Application of triflorin-7-O-glucopyranoside in prevention and treatment of sarcopenia

Abstract

The invention discloses application of a glucoside derivative of trifolium pretense or a salt of the glucoside derivative to preparation of a medicine for preventing and / or treating sarcopenia. The glucoside derivative is a monoglucoside flavonoid compound. Wherein the glycoside derivative is a compound formed by connecting a glycosyl group to a 7-O site of trifolium pretense. In in-vivo experiments, the glucoside derivative of triflorin or the salt thereof can significantly improve skeletal muscle mass of sarcopenia model animals, reduce body fat content, improve muscle strength and muscle functions, increase a skeletal muscle wet-weight ratio, recover skeletal muscle pathological damage, increase muscle fiber cross-sectional area and promote expression of myoblastoma-related factors, and can be used for treating sarcopenia. And the expression of atrophy-related markers is reduced, so that the glycoside derivative or the salt thereof has application potential as a medicine for preventing and / or treating sarcopenia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of red clover extract-7-O-glucopyranoside in the prevention and treatment of sarcopenia. Background Technology

[0002] Sarcopenia is a common disease among middle-aged and elderly people. Its main clinical manifestation is a chronic, progressive decrease in muscle mass and quantity with age, leading to a 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 can cause adverse consequences and is closely related to limb dysfunction, reduced self-care ability, and a significantly increased risk of fractures in the elderly, resulting in 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 mass reaches its peak at age 40, after which it gradually declines.The 2016 Asian Sarcopenia Management Guidelines estimated the prevalence of sarcopenia in middle-aged and elderly people aged 60 to 70 in China to be around 4.1% to 11.5%, while the prevalence in men and women aged 70 and above was 13.2% and 4.8%, respectively. The incidence rate increased significantly in people over 80 years of age, reaching as high as 50% [Cruz-Jentoft AJ, Bahat G, Bauer J, Boirie Y, Bruyère O, Cederholm T, CooperC, Landi F, Rolland Y, Sayer AA, Schneider SM, Sieber CC, Topinkova E, Vandewoude M, Visser M, Zamboni M; Writing Group for the European WorkingGroup 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 Juan, Ding Qingqing, Zhou Baiyu, Liu Xiang, Liu Jingmin, Liu Yongming, Ding Guoxian, Zhang Cuntai, Wang Jianye, Yu Pulin, Chinese Society of Geriatrics, Editorial Board of Chinese Journal of Geriatrics. Expert consensus on the diagnosis and treatment of sarcopenia in the elderly 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 received widespread attention from scholars at home and abroad, and extensive research has been carried out in the fields of risk factors, pathogenesis, and treatment of the disease. However, there are still differences in the understanding of the disease between China and other countries. Furthermore, since the etiology of sarcopenia is still unknown, it is related to multiple factors and involves complex molecular mechanisms, which makes the treatment of the disease difficult. Currently, there is still a lack of single effective drugs for sarcopenia. Resistance exercise is the main non-pharmacological therapy for improving muscle quality and function. However, in reality, elderly patients have poor adherence to resistance exercise. Therefore, exploring effective treatment methods is an important direction in the current research field of sarcopenia treatment.

[0003] There is no existing technology showing the application of the red clover extract-7-O-glucopyranoside claimed in this invention for the prevention and treatment of sarcopenia. Summary of the Invention

[0004] Based on this, the present invention provides the use of a glycoside derivative of red cloverin or a salt thereof in the preparation of a medicament for the prevention and / or treatment of sarcopenia, wherein the glycoside derivative is a monosaccharide flavonoid compound. This glycoside derivative is a compound formed by linking a sugar group to the 7-O position of red cloverin.

[0005] According to another aspect of the invention, there is a use of a composition comprising a glycoside derivative of tsacolin or a salt thereof in the preparation of a medicament for the prevention and / or treatment of sarcopenia, wherein the glycoside derivative is a monosaccharide flavonoid compound; This glycoside derivative is a compound formed by linking a sugar group at the 7-O position to red cloverin.

[0006] Furthermore, the glycosyl group is only glucose (glc).

[0007] Furthermore, the glucosyl (glc) is β-D-glucosyl (β-D-glc).

[0008] Furthermore, the glycoside derivative of this prasein is only prasein 7-O-glucopyranoside (PRA).

[0009] Furthermore, the chemical structural formula of this red cloverin-7-O-glucopyranoside is shown in Formula I below:

[0010] Formula I.

[0011] Furthermore, sarcopenia is sarcopenia caused by age, tumors, and / or malnutrition.

[0012] Furthermore, the composition further includes one or more other drugs or other extracts for the prevention and / or treatment of sarcopenia.

[0013] Furthermore, the other drug is selected from one or more of the following: myostatin inhibitory protein antibody, nuclear factor-κB receptor activator ligand inhibitor, vitamin D, calcium, recombinant human growth hormone, testosterone, selective estrogen receptor modulator, rapamycin, fatty acid synthase inhibitor, adiponectin, tanshinone, and coenzyme Q.

[0014] Furthermore, the composition further includes one or more pharmaceutically acceptable excipients.

[0015] Furthermore, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, opacifiers, and antioxidants.

[0016] Furthermore, the dosage form of the composition is tablets, pellets, capsules, powders, injections, films, lozenges, granules, or oral liquids.

