, Bruyère O, Cederholm T, Cooper C, Landi F, Rolland Y, Sayer AA, Schneider SM, Sieber CC, Topinkova E, Vandewoude M, Visser M, Zamboni M; Writing Group for the European Working Group on Sarcopenia in Older People 2 (EWGSOP2), and the Extended Group for EWGSOP2. Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing. 2019 Jan 1; 48(1): 16-31. Liu J, Ding QQ, Zhou BY, Liu X, Liu JM, Liu YM, Ding GX, Zhang CT, Wang JY, Yu PL, Chinese Society of Geriatrics, Editorial Committee of Chinese Journal of Geriatrics. Expert consensus on diagnosis and treatment of sarcopenia in China (2021) [J]. Chinese Journal of Geriatrics, 2021, 40(8):943-952.]. With the arrival of an aging society, the incidence of sarcopenia is increasing year by year, seriously affecting the quality of life and health of the elderly. Therefore, it has attracted widespread attention from scholars at home and abroad, and extensive research has been conducted on the risk factors, pathogenesis, treatment and other fields of the disease. However, there are still differences in the understanding of the disease at home and abroad. And because the cause of sarcopenia is still unknown, it is related to many factors and involves complex molecular mechanisms that are interrelated, making it difficult to treat the disease. At present, there is still a lack of a single effective treatment drug for sarcopenia, and resistance exercise is the main non-drug therapy for improving muscle mass and function, but the compliance of elderly patients to resistance exercise is poor in reality. Therefore, exploring effective treatment methods is an important direction in the field of sarcopenia treatment research.

[0003] SUMMARY

[0004] Therefore, the application provides a use of a flavonoid derivative or a salt thereof represented by formula I in the preparation of a drug for preventing and / or treating sarcopenia, and the chemical structural formula of the formula I is as follows:

[0005] wherein R1, R2 is a monosaccharide or oligosaccharide composed of glucose (glc) and / or rhamnose (rha) in different amounts and ways.

[0006] According to another aspect of the present application, there is provided a use of a composition comprising a flavonoid derivative of Formula I or a salt thereof in the manufacture of a medicament for preventing and / or treating sarcopenia, the chemical structure of Formula I being shown as follows:

[0007] wherein R1, R2 is a monosaccharide or oligosaccharide composed of glucose (glc) and / or rhamnose (rha) in different amounts and ways.

[0008] Further, the oligosaccharide is a disaccharide.

[0009] Further, the glucose is β-D-glucose (β-D-glc).

[0010] Further, the rhamnose is α-L-rhamnose (α-L-rha).

[0011] Further, R1 is β-D-glc (1→3) α-L-rha, β-D-glc (1→2) α-L-rha, α-L-rha (1→2) α-L-rha, α-L-rha.

[0012] Further, R2 is β-D-glucose (β-D-glc).

[0013] Further, the flavonoid derivative is icariin, epimediumin A, epimediumin A1 or epimediumin C.

[0014] Further, the sarcopenia is age-, tumor- and / or malnutrition-induced sarcopenia.

[0015] Further, the composition further comprises one or more other drugs or other extracts for preventing and / or treating sarcopenia.

[0016] Further, the other drug is selected from one or more of the following: myostatin antibody, inhibitor of receptor activator of nuclear factor-κB ligand, vitamin D, calcium agent, recombinant human growth hormone, testosterone, selective estrogen receptor modulator, rapamycin, fatty acid synthase inhibitor, adiponectin, danshensu and coenzyme Q.

[0017] Further, the composition further comprises one or more pharmaceutically acceptable excipients.

[0018] Further, the adjuvant is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained release agents, retention aids, lubricants, dispersants, plasticizers, opacifiers, and antioxidants.

[0019] Further, the dosage form of the composition is a tablet, a drop pill, a capsule, a powder, an injection, a film, a lozenge, a granule, or an oral solution.

[0020] Further, the R1 is not selected from β-D-xyl(1→2)α-L-rha or H.

[0021] Further, the R2 is not H.

[0022] Further, the compound is not damianin B, icariside I, and icariside II.

[0023] Further, the effect of the flavone derivative or salt thereof or the composition in preventing and / or treating sarcopenia is by one or both of the following: improving abnormal myoblast differentiation and increasing mRNA expression of MRFs family factors.

[0024] Further, the abnormal myoblast differentiation is selected from one or more of the following: reduced nuclear fusion, cell morphological atrophy, cell length reduction, and cell diameter reduction.

