Use of strong loquat extract in the preparation of a drug for improving and / or treating muscle atrophy

Abstract

The present application relates to the technical field of medicine, and provides application of strong loquat juice in preparation of a medicine for improving and / or treating muscle atrophy. The strong loquat juice is applied in preparation of the medicine for improving and / or treating muscle atrophy, in particular age-related muscle atrophy or inflammation factor-induced muscle atrophy, and the strong loquat juice improves and / or treats muscle atrophy by improving motor function, promoting myotube differentiation and hypertrophy, and maintaining muscle fiber structural integrity. The present application discloses the improvement effect of the strong loquat juice on muscle structure and function in cell and animal models: in a TNF-alpha-induced C2C12 myotube atrophy model, the strong loquat juice can significantly increase myotube diameter and improve myosin heavy chain expression; in an aging-related Caenorhabditis elegans model, the strong loquat juice can significantly enhance body bending ability, pharyngeal pump movement rate and muscle fiber structural integrity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of strong loquat syrup in the preparation of drugs for improving and / or treating muscle atrophy. Background Technology

[0002] Muscle atrophy is a pathological condition characterized by a decrease in the cross-sectional area of ​​muscle fibers and a progressive loss of muscle mass and function. This condition can be induced by a variety of factors, including but not limited to systemic diseases such as heart failure and chronic kidney disease, acute injuries such as severe trauma and extensive burns, malignant tumors and related cachexia, and age-related physiological decline. Muscle atrophy not only directly leads to decreased muscle strength and motor dysfunction, but also significantly increases the risk of falls, metabolic disorders, and complications, severely impairing patients' ability to live independently and their overall quality of life. Currently, clinical interventions for muscle atrophy remain very limited; besides basic exercise rehabilitation training and nutritional support, there is a lack of safe and effective specific drug treatment options. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the present invention provides the application of strong loquat syrup in the preparation of drugs for improving and / or treating muscle atrophy, the purpose of which is to solve the problems mentioned in the background art.

[0004] This invention provides the use of a potent loquat syrup in the preparation of a drug for improving and / or treating muscle atrophy. The potent loquat syrup comprises loquat leaf, poppy husk, stemona root, cynanchum paniculatum, mulberry bark, platycodon grandiflorus, and menthol, in a mass ratio of loquat leaf:poppy husk:stemon root:cynanchum paniculatum:mulberry bark:platycodon grandiflorus:menthol = 69:50:15:9:6:6:0.15.

[0005] Furthermore, the muscle atrophy includes age-related muscle atrophy or inflammatory factor-induced muscle atrophy.

[0006] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0007] Furthermore, the excipients are one or more of the following: diluents, buffers, adhesives, flavoring agents, and fillers.

[0008] Furthermore, the drug is formulated into a clinically acceptable dosage form.

[0009] Furthermore, the dosage form of the drug is an oral liquid.

[0010] The present invention has the following beneficial effects: (1) Strong loquat syrup is used in the preparation of drugs to improve and / or treat muscle atrophy, especially age-related muscle atrophy or inflammatory factor-induced muscle atrophy. Strong loquat syrup improves and / or treats muscle atrophy by improving motor function, promoting myotube differentiation and hypertrophy, and maintaining the integrity of muscle fiber structure.

[0011] (2) Revealing the effects of strong loquat extract on muscle structure and function in cell and animal models: In the TNF-α-induced C2C12 myotube atrophy model, strong loquat extract can significantly increase the diameter of myotubes and enhance the expression of myosin heavy chain; In the aging-related Caenorhabditis elegans model, strong loquat extract can significantly enhance the body bending ability, pharyngeal pump movement rate and muscle fiber structure integrity. Attached Figure Description

[0012] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 This is a chromatogram of the chemical composition analysis of the strong loquat syrup using liquid chromatography-mass spectrometry in Example 1 of the present invention, wherein: Figure 1 In this chromatogram, 'a' represents the total ion current chromatogram in positive ion mode. Figure 1 In the figure, b represents the total ion chromatogram in negative ion mode.

[0013] Figure 2 The figure shows the effect of potent loquat extract on the proliferation and cytotoxicity of C2C12 myoblasts in Example 2. Figure 2 In the figure, 'a' represents the absorbance of C2C12 cells treated with different concentrations of strong loquat extract for 48 hours at 490 nm. ** indicates p < 0.01, **** indicates p < 0.0001. Figure 2 In the figure, b represents the absorbance of C2C12 cells treated with Strong Loquat Dew at 490 nm, as detected by the MTS method at different time periods.

[0014] Figure 3 The graph shows the effect of strong loquat extract on C2C12 myoblast differentiation in Example 2. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Figure 3 In the diagram, 'a' represents a schematic diagram of the C2C12 myoblast differentiation experiment. Figure 3 In the figure, b represents the relative quantitative statistical graph of protein expression of the myoblast differentiation marker gene MyoD; Figure 3 In the figure, 'c' represents the relative quantitative statistical graph of protein expression of Myogenin, a marker gene for myoblast differentiation. Figure 3 In the figure, d represents the relative quantitative statistics of myosin heavy chain (MyHC) protein expression; Figure 3 In the figure, 'e' represents the morphological characteristics and distribution of myosin heavy chain (MyHC) in differentiated