[0017] Furthermore, the glycoside derivatives of red cloverin or their salts, or the composition thereof, prevent and / or treat sarcopenia through one or more of the following mechanisms: activating the Nfr2 target, specifically binding to KEAP1, improving C2C12 cell survival, improving C2C12 myotube atrophy, improving body composition, increasing skeletal muscle wet weight ratio, improving muscle strength, improving physical function, improving skeletal muscle pathological damage, promoting the protein and / or mRNA expression of myoblasts, and reducing the protein and / or mRNA expression of muscle atrophy-related markers.

[0018] Furthermore, this improvement in C2C12 myotube atrophy includes improving myotube diameter and / or enhancing MyHC expression.

[0019] Furthermore, this improvement in body composition includes increased muscle mass and / or decreased fat mass.

[0020] Furthermore, the skeletal muscle wet weight ratio includes the tibialis anterior muscle wet weight ratio, gastrocnemius muscle wet weight ratio, soleus muscle wet weight ratio, and / or quadriceps muscle wet weight ratio.

[0021] Furthermore, the skeletal muscle wet weight ratio is the ratio of the wet weight of the skeletal muscle to the animal's body weight.

[0022] Furthermore, the animal is a mammal, such as a human.

[0023] Furthermore, the skeletal muscle pathological injury includes tibialis anterior muscle pathological injury and / or gastrocnemius muscle pathological injury.

[0024] Furthermore, this pathological damage includes abnormal muscle fiber morphology, uneven muscle fiber arrangement, widening of intermuscular space, and / or reduction in the cross-sectional area of ​​muscle fibers.

[0025] Furthermore, the myoblastic factor includes MyoD and / or MyoG.

[0026] Furthermore, the markers associated with muscle atrophy include MuRF-1 and / or Atrogin-1.

[0027] The beneficial effects of this invention are: The glycoside derivatives of red cloverin or their salts of the present invention can significantly improve skeletal muscle mass, reduce body fat content, improve muscle strength and function, increase skeletal muscle wet weight ratio, restore skeletal muscle pathological damage, increase muscle fiber cross-sectional area, promote the expression of myogenic factors, and reduce the expression of atrophy-related markers in in vivo experiments, indicating that the glycoside derivatives or their salts have the potential to be used as drugs for the prevention and / or treatment of sarcopenia. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0029] Figure 1 This diagram illustrates the results of the monomeric components' resistance to dexamethasone-induced proliferation activity in C2C12 cells. A represents the experimental results of gentianin, B represents the experimental results of red cloverin-7-O-glucopyranoside, and C represents the experimental results of verbenafiloside.

[0030] Figure 2 This is a schematic diagram showing the results of myotube atrophy in C2C12 cells induced by resistance to dexamethasone by monomeric components. A is a MyHC immunofluorescence image, and B is a quantitative analysis of the average cross-sectional area of ​​the myotubes.

[0031] Figure 3 This is a schematic diagram showing the result of covalent binding between the monomeric component and KEAP1.

[0032] Figure 4 Flowchart of the experiment on dexamethasone-induced sarcopenia model mice using red cloverin-7-O-glucopyranoside Figure 5 This is a schematic diagram showing the effect of trefoilin-7-O-glucopyranoside on the body weight of dexamethasone-induced sarcopenia model mice.

[0033] Figure 6 This diagram illustrates the effects of trefoilin-7-O-glucopyranoside on fat content, muscle content, and body mass index (BMD) in dexamethasone-induced sarcopenia model mice. A represents the fat content results, B represents the muscle content results, and C represents the BMD results.

[0034] Figure 7This is a schematic diagram showing the effect of red clover extract-7-O-glucopyranoside on the wet weight ratio of skeletal muscle in a dexamethasone-induced sarcopenia model mouse. A shows radiographs of the tibialis anterior, gastrocnemius, soleus, and quadriceps muscles; B shows the experimental results of the wet weight ratio of the tibialis anterior, gastrocnemius, soleus, and quadriceps muscles.

[0035] Figure 8 This diagram illustrates the effects of red clover extract-7-O-glucopyranoside on running time, running distance, and muscle strength in a dexamethasone-induced sarcopenia model mouse. A represents the results of the running distance experiment, B represents the results of the running time experiment, and C represents the results of the muscle strength experiment.

[0036] Figure 9 This diagram illustrates the effects of red clover extract-7-O-glucopyranoside on the pathological damage to the tibialis anterior muscle and the size and distribution of muscle fiber cross-sectional area in a dexamethasone-induced sarcopenia model mouse. A shows an HE-stained section of the tibialis anterior muscle; B shows the experimental results of the average cross-sectional area of ​​muscle fibers; and C shows the experimental results of the percentage distribution of muscle fiber cross-sectional area.

[0037] Figure 10 This diagram illustrates the effects of red clover extract-7-O-glucopyranoside on the expression levels of myoblastic factor and atrophy marker genes in the tibialis anterior muscle of a dexamethasone-induced sarcopenia model mouse. A represents the MyoD experimental results, B represents the MyoG experimental results, C represents the Atrogin-1 experimental results, and D represents the MuRF-1 experimental results.