[0025] Further, the MRFs family factor is selected from one or more of the following: MyoD protein, Mrf5 protein, Mygenin protein, and Myf6 protein.

[0026] Further, the effect of the flavone derivative or salt thereof or the composition in preventing and / or treating sarcopenia is one or more of the following: delaying apparent aging, increasing muscle strength, reducing body fat content, reducing bone mineral content, increasing muscle content, and improving abnormal skeletal muscle.

[0027] Further, the degree of apparent aging is evaluated using an aging degree rating scale.

[0028] Further, the evaluation index of the scale is selected from one or more of the following: reactivity, passivity, degree of skin aging, degree of hair aging, degree of eye aging, and degree of spinal column aging.

[0029] Further, the skeletal muscle is the tibialis anterior muscle.

[0030] Further, the abnormality is selected from one or more of the following: uneven arrangement of skeletal muscle tissue, reduced cross-sectional area, and increased intermuscular space.

[0031] Advantages of the present application:

[0032] The application discloses a single component of Epimedium for preventing and / or treating sarcopenia. The single component of Epimedium (icariin, epimediumin A, epimediumin A1 and epimediumin C) of the application can significantly promote myoblast differentiation in in-vivo experiments, can improve muscle strength, muscle content and muscle function, reduce body fat content and delay aging process in in-vivo experiments, and therefore can be used as a medicine for treating sarcopenia. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art based on these drawings without exceeding the scope of the present application.

[0034] Fig. 1 is the effect of the single component of Epimedium on skeletal muscle of SAMP8 sarcopenia model mice. A. HE staining of tibialis anterior muscle; B. DEXA graph of body composition. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described clearly and completely in the following with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0036] Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the meanings that are commonly understood by one of ordinary skill in the art in the field of the application, or that are given in the specification. Although any methods, conditions, materials, and materials similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods, conditions, materials, and materials are described herein.

[0037] The present application is intended to encompass all alternatives, modifications and equivalents that can be included within the scope of the present application as defined by the claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The application is not intended to be limited to the embodiments described.

[0038] As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0039] In the present invention, the term "comprising" is synonymous with "including," "containing," or "comprehending." As used herein, the terms "comprise", "comprising", "include", "including", "contain", "containing", "have", "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0040] As described in the background section, there is still a lack of a single effective therapeutic drug for sarcopenia. In order to solve the above-mentioned problem, the present invention provides a use of a flavonoid derivative or a salt thereof represented by Formula I in the preparation of a drug for preventing and / or treating sarcopenia, and the chemical structure of Formula I is as follows:

[0041] wherein R1, R2 is a monosaccharide or oligosaccharide composed of glucose (glc) and / or rhamnose (rha) in different quantities and combinations.

[0042] According to another aspect of the present invention, there is provided a use of a composition comprising a flavonoid derivative or a salt thereof represented by Formula I in the preparation of a drug for preventing and / or treating sarcopenia, and the chemical structure of Formula I is as follows:

[0043] wherein R1, R2 is a monosaccharide or oligosaccharide composed of glucose (glc) and / or rhamnose (rha) in different quantities and combinations.

[0044] In a preferred embodiment, the oligosaccharide is a disaccharide.

[0045] In a preferred embodiment, the glucose is β-D-glucose (β-D-glc).

[0046] In a preferred embodiment, the rhamnose is α-L-rhamnose (α-L-rha).

[0047] In a preferred embodiment, R1 is β-D-glc (1→3) α-L-rha, β-D-glc (1→2) α-L-rha, α-L-rha (1→2) α-L-rha, α-L-rha.

[0048] In a preferred embodiment, R2 is β-D-glucose (β-D-glc).

[0049] In a preferred embodiment, the flavonoid derivative is icariin, epimediumin A, epimediumin A1, or epimediumin C.

[0050] In a preferred embodiment, the sarcopenia is age-, tumor- and / or malnutrition-induced sarcopenia.

[0051] In a preferred embodiment, the composition further comprises one or more other drugs or other extracts for preventing and / or treating sarcopenia.

[0052] In a preferred embodiment, the other drug is selected from one or more of the following: myostatin antibody, nuclear factor-kappa B receptor activator ligand inhibitor, vitamin D, calcium agent, recombinant human growth hormone, testosterone, selective estrogen receptor modulator, rapamycin, fatty acid synthase inhibitor, adiponectin, danshensu, and coenzyme Q.