[0038] Figure 11 This is a schematic diagram showing the effect of red clover extract-7-O-glucopyranoside on the expression levels of myoblastic factor and atrophy marker proteins in the tibialis anterior muscle of dexamethasone-induced sarcopenia model mice.

[0039] Figure 12 This diagram illustrates the effects of red clover extract-7-O-glucopyranoside on the pathological damage to the gastrocnemius muscle, as well as the size and distribution of muscle fiber cross-sectional area, in a dexamethasone-induced sarcopenia model mouse. A shows the hematoxylin and eosin (HE) staining of the gastrocnemius muscle; B shows the experimental results of the average cross-sectional area of ​​muscle fibers; and C shows the percentage distribution of muscle fiber cross-sectional area.

[0040] Figure 13 This diagram illustrates the effects of red clover extract-7-O-glucopyranoside on the expression levels of myoblastic factor and atrophy marker genes in the gastrocnemius muscle of a dexamethasone-induced sarcopenia model mouse. A represents the MyoD experimental results, B represents the MyoG experimental results, C represents the Atrogin-1 experimental results, and D represents the MuRF-1 experimental results.

[0041] Figure 14This is a schematic diagram showing the effect of red clover extract-7-O-glucopyranoside on the expression levels of myoblastic factors and atrophy marker proteins in the gastrocnemius muscle of a dexamethasone-induced sarcopenia model.

[0042] In the above appendix Figures 1-14 Among them, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05 vs model group. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0045] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0046] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0047] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0048] As described in the background section, the application of the claimed red cloverin-7-O-glucopyranoside in the prevention and treatment of sarcopenia is not found in the prior art. To address the above problem, the present invention provides the use of a glycoside derivative of red cloverin or a salt thereof in the preparation of a medicament for the prevention and / or treatment of sarcopenia, wherein the glycoside derivative is a monosaccharide flavonoid compound; This glycoside derivative is a compound formed by linking a sugar group at the 7-O position to red cloverin.

[0049] According to another aspect of the invention, there is a use of a composition comprising a glycoside derivative of tsacolin or a salt thereof in the preparation of a medicament for the prevention and / or treatment of sarcopenia, wherein the glycoside derivative is a monosaccharide flavonoid compound; This glycoside derivative is a compound formed by linking a sugar group at the 7-O position to red cloverin.

[0050] In a preferred embodiment, the glycosyl group is only glucose (glc).

[0051] In a preferred embodiment, the glucosyl (glc) is β-D-glucosyl (β-D-glc).

[0052] In a preferred embodiment, the glycoside derivative of the pratidin is only pratidin 7-O-glucopyranoside (PRA).

[0053] In a preferred embodiment, the chemical structural formula of the red cloverin-7-O-glucopyranoside is shown in Formula I below:

[0054] Formula I.

[0055] In a preferred embodiment, the sarcopenia is sarcopenia caused by age, tumors, and / or malnutrition.

[0056] In a preferred embodiment, the composition further comprises one or more other drugs or other extracts for the prevention and / or treatment of sarcopenia.

[0057] In a preferred embodiment, the other drug is selected from one or more of the following: myostatin inhibitory protein antibody, nuclear factor-κB receptor activator ligand inhibitor, vitamin D, calcium, recombinant human growth hormone, testosterone, selective estrogen receptor modulator, rapamycin, fatty acid synthase inhibitor, adiponectin, tanshinone, and coenzyme Q.

[0058] In a preferred embodiment, the composition further comprises one or more pharmaceutically acceptable excipients.

[0059] In a preferred embodiment, the pharmaceutical composition of the present invention contains at least one pharmaceutically acceptable excipient in an 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.% relative to the total weight of the pharmaceutical composition.

[0060] In this invention, the term "pharmaceutically acceptable" means a substance, such as a carrier or diluent, that does not destroy the biological activity or properties of a compound and is relatively non-toxic, such that, when administered to an individual, it will not cause unwanted biological effects or interact with any of its constituent components in a harmful manner.

[0061] In this invention, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0062] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants.

[0063] Those skilled in the art will know how to select specific chemical substances within the aforementioned excipient categories. For example, the diluent may 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 may be selected from one or more of the following: polyoxymethylene stearate, poloxamer, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ethers, polysorbate esters such as polysorbate 80, cetyl alcohol, glyceryl fatty acid esters (such as triacetin, glyceryl monostearate, and the like), polyoxyethylene fatty acid esters, polyethylene glycol fatty acid esters, sodium lauryl sulfate, sorbitol fatty acid esters, sucrose fatty acid esters, polyoxyethylene ethers, benzalkonium chloride, polyoxyethylene castor oil, and sodium docusate. The binder may be selected from one or more of the following: polyvinylpyrrolidone, hydroxypropyl cellulose, polyethylene glycol, and methylcellulose. The disintegrant may be selected from one or more of the following: carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose. The flavoring agent may be selected from one or more of the following: sorbitol, glucose, mannose, sucrose, and lactose. The dispersant may be selected from one or more of the following: croscarmellose sodium, sodium starch glycolate, and pregelatinized corn starch. The plasticizer may be dibutyl sebacate and / or various citrate esters. The sustained-release agent may 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 may be selected from one or more of the following: sodium bisulfite, sodium metabisulfite, sodium sulfite, and sodium thiosulfate. The lubricant may be selected from one or more of the following: calcium stearate, talc, magnesium stearate, stearic acid, and colloidal silica.