[0053] In a preferred embodiment, the composition further comprises one or more pharmaceutically acceptable excipients.

[0054] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0055] In a preferred embodiment, the pharmaceutical composition of the present application contains at least one pharmaceutically acceptable excipient in a total amount of 0.00001 to 50 wt.%, or 0.0001 to 10 wt.%, or 0.0001 to 5 wt.%, or 0.005 to 1 wt.%, or 0.1 to 20 wt.%, or 0.5 to 15 wt.%, or 1 to 5 wt.%, calculated relative to the weight of the pharmaceutical composition.

[0056] In the present application, the term "pharmaceutically acceptable" refers herein to a substance, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material is not biologically or otherwise undesirable, i.e., the material is not harmful when administered in a subject.

[0057] In the present application, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible with the subject and active ingredient, i.e., not biologically or otherwise undesirable, and that does not abrogate the biological activity or properties of the active ingredient (i.e., is able to induce the desired therapeutic effect without causing any undesirable local or systemic effects) and is known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0058] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, complexing agents, flavoring agents, sustained release agents, retention aids, lubricants, dispersing agents, plasticizers, opacifiers, and antioxidants.

[0059] One skilled in the art will know how to select a particular chemical within the above-mentioned excipient categories. For example, the diluent can be selected from one or more of the following: powdered sugar, starch, compressible starch, lactose, dextrin, mannitol, sorbitol, microcrystalline cellulose, calcium sulfate, and calcium carbonate. The wetting agent can be selected from one or more of the following: polyoxyl stearate, poloxamer, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether, polysorbate such as polysorbate 80, cetyl alcohol, glycerol fatty acid ester (such as triacetin, glycerol monostearate, and the like), polyoxyethylene fatty acid ester, polyethylene glycol fatty acid ester, sodium dodecyl sulfate, sorbitol fatty acid ester, sucrose fatty acid ester, polyoxyethylene ether, benzalkonium chloride, polyoxyethylene castor oil, and sodium docusate. The binder can be selected from one or more of the following: polyvinylpyrrolidone, hydroxypropyl cellulose, polyethylene glycol, and methyl cellulose. The disintegrant can be selected from one or more of the following: carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose. The flavoring agent can be selected from one or more of the following: sorbitol, glucose, mannose, sucrose, and lactose. The dispersing agent can be selected from one or more of the following: sodium croscarmellose, sodium starch glycolate, and pregelatinized corn starch. The plasticizer can be dibutyl sebacate and / or various citric acid esters. The sustained release agent can be selected from one or more of the following: sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, hydroxyethyl cellulose, gum arabic, gelatin, and shellac. The antioxidant can be selected from one or more of the following: sodium bisulfite, sodium metabisulfite, sodium sulfite, and sodium thiosulfate. The lubricant can be selected from one or more of the following: calcium stearate, talc, magnesium stearate, stearic acid, and colloidal silicon dioxide.

[0060] The excipients are preferably pharmaceutically inert, or can have a synergistic or additive effect to enhance the therapeutic activity of the pharmaceutical composition, and the above-mentioned excipients are only illustrative, and the excipients actually used in the present application are not limited to the above-mentioned excipients, and can be adjusted according to the actual situation, and all can achieve the effect of the present application.

[0061] In a preferred embodiment, the dosage form of the composition is a tablet, a dripping pill, a capsule, a powder, an injection, a film, a lozenge, a granule, or an oral liquid.

[0062] In a preferred embodiment, R1is not selected from β-D-xyl(1→2)α-L-rha or H.

[0063] In a preferred embodiment, R2 is not H.

[0064] In a preferred embodiment, the compound is not dammarane-type triterpene, dammarane-type triterpene, and dammarane-type triterpene.

[0065] In a preferred embodiment, the effect of the flavone derivative or salt thereof or the composition in preventing and / or treating sarcopenia is by one or both of the following: improving abnormal myoblast differentiation and increasing mRNA expression of MRFs family factors.

[0066] In a preferred embodiment, the abnormal myoblast differentiation is selected from one or more of the following: reduced nuclear fusion, cell morphological atrophy, cell length reduction, and cell diameter reduction.

[0067] In a preferred embodiment, the MRFs family factor is selected from one or more of the following: MyoD protein, Mrf5 protein, Mygenin protein, and Myf6 protein.

[0068] In a preferred embodiment, the effect of the flavone derivative or salt thereof or the composition in preventing and / or treating sarcopenia is one or more of the following: delaying apparent aging, increasing muscle strength, reducing body fat content, reducing bone mineral content, increasing muscle content, and improving abnormal skeletal muscle.

[0069] In a preferred embodiment, the degree of apparent aging is evaluated using an aging degree rating scale.

[0070] In a preferred embodiment, the evaluation index of the scale is selected from one or more of the following: reactivity, passivity, degree of skin aging, degree of hair aging, degree of eye aging, and degree of spinal column aging.

[0071] In a preferred embodiment, the skeletal muscle is tibialis anterior muscle.

[0072] In a preferred embodiment, the abnormality is selected from one or more of the following: uneven arrangement of skeletal muscle tissue, reduced cross-sectional area, and increased intermuscular space.

[0073] According to another aspect of the present application, there is provided a flavone derivative or salt thereof represented by the above Formula I or the above pharmaceutical composition for use in preventing and / or treating sarcopenia in a subject.

[0074] According to another aspect of the present application, there is provided a method of preventing and / or treating sarcopenia in a subject, comprising administering to the subject an effective amount of a flavone derivative or salt thereof represented by the above Formula I or the above pharmaceutical composition.

[0075] In the present application, the term "subject" is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing a model of sarcopenia. Preferably, the subject is a human.

[0076] An "effective amount" of a pharmaceutical composition or formulation used in the present application can achieve the desired therapeutic and / or prophylactic effect. The amount effective for this use will depend on, for example, the pharmaceutical composition, the mode of administration, the stage and severity of the disease being treated, the individual's body weight and general health status, and the judgment of the prescribing physician. The administration of the dose can be once a week, or two days or once a day, or even several times a day. The dose unit can be administered for a short period (e.g., several weeks to several months) or for a longer period (several months to several years).

[0077] The present application is further illustrated by the following specific examples. It is to be understood that these examples are merely for the purpose of illustration and are not to be taken as limiting the scope of the present application. The experimental methods in the following examples, unless otherwise specified, are generally performed according to conventional conditions or as suggested by the manufacturer.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are thus meant to be illustrative only and not limiting.

[0079] The above features mentioned in the present application, or the features mentioned in the examples, can be combined in any combination. All features disclosed in the present patent specification can be used in any combination, and each feature disclosed in the present specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless specifically stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

[0080] Examples

[0081] Example 1 Effect of Epimedium monomers on myoblast cells

[0082] 1. Purpose of the experiment

[0083] By investigating the effect of drugs on the differentiation of myoblast cells, the effect of different Epimedium monomers on the myogenic differentiation of C2C12 cells was evaluated.

[0084] 2. Experimental methods

[0085] 2.1 Induction of myogenic differentiation culture of C2C12 cells

[0086] When C2C12 myoblast cells grow and fuse to 70% to 80%, discard the growth medium, add serum-free medium to starve overnight, then replace the differentiation medium (DMEM high-sugar medium + 2% horse serum + double antibody) to induce muscle differentiation for 6 days to form myotubes, and replace the new differentiation medium every 1 day to continue the culture.

[0087] 2.2, cell grouping and administration

[0088] C2C12 myoblast cells were randomly divided into a control group, a TNF-α / IFN-γ stimulation group, and an Epimedium monomer group including Icaritin, Damiacin A, Damiacin A1, Damiacin B, Damiacin C, Icaritin B, and Icaritin C (10 μM and 50 μM), with 3 replicate holes in each group. The control group was cultured in the growth medium for 24 h; the stimulation group was cultured in the growth medium containing 10 ng / mL TNF-α and 10 ng / mL IFN-γ for 24 h; and each Epimedium monomer group was cultured in the growth medium containing 10 ng / mL TNF-α, 10 ng / mL IFN-γ, and Epimedium monomers (10 μM and 50 μM) for 24 h. On the 5th day of muscle differentiation induction, each group was cultured in the differentiation medium containing or not containing drugs for 24 h, and the myotube cell morphology of each group was observed in real time under a fluorescence inverted microscope and photographed.