[0064] These excipients are preferably drug-inert, or may have synergistic or enhancing effects to enhance the therapeutic activity of the drug composition. The above excipients are merely examples, and the excipients actually used in this invention are not limited to the above excipients. They can be adjusted according to the actual situation to achieve the effects of this invention.

[0065] In a preferred embodiment, the dosage form of the composition is tablets, pellets, capsules, powders, injections, films, lozenges, granules, or oral liquids.

[0066] In a preferred embodiment, the glycoside derivative of red cloverin or its salt, or the composition thereof, prevents and / or treats sarcopenia through one or more of the following mechanisms: activating the Nfr2 target, specifically binding to KEAP1, improving C2C12 cell survival, improving C2C12 myotube atrophy, improving body composition, increasing skeletal muscle wet weight ratio, improving muscle strength, improving physical function, improving skeletal muscle pathological damage, promoting the protein and / or mRNA expression of myogenic factors, and reducing the protein and / or mRNA expression of muscle atrophy-related markers.

[0067] In a preferred embodiment, the improvement of C2C12 myotube atrophy includes improving myotube diameter and / or enhancing MyHC expression.

[0068] In a preferred embodiment, the improvement in body composition includes an increase in muscle mass and / or a decrease in fat mass.

[0069] In a preferred embodiment, the skeletal muscle wet weight ratio includes the tibialis anterior muscle wet weight ratio, the gastrocnemius muscle wet weight ratio, the soleus muscle wet weight ratio, and / or the quadriceps femoris muscle wet weight ratio.

[0070] In a preferred embodiment, the skeletal muscle wet weight ratio is the ratio of the wet weight of the skeletal muscle to the body weight of the animal.

[0071] In a preferred embodiment, the animal is a mammal, such as a human.

[0072] In a preferred embodiment, the skeletal muscle pathological injury includes tibialis anterior muscle pathological injury and / or gastrocnemius muscle pathological injury.

[0073] In a preferred embodiment, the pathological damage includes abnormal muscle fiber morphology, uneven muscle fiber arrangement, widening of intermuscular space, and / or reduction in the cross-sectional area of ​​muscle fibers.

[0074] In a preferred embodiment, the myogenic factor includes MyoD and / or MyoG.

[0075] In a preferred embodiment, the muscle atrophy-related markers include MuRF-1 and / or Atrogin-1.

[0076] According to another aspect of the present invention, there is provided a red clover extract-7-O-glucopyranoside of Formula I or a salt thereof or the above pharmaceutical composition for the prevention and / or treatment of sarcopenia in a subject.

[0077] According to another aspect of the invention, a method for preventing and / or treating sarcopenia in a subject is provided, comprising administering to the subject an effective amount of the above-described thymol-7-O-glucopyranoside or a salt thereof or the above-described pharmaceutical composition as shown in Formula I.

[0078] In this invention, the term "subject" refers to a mammal. A 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 sarcopenia models. Preferably, the subject is a human.

[0079] The “effective amount” of the pharmaceutical composition or formulation used in this invention can achieve the desired therapeutic and / or preventative effects. The effective amount for this purpose will depend on factors such as the pharmaceutical composition, the route of administration, the stage and severity of the disease being treated, the individual’s weight and overall health status, and the judgment of the prescribing physician. The dose can be administered once a week, every two days, or daily, or even several times a day. Dosage units can be administered over a short period (e.g., weeks to months) or a longer period (months to years).

[0080] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0082] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0083] Example 1: Efficient discovery of Nrf2-keap1 covalently bound monomer components 1. Experimental Methods 1.1 Experimental Instruments and Materials The instruments required for the experiment are shown in Table 1.

[0084] Table 1. Instruments required for the experiment

[0085] The purity of the reference standards used in the experiment, namely, tufted rosin (CAS No.: 20633-67-4), red cloverin-7-O-glucopyranoside (CAS No.: 36191-03-4), and styracin (CAS No.: 486-62-4), was ≥98%, and they were purchased from Chengdu Alpha Biotechnology Co., Ltd.

[0086] calycosin-7-O-glucoside

[0087] Pratensein 7-O-glucopyranoside

[0088] Ononin

[0089] 1.2 Cell Culture Methods The C2C12 myoblast cell line was purchased from the China Center for Type Culture Collection. Cells were cultured in a high-glucose Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine mixed antibiotics. Cells were incubated at 37 °C in a 5% CO2 incubator, and cell growth was observed regularly. When cell confluence reached 70%–80%, cells were passaged using standard methods. The C2C12 cells used for subsequent experiments were passaged 15–20 times. All cells underwent multiple population doubling and exhibited stable growth.

[0090] 1.3. CCK8 assay for cell viability C2C12 cells were treated with different concentrations of monomeric components and cultured with or without 150 μM dexamethasone treatment. Cell viability was assessed using a CCK-8 assay kit (Abcam, ab228554), and absorbance was measured at 450 nm using a microplate reader (BioTek Synergy HT).

[0091] 1.4. C2C12 cell-induced myodifferentiation culture To establish a dexamethasone (Dexa)-induced muscle atrophy model, C2C12 myoblasts were cultured in differentiation medium, specifically DMEM supplemented with 2% horse serum (HS) and antibiotics. On day 5 of differentiation, 100 μM dexamethasone was added, along with 25 μM of various traditional Chinese medicine monomers, to evaluate the protective effect of the monomer components against dexamethasone-induced myoblast atrophy. The experimental groups were as follows: untreated control group (N), Dexa-treated group (M), and Dexa + various traditional Chinese medicine monomer treatment groups (48 h intervention).

[0092] 1.5 Immunofluorescence staining of C2C12 myotubes C2C12 myotube cells were cultured and treated according to the above method. After intervention, the cells were washed, fixed, and incubated overnight at 4 °C with anti-myosin heavy chain (MHC) primary antibody (1:100; A25464, Abclonal, USA). The next day, the cells were incubated with goat anti-mouse IgG H&L secondary antibody (1:1000) at room temperature for 90 min. Subsequently, the cell nuclei were counterstained with DAPI (300 nM). After washing three times with PBST, the stained cells were observed and imaged using a fluorescence microscope (BioTek Instruments, Inc.).

[0093] 1.6 Screening Experiment for Fluorescent Nanoantennas 0.2 mg / mL of 6×His-Smt3-human Keap1 recombinant protein was dissolved in binding buffer (25 mM Tris-HCl, pH 8.0). The experiment was performed using 96-well black microplates. 50 μL of engineered Keap1 protein solution and 50 μL of 40 μM fluorescent probe solution were added to each well, and the plates were incubated at 37 °C for 2 h to form the protein-probe reaction system. After incubation, the fluorescence intensity of each well was continuously measured using a multi-mode microplate reader (Agilent BioTek Synergy H1) at the maximum excitation and emission wavelengths corresponding to the probe. To eliminate interference from non-specific fluorescence signals, a probe solution without Keap1 protein was used as a background control, and the fluorescence values ​​obtained were subtracted from the corresponding background signal before being used for subsequent analysis.

[0094] 2. Research Results 2.1. Validation of monomeric activity using a Dexa-induced C2C12 myotube atrophy model: Efficacy assessed by CCK-8 and MyHC immunofluorescence assays. This study used a dexamethasone (Dexa)-induced C2C12 myotube atrophy model to systematically evaluate its anti-atrophy ability from two aspects: cell viability and muscle characteristic proteins. CCK-8 results showed that Dexa significantly reduced C2C12 cell viability, while red cloverin-7-O-glucopyranoside significantly improved cell survival rate after treatment. P <0.05 or P <0.01), while the other two compounds showed no significant changes compared to the model group, demonstrating that thymol-7-O-glucopyranoside has significant anti-damage and activity-promoting effects (e.g., Figure 1 As shown). Further MyHC (myosin heavy chain) immunofluorescence assays showed that Dexa treatment resulted in thinner myotubes and decreased fluorescence intensity, while monomeric intervention improved myotube morphology to varying degrees, manifested as increased myotube diameter and significantly enhanced MyHC fluorescence intensity. Quantitative analysis of cell cross-sectional area revealed that red cloverin-7-O-glucopyranoside and other compounds significantly resisted DEXA-induced C2C12 myotube atrophy (as shown). P <0.0001), while the other two compounds showed no change relative to the model group (e.g., Figure 2 (As shown).

[0095] 2.2. Monomer compound screening based on fluorescent probes: Identification of covalently bound components of KEAP1 To further elucidate the molecular mechanism by which the screened key monomers activate NRF2, this study employed a specific fluorescent probe labeling method to experimentally verify the direct binding relationship between the monomers and the KEAP1 protein. Fluorescent probes can generate detectable fluorescence signals when monomers undergo a covalent reaction with KEAP1, providing a reliable tool for confirming KEAP1 modification and the formation of covalent bonds. Experimental results showed that rutabagin-7-O-glucopyranoside exhibited significant fluorescence enhancement after co-incubation with recombinant KEAP1 protein, while no corresponding fluorescence changes were observed in the negative control and the other two compounds. This suggests that rutabagin-7-O-glucopyranoside specifically acts on the active site of KEAP1 (e.g., NRF2 activation). Figure 3 (As shown).

[0096] 3. Conclusion The results showed that red cloverin-7-O-glucopyranoside could significantly activate the Nrf2 target of the oxidative stress signaling pathway and specifically bind to KEAP1; and resist the dexamethasone-induced decrease in C2C12 cell proliferation activity and myotube atrophy, while the other two compounds showed no change compared to the model group.

[0097] Example 2: Effects of red clover extract-7-O-glucopyranoside on dexamethasone-induced sarcopenia model mice 1. Materials 1.1 Laboratory Animals Eight-week-old male C57 mice, weighing 25±2g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. The animals were housed in the SPF-grade animal facility of the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine. The facility temperature was (24±1)℃, relative humidity was 50%–70%, and the light / dark cycle was 12h / 12h. The experimental procedures strictly followed the relevant regulations and rules of the Ethics Committee of the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine.

[0098] 1.2 Experimental Drugs The red cloverin-7-O-glucopyranoside monomer used in the embodiments of the present invention was purchased from Chengdu Alpha Biotechnology Co., Ltd.