[0089] 2.3, CCK8 method for detecting cell viability

[0090] Logarithmic growth period C2C12 myoblast cells were inoculated in a 96-well plate at a density of 5 × 10 3 cells per well, cultured overnight to adhere, incubated with different concentrations of Epimedium monomers (0, 10, 20, 50, and 100 μM) for 24 h, 20 μL of CCK8 reagent was added to each well for 1 h of continuous culture, and the optical density (OD) value of each well was detected at 450 nm wavelength by using an enzyme-labeled instrument, and the cell viability was calculated, with 3 replicate holes in each group.

[0091] 2.4, RT-qPCR method for detecting mRNA expression of MRFs family factors of myoblast cell differentiation myotube cells

[0092] The cells were treated according to the method in item 2.2, C2C12 myoblast cells and differentiation myotube cells were collected, RZ lysis solution was added, total RNA was extracted by using a total RNA extraction kit, and cDNA was obtained by reverse transcription, and the mRNA expression of inflammatory factors of myoblast cells and the mRNA expression of MRFs family factors of differentiation myotube cells were detected. GAPDH was used as an internal reference, and 2-ΔΔCT method was used for analysis. The primer sequences were derived from PrimerBank (as shown in Table 1) and synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0093] Table 1 primer sequences

[0094] 3. Results

[0095] 3.1 Effects of different Epimedium extracts on the viability of C2C12 myoblasts

[0096] As shown in Table 2, compared with the control group, 100 μM of different Epimedium extracts inhibited the viability of C2C12 myoblasts (P < 0.001), and therefore, concentrations below 50 μM were selected for subsequent experiments.

[0097] Table 2 Effects of different Epimedium extracts on the viability of C2C12 myoblasts

[0098] 3.2 Effects of different Epimedium extracts on the morphology of differentiated myotubes and the expression of MRFs genes

[0099] Compared with the control group, the nuclei of the myotubes in the stimulation group fused less, the morphology was atrophic, and the diameter and length were reduced. The nuclei of the myotubes in the 50 μM Epimedium extract icariin, epimedium-2, epimedium-3, and epimedium-4 groups fused more, the morphology was normal, and the diameter and length were not significantly changed. Epimedium-1, epimedium-5, and epimedium-6 had no improving effect.

[0100] Compared with the control group, the expression of MyoD, Mrf5, Mygenin, and Myf6 mRNA in the stimulation group was reduced (P < 0.05, P < 0.01). Compared with the stimulation group, 10 μM and 50 μM epimedium-2 significantly increased the expression of MyoD, Mrf5, Mygenin, and Myf6 mRNA (P < 0.05). 50 μM icariin, epimedium-3, and epimedium-4 increased the expression of MyoD, Mrf5, Mygenin, and Myf6 mRNA, and other extracts had no effect on the expression of MyoD, Mrf5, Mygenin, and Myf6 mRNA.

[0101] Table 3 Effects of different Epimedium extracts on the expression of MyoD, Mrf5, Mygenin, and Myf6 mRNA in C2C12 myoblasts Note: * indicates P < 0.05 compared with the stimulation group; ** indicates P < 0.01 compared with the stimulation group; *** indicates P < 0.001 compared with the stimulation group.

[0102] 4. Conclusion

[0103] The results of the cell experiments showed that Epimedium extracts (icariin, epimedium-2, epimedium-3, and epimedium-4) significantly promoted the differentiation of myoblasts and had the best effect.

[0104] Effect of icariin, epimedium-derived compounds C, A and A1 on SAMP8 mice with muscle atrophy

[0105] 1. Materials

[0106] 1.1. Experimental animals

[0107] SAMP8 male mice, 16 weeks old, weighing 30±2g; SAMR1 male mice, 16 weeks old, weighing 30±2g; purchased from Jiangsu Ailinfeng Biotechnology Co., Ltd.

[0108] The experimental animals were fed in the SPF animal room of Shanghai Experimental Animal Center, with a temperature of (24±1)℃, a relative humidity of 50%-70%, and a light-dark cycle of 12h / 12h. The experimental operation process strictly followed the relevant regulations and provisions of the Ethics Committee of Shanghai Experimental Animal Center.

[0109] 1.2. Experimental drugs

[0110] The icariin monomers used in the embodiments of the application were self-made in the laboratory.

[0111] 1.3. Experimental instruments

[0112] Dual-energy X-ray bone densitometer (iNSiGHT VET DXA, OsteoSys, KOREAN); YLS-13A muscle strength tester, Jinan Yijian Technology Co., Ltd.