[0099] 1.3 Experimental Apparatus Dual-energy X-ray absorptiometry (iNSiGHT VET DXA, OsteoSys, KOREAN); YLS-13A muscle strength tester, Jinan Yiyan Technology Co., Ltd.

[0100] 2. Experimental Methods 2.1 Preparation of the sarcopenia model In this embodiment, the sarcopenia model was induced using dexamethasone. The experimental animals were injected intraperitoneally with 20 mg / kg of dexamethasone daily for 2 weeks, while the normal group mice were injected intraperitoneally with an equal amount of physiological saline daily.

[0101] 2.2 Grouping and Administration Fifty C57 mice were randomly divided into a normal control group, a model group, a low-dose group (15 mg / kg) of trifolin-7-O-glucopyranoside, a high-dose group (30 mg / kg) of trifolin-7-O-glucopyranoside, and a vitamin D group (0.1 μg / kg). Trifolin-7-O-glucopyranoside was dissolved in 0.3% sodium carboxymethyl cellulose solution. Each treatment group was administered different concentrations of trifolin-7-O-glucopyranoside solution by gavage once daily for two weeks, with a gavage volume of 0.1 mL / 10 g. The negative control group and the model group were administered 0.3% sodium carboxymethyl cellulose solution by gavage once daily for two weeks, with a gavage volume of 0.1 mL / 10 g.

[0102] 2.3 Detection Indicators and Methods 2.3.1. Measurement of mouse body composition Three days before sacrifice, mice were scanned using dual-energy X-ray absorptiometry (DEXA) to determine their body composition, including body fat, muscle mass, and bone mineral content. The data were then analyzed using software provided by the manufacturer.

[0103] 2.3.2 Measurement of Mouse Muscle Strength Forelimb grip strength in mice was measured using a YLS-13A muscle strength testing instrument (Yiyan Technology Co., Ltd., Jinan, China). Mice were lifted and secured by their tails so that their limbs could grip a wire mesh. The mice's tails were then gently pulled back until they were parallel to the table surface, until they released the mesh. Peak forces exerted by the mice's limbs were recorded in grams (g). Evaluations were conducted at 0, 4, 8, and 12 weeks, with three tests per mouse. The average values ​​were taken and weighted for statistical analysis.

[0104] 2.3.3. Mouse muscle function assay Three days before sacrifice, muscle function was assessed using a treadmill. Mice were placed on the track with an initial speed of 5 m / min and an incline of 0 degrees. The speed and incline were increased by 5 m / min and 5 degrees every 5 minutes until a speed of 20 m / min and an incline of 15 degrees were reached. The time to exhaustion was recorded. (Exhaustion was defined as a mouse not moving from the track for at least 20 seconds, indicating a significant decrease in its response to external stimuli. If a mouse did not reach exhaustion after 3 hours, the time to exhaustion was recorded as 3 hours.) 2.3.4 Determination of skeletal muscle wet weight ratio Mice were deeply anesthetized with 0.3% sodium pentobarbital and then dissected to obtain bilateral tibialis anterior, gastrocnemius, soleus, and quadriceps muscles. The body weight and wet weight of the skeletal muscles were recorded at the time of sampling.

[0105] 2.3.5 Skeletal muscle pathological examination (HE staining) The tibialis anterior and gastrocnemius muscles on one side were soaked in fresh 4% paraformaldehyde at room temperature for 3-5 days, dehydrated using an alcohol gradient (75% ethanol, 2 hours; 85% ethanol, 1 hour; 95% ethanol, 1 hour; 95% ethanol, 1 hour; 100% ethanol, 0.5 hours; 100% ethanol), and then embedded in paraffin. 5 μm paraffin sections were prepared from the tibialis anterior and gastrocnemius muscles for histological analysis. The paraffin sections were first deparaffinized, soaked in xylene, and then rehydrated in a gradient of alcohols. The sections were stained with hematoxylin for 10 minutes, rinsed with tap water for 10 minutes, and stained with eosin for 1 minute. After thoroughly rinsing in tap water, the sections were cleared with ethanol and xylene, and finally covered with a slide. The stained sections were examined under a microscope, and images were captured using a Leica Aperio GT450 scanner (Leica, Germany). In addition, sections were stained with multiplex immunohistochemicals using a commercial kit (FH33020R, Foregene, China), and immunofluorescence was performed using laminin antibody (1:200; 23498-1-AP, Proteintech, China). Subsequently, the cross-sectional area of ​​myofibrils (CSA) was quantified using ImageJ software (NIH, Maryland, USA).

[0106] 2.3.6 Detection of skeletal muscle myogenic factors (MyoD, MyoG, Murf-1, Atrogin-1) mRNA expression by RT-qPCR Total RNA was extracted from skeletal muscle tissue using Trizol™ reagent (B511311-0100, Sangon Biotech, China), and reverse transcribed into complementary DNA (cDNA) using a PrimeScript RT kit (RR047A, Takara Bio, Japan). Real-time quantitative PCR (RT-qPCR) amplification was then performed using TB Green Premix Ex Taq™ (RR420A, Takara Bio, Japan). The mRNA expression levels of the target gene were normalized using Gapdh as an internal reference gene. The primer sequences used are shown in Table 2.