[0113] 2. Methods

[0114] 2.1. Grouping and administration

[0115] The common feed was adaptively fed for 1 week, and among them, 128 SAMP8 mice were randomly divided into a model group, an icariin low-dose group (5mg / kg), a medium-dose group (10mg / kg), an epimedium-derived compound A low-dose group (5mg / kg), a medium-dose group (10mg / kg), an epimedium-derived compound A1 low-dose group (5mg / kg), a medium-dose group (10mg / kg), an epimedium-derived compound B low-dose group (5mg / kg), a medium-dose group (10mg / kg), an epimedium-derived compound C low-dose group (5mg / kg), a medium-dose group (10mg / kg), icariin secondary glycoside I low-dose group (5mg / kg), a medium-dose group (10mg / kg), icariin secondary glycoside II low-dose group (5mg / kg), a medium-dose group (10mg / kg), each group containing 8 mice; 8 SAMR1 mice of the same age were used as a negative control group.

[0116] Epimedium monomer was dissolved in 0.3% sodium carboxymethylcellulose solution. Each administration group was administered with different concentrations of epimedium monomer solution by gavage, once a day for 12 consecutive weeks, with a gavage volume of 0.1 mL / 10 g. The negative control group and the model group were administered with 0.3% sodium carboxymethylcellulose solution by gavage, once a day for 12 consecutive weeks, with a gavage volume of 0.1 mL / 10 g.

[0117] 2.2, detection index and method

[0118] 2.2.1, aging degree score

[0119] The degree of apparent aging was evaluated by using the aging degree score scale. The scale includes reactivity, passivity, skin and hair, eyes and spine-related aging, etc., a total of 11 categories, and each category is scored from 0 to 4 points. Evaluation was performed once every 0, 4, 8, and 12 weeks.

[0120] 2.2.2, determination of mouse muscle strength

[0121] The forelimb grip strength of mice was measured by using a YLS-13A type muscle strength tester (Jinan Yijian Technology Co., Ltd., Jinan, China). The rats were lifted and fixed with their tails so that their limbs could hold a wire mesh. Then, the mice were gently pulled backward by the tail, with their posture parallel to the surface of the table, until they released the grid. The peak force exerted by the mouse limbs was recorded in grams (g). Evaluation was performed once every 0, 4, 8, and 12 weeks, with three tests for each mouse, and the average value was used for statistical analysis after body weight weighting.

[0122] 2.2.3, determination of mouse body composition

[0123] Three days before the mice were sacrificed, a dual-energy X-ray bone densitometer (DEXA) was used for scanning to determine the body composition, including the fat content, muscle content, and bone mineral content of the mice, and the data were analyzed using the software provided by the manufacturer.

[0124] 2.2.4, skeletal muscle pathology detection

[0125] The mice were sacrificed after the intervention, and the tibialis anterior muscle was obtained. The tibialis anterior muscle was fixed with 4% paraformaldehyde for 24 h, dehydrated, and embedded in paraffin, and then cut into 5 μm paraffin sections. For skeletal muscle tissue sections, HE staining was used to detect the morphology and number of mouse skeletal muscle fibers.

[0126] 2.2.5, statistical method

[0127] The experimental data were analyzed by using SPSS 21.0 software, and the measurement data were represented by mean ± standard deviation. One-way ANOVA analysis was used for comparison between groups, and P<0.05 was considered statistically significant.

[0128] 3、Results

[0129] 3.1 Effects of Epimedium Monomers on Aging Degree of SAMP8 Sarcopenia Model Mice

[0130] The results are shown in Table 4. There was no significant difference in the aging degree score of SAMP8 mice among the groups before administration, indicating that the groups were comparable. After 4 weeks of administration, the aging degree score of the model group was significantly higher than that of the control group (P<0.01). Compared with the model group, the aging degree score of the middle dose group was decreased (P<0.05), and the low dose group showed a decreasing trend. Among them, the aging degree scores of Chailin A1 and Chailin C decreased most significantly. After 8 weeks of administration, the aging degree score of the model group was significantly higher than that of the control group (P<0.01). Compared with the model group, the aging degree score of the middle dose group was significantly decreased (P<0.05), and the low dose group showed a decreasing trend. There was no significant difference among the groups with the same dose. After 12 weeks of administration, the aging degree score of the model group was significantly higher than that of the control group (P<0.01). Compared with the model group, the aging degree score of the middle dose group was significantly decreased (P<0.05), and the low dose group showed a decreasing trend. There was no significant difference among the groups with the same dose. Among them, Chailin A1 and Icaritin had the most significant and dose-dependent effects on the aging degree score. Other monomers had no effect on the aging degree score.

[0131] Table 4 Effects of Epimedium Monomers on Aging Degree of SAMP8 Sarcopenia Model Mice Note: Compared with the control group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01. # P<0.05, ## P<0.01.

[0132] 3.2 Effects of Epimedium Monomers on Muscle Strength of SAMP8 Sarcopenia Model Mice

[0133] Results are shown in Table 5, before administration, compared with the control group, the muscle strength of each group decreased significantly (P<0.01); there was no significant difference in muscle strength between SAMP8 mice in each group, suggesting that the experimental system was reliable and comparable between groups. After 4 weeks of administration, compared with the control group, the muscle strength of the model group decreased significantly (P<0.01); compared with the model group, the muscle strength of the medium-dose group of icariin and epimedin A1 increased significantly (P<0.01), the muscle strength of epimedin A and epimedin C increased significantly (P<0.05), the muscle strength of each low-dose group had a rising trend, and there was no significant difference between them. After 8 weeks of administration, compared with the control group, the muscle strength of the model group decreased significantly (P<0.01); compared with the model group, the muscle strength of the medium-dose group of icariin and epimedin A1 increased significantly (P<0.01), the muscle strength of epimedin A and epimedin C increased significantly (P<0.05), the muscle strength of the low-dose group of icariin increased significantly (P<0.05), and the muscle strength of the other groups had a rising trend, and there was no significant difference between each administration group. After 12 weeks of administration, compared with the control group, the muscle strength of the model group decreased significantly (P<0.01); compared with the model group, the muscle strength of the medium-dose group of icariin, epimedin C and epimedin A1 increased significantly (P<0.01), the muscle strength of the low-dose group of icariin increased significantly (P<0.05), and the muscle strength of the other groups had a rising trend, and there was no significant difference between each administration group. Other monomers had no effect on the muscle strength of mice.

[0134] Table 5 Effect of Icaria Monomers on Muscle Strength of SAMP8 Myopathy Model Mice Note: Compared with the control group, the model group *P<0.05, **P<0.01; compared with the model group, the drug group # P<0.05, ## P<0.01.

[0135] 3.3, Effect of Icaria Monomers on Body Fat Content and Muscle Content of SAMP8 Myopathy Model Mice

[0136] The results after 12 weeks of administration are shown in Table 6. Compared with the control group, the body fat content of the model group increased significantly (P<0.05); compared with the model group, the body fat content of the medium-dose group of icariin decreased significantly (P<0.01), the muscle content of icariin and epimedin A1 increased significantly (P<0.01), the muscle content of epimedin A increased significantly (P<0.05), the body fat content of the low-dose group had a decreasing trend, and the muscle content had an increasing trend, and other monomers had no effect on the body composition of mice. The DEXA graph of the body composition of SAMP8 mice is shown in Figure 1(B).

[0137] Table 6 Effect of Monomers on Body Fat Content and Muscle Content of SAMP8 Myopathy Model Mice Note: compared with the control group, the model group *P<0.05, **P<0.01; compared with the model group, the drug group # P<0.05, ## P<0.01.

[0138] 3.6, the effect of icariin monomers on skeletal muscle sections of SAMP8 muscle atrophy model mice

[0139] HE-stained sections were used to analyze the pathological damage of skeletal muscle in SAMP8 mice. The skeletal muscle tissue of normal SAMR1 mice was arranged in complete, regular and clear. However, in the SAMP8 mouse model, the skeletal muscle was abnormal and uneven, with smaller cross-sectional area and wider intermuscular space. However, icariin monomers (icariin, doriden A, doriden A1, doriden C) significantly reversed these changes. After icariin monomer intervention, the muscle fiber morphology of mice was improved compared with the model group, arranged more regularly, with smaller intermuscular space and larger cross-sectional area, indicating that icariin monomers help to restore the pathological damage of skeletal muscle in aging mice, and the medium dose group is particularly prominent, while other monomers have no effect on the pathological loss of skeletal muscle in mice. The tibialis anterior muscle sections of SAMP8 mice are shown in Figure 1(A).

[0140] 4, Summary

[0141] Icariin, doriden A, doriden A1 and doriden C can increase the muscle strength and muscle content of SAMP8 muscle atrophy model mice, reduce body weight, body fat and BMC content, and delay the aging process, indicating that icariin, doriden A, doriden A1 and doriden C can be used as drugs for treating muscle atrophy.