[0107] Table 2 Primer sequences

[0108] 2.3.7 Western blot analysis of skeletal muscle myogenic factor (MyoD, MyoG, Murf-1, Atrogin-1) protein expression Total protein was extracted from tibialis anterior and gastrocnemius muscle tissues using RIPA lysis buffer (P0013B, Beyotime, China) containing a protease / phosphatase inhibitor (P1045, Beyotime, China). Protein samples were separated by SDS-PAGE (10–12.5% ​​separating gel) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% BSA at room temperature for 1 h and then incubated overnight at 4 °C with primary antibody (details in Table S4). Subsequently, the membranes were washed three times with phosphate-buffered saline (PBST) containing Tween-20 and incubated for 1 h at room temperature with HRP-labeled secondary antibody (1:5000). Protein bands were visualized by enhanced chemiluminescence (ECL) reagent. The experimental materials used are shown in Table 3.

[0109] Table 3 Experimental Materials

[0110] 2.4 Statistical Methods Experimental data were analyzed using SPSS 21.0 software. Quantitative data were expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0111] 3. Experimental Results 3.1 Effect of red clover extract-7-O-glucopyranoside on body weight in a dexamethasone-induced sarcopenia model Animal experimental protocols, such as Figure 4 As shown. The results of the weight experiment are as follows. Figure 5 As shown, two weeks after modeling and administration, there was no significant difference in body weight among the normal group, model group, and low- and high-dose groups of thymol-7-O-glucopyranoside. The body weight of the vitamin D group was significantly lower than that of the model group. P <0.01).

[0112] 3.2 Effects of red clover extract-7-O-glucopyranoside on body composition in a dexamethasone-induced sarcopenia model The results are as follows Figure 6 As shown, two weeks after drug administration, the muscle mass in the model group was significantly lower than that in the normal group. P <0.0001, fat content was significantly higher than that of the normal group ( P <0.0001, BMD decreased significantly ( P <0.01%. Two weeks after administration, the low-dose group of red clover extract-7-O-glucopyranoside showed an increasing trend in muscle mass and a decreasing trend in fat mass; the high-dose group of red clover extract-7-O-glucopyranoside showed a significant increase in muscle mass ( P<0.001, fat content decreased significantly ( P <0.001, had no significant effect on BMD; vitamin D had no significant effect on muscle mass and fat mass, but could significantly increase BMD levels ( P <0.01).

[0113] 3.3 Effect of red clover extract-7-O-glucopyranoside on the wet weight ratio of skeletal muscle in a dexamethasone-induced sarcopenia model The results are as follows Figure 7 As shown, two weeks after drug administration, the wet weight ratios of the tibialis anterior, gastrocnemius, soleus, and quadriceps muscles in the model group were significantly lower than those in the normal group. P <0.05); After 2 weeks of administration, the skeletal muscle wet weight ratio in the low-dose group of red clover extract-7-O-glucopyranoside showed an increasing trend compared with the model group; the tibialis anterior muscle wet weight ratio in the high-dose group of red clover extract-7-O-glucopyranoside was significantly increased ( P <0.05, the wet weight ratio of the gastrocnemius muscle increased significantly ( P <0.01), the wet weight ratio of soleus muscle increased significantly ( P <0.05, the wet weight ratio of the quadriceps femoris muscle increased significantly ( P <0.05); the wet weight ratio of the tibialis anterior muscle was significantly increased in the vitamin D group ( P <0.05, the wet weight ratio of the gastrocnemius muscle increased significantly ( P <0.01), the wet weight ratio of soleus muscle and quadriceps muscle showed an increasing trend.

[0114] 3.4 Effects of red clover extract-7-O-glucopyranoside on muscle strength and function in a dexamethasone-induced sarcopenia model The results are as follows Figure 8 As shown, two weeks after drug administration, the running time and distance in the model group decreased significantly. P <0.0001), muscle strength significantly decreased ( P <0.001); After 2 weeks of administration, the running time and running distance of the low-dose group of red clover extract-7-O-glucopyranoside were significantly improved ( P <0.05%, muscle strength showed a trend of improvement; the high-dose group of red clover extract-7-O-glucopyranoside showed significant improvements in running time and running distance ( P <0.001), muscle strength significantly improved ( P <0.05); the running time and running distance were significantly improved in the vitamin D group ( P <0.001), muscle strength significantly improved ( P <0.05).

[0115] 3.5 Effects of red clover extract-7-O-glucopyranoside on tibialis anterior and gastrocnemius muscle sections in a dexamethasone-induced sarcopenia model The results are as follows Figure 9 and Figure 12 As shown, HE-stained sections were used to analyze the pathological damage of skeletal muscle in a dexamethasone-induced sarcopenia model mouse. In normal mice, skeletal muscle tissue is intact, regular, and clearly arranged. However, in the sarcopenia mouse model, skeletal muscle is abnormal and heterogeneous, with a smaller cross-sectional area and wider intermuscular spaces. However, red clover extract-7-O-glucopyranoside and vitamin D significantly reversed these changes. After intervention, the muscle fiber morphology of the mice was improved compared to the model group, with a more orderly arrangement, smaller intermuscular spaces, and a larger cross-sectional area, indicating that red clover extract-7-O-glucopyranoside helps restore the pathological damage of skeletal muscle in aging mice, with the high-dose group showing particularly outstanding results.