[0142] The above describes the embodiments of the present application in detail, and the specific examples are applied to explain the principles and implementation modes of the present application. The above examples are only used to help understand the method and core idea of the present application. Meanwhile, the changes or deformations made by the skilled in the art according to the idea of the present application, based on the specific implementation mode and application range of the present application, all belong to the protection range of the present application. In summary, the content of the specification should not be understood as a limitation of the present application.

Claims

1. Use of a flavone derivative represented by the formula I or a salt thereof in the manufacture of a medicament for the prevention and / or treatment of sarcopenia, characterized in that, The chemical structure of Formula I is shown below: wherein R1, R2 are monosaccharides or oligosaccharides composed of glucose (glc) and / or rhamnose (rha) in different quantities and combinations.

2. Use of a composition comprising a flavone derivative represented by Formula I or a salt thereof in the manufacture of a medicament for preventing and / or treating sarcopenia, characterized in that, The chemical structure of Formula I is shown below: wherein R1, R2 are monosaccharides or oligosaccharides composed of glucose (glc) and / or rhamnose (rha) in different quantities and combinations.

3. Use according to claim 1 or 2, characterized in that, The oligosaccharide is a disaccharide; Preferably, the glucose is β-D-glucose (β-D-glc); Preferably, the rhamnose is α-L-rhamnose (α-L-rha); Preferably, R1 is β-D-glc (1→3) α-L-rha, β-D-glc (1→2) α-L-rha, α-L-rha (1→2) α-L-rha, α-L-rha; Preferably, R2 is β-D-glucose (β-D-glc); Preferably, the flavonoid derivative is icariin, damianin A, damianin A1 or damianin C.

4. Use according to claim 1 or 2, characterized in that, The sarcopenia is caused by age, tumor and / or malnutrition.

5. Use according to claim 2, characterized in that, The composition further comprises one or more other drugs or other extracts for preventing and / or treating sarcopenia; Preferably, the other drug is selected from one or more of the following: myostatin antibody, inhibitor of receptor activator of nuclear factor-κB ligand, vitamin D, calcium agent, recombinant human growth hormone, testosterone, selective estrogen receptor modulator, rapamycin, fatty acid synthase inhibitor, adiponectin, danshensu and coenzyme Q; More preferably, the composition further comprises one or more pharmaceutically acceptable excipients; Still more preferably, the excipient is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, retention aid, lubricant, dispersant, plasticizer, opacifier and antioxidant; Still more preferably, the dosage form of the composition is tablet, drop pill, capsule, powder, injection, film, lozenge, granule or oral liquid.

6. Use according to claim 1 or 2, characterized in that, R1 is not selected from β-D-xyl (1→2) α-L-rha or H; Preferably, R2 is not H; Preferably, the compound is not damianin B, icariin I or icariin II.

7. Use according to claim 1 or 2, characterized in that, The effect of the flavonoid derivative or its salt or the composition on preventing and / or treating sarcopenia is by one or both of the following: improving abnormal myoblast differentiation and increasing mRNA expression of MRFs family factors.

8. Use according to claim 7, characterized in that, The abnormal myoblast differentiation is selected from one or more of the following: reduced nuclear fusion, cell morphological atrophy, cell length reduction and cell diameter reduction; Preferably, the MRFs family factor is selected from one or more of the following: MyoD protein, Mrf5 protein, Mygenin protein and Myf6 protein.

9. Use according to claim 1 or 2, characterized in that, The effect of the flavonoid derivative or its salt or the composition on preventing and / or treating sarcopenia is one or more of the following: delaying apparent aging, increasing muscle strength, reducing body fat content, reducing bone mineral content, increasing muscle content and improving abnormal skeletal muscle.

10. Use according to claim 9, characterized in that, The degree of apparent aging is evaluated by using an aging degree score scale; The degree of apparent aging is evaluated by using an aging degree score scale; Preferably, the evaluation indicator of the scale is selected from one or more of the following: reactivity, passivity, degree of skin aging, degree of hair aging, degree of eye aging, and degree of spinal aging; Preferably, the skeletal muscle is the tibialis anterior muscle; More preferably, the abnormality is selected from one or more of the following: uneven arrangement of skeletal muscle tissue, reduced cross-sectional area, and increased intermuscular space.

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