[0116] 3.6 Effects of red clover extract-7-O-glucopyranoside on the mRNA and protein expression levels of myogenic factor and atrophic factor in the tibialis anterior and gastrocnemius muscles of a dexamethasone-induced sarcopenia model. The results are as follows Figure 10 , Figure 11 , Figure 13 and Figure 14 As shown, in dexamethasone-induced sarcopenia model mice, the gene and protein expression levels of myogenic factors (MyoD and MyoG) in the tibialis anterior and gastrocnemius muscles were significantly decreased, while the gene and protein expression levels of muscle atrophy-related markers (MuRF-1 and Atrogin-1) were significantly increased. Two weeks after administration, all treatment groups showed improvement in the above indicators, with the high-dose group of cloverin-7-O-glucopyranoside showing the most significant effect. The high-dose group of cloverin-7-O-glucopyranoside significantly increased the gene and protein expression levels of MyoD and MyoG, and significantly decreased the gene and protein expression levels of MuRF-1 and Atrogin-1.

[0117] 4. Experiment Summary Red clover extract-7-O-glucopyranoside can increase muscle mass, muscle strength, running time, and running distance in dexamethasone-induced sarcopenia model mice, increase the wet weight ratio of the tibialis anterior, gastrocnemius, soleus, and quadriceps muscles, repair pathological damage to skeletal muscle, increase the cross-sectional area of ​​muscle fibers, promote the protein and gene expression levels of myogenic factors, reduce fat content, and reduce the protein and gene expression levels of muscle atrophy-related markers, indicating that red clover extract-7-O-glucopyranoside can be used as a drug for the treatment of sarcopenia.

[0118] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. The use of a glycoside derivative of red cloverin or a salt thereof in the preparation of a medicament for the prevention and / or treatment of sarcopenia, characterized in that, The glycoside derivatives are monosaccharide flavonoid compounds; The glycoside derivative is a compound formed by linking a sugar group to the 7-O position of red cloverin.

2. Use of a composition comprising a glycoside derivative of red cloverin or a salt thereof in the preparation of a medicament for the prevention and / or treatment of sarcopenia, characterized in that, The glycoside derivatives are monosaccharide flavonoid compounds; The glycoside derivative is a compound formed by linking a sugar group to the 7-O position of red cloverin.

3. The use according to claim 1 or 2, characterized in that, The glycosyl group is only glucosyl (glc); Preferably, the glucosyl (glc) is β-D-glucosyl (β-D-glc); More preferably, the glycoside derivative of thymol is only thymol-7-O-glucopyranoside; Preferably, the chemical structural formula of the red cloverin-7-O-glucopyranoside is shown in Formula I below: ， Formula I.

4. The use according to claim 1 or 2, characterized in that, The sarcopenia mentioned refers to sarcopenia caused by age, tumors, and / or malnutrition.

5. The use according to claim 2, characterized in that, The composition further comprises one or more other drugs or other extracts for the prevention and / or treatment of sarcopenia.

6. The use according to claim 5, characterized in that, The other drugs are selected from one or more of the following: myostatin inhibitory protein antibody, nuclear factor-κB receptor activator ligand inhibitor, vitamin D, calcium, recombinant human growth hormone, testosterone, selective estrogen receptor modulator, rapamycin, fatty acid synthase inhibitor, adiponectin, tanshinone, and coenzyme Q. More preferably, the composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the excipients are selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants; Preferably, the dosage form of the composition is tablets, pellets, capsules, powders, injections, films, lozenges, granules, or oral liquids.

7. The use according to claim 1 or 2, characterized in that, The glycoside derivatives of red cloverin or their salts, or the composition thereof, prevent and / or treat sarcopenia through one or more of the following mechanisms: activating the Nfr2 target, specifically binding to KEAP1, improving C2C12 cell survival, improving C2C12 myotube atrophy, improving body composition, increasing skeletal muscle wet weight ratio, improving muscle strength, improving physical function, improving skeletal muscle pathological damage, promoting the protein and / or mRNA expression of myoblastic factors, and reducing the protein and / or mRNA expression of muscle atrophy-related markers.

8. The use according to claim 7, characterized in that, The improvement of C2C12 myotube atrophy includes improving myotube diameter and / or enhancing MyHC expression.

9. The use according to claim 7, characterized in that, The improvement in body composition includes increased muscle mass and / or decreased fat mass; Preferably, the skeletal muscle wet weight ratio includes the tibialis anterior muscle wet weight ratio, gastrocnemius muscle wet weight ratio, soleus muscle wet weight ratio and / or quadriceps muscle wet weight ratio; Preferably, the skeletal muscle wet weight ratio is the ratio of the wet weight of the skeletal muscle to the body weight of the animal; Preferably, the animal is a mammal, such as a human; Preferably, the skeletal muscle pathological injury includes tibialis anterior muscle pathological injury and / or gastrocnemius muscle pathological injury; Preferably, the pathological damage includes abnormal muscle fiber morphology, uneven muscle fiber arrangement, widening of intermuscular space and / or reduction of muscle fiber cross-sectional area.

10. The use according to claim 7, characterized in that, The myogenic factors include MyoD and / or MyoG; Preferably, the muscle atrophy-related markers include MuRF-1 and / or Atrogin-1.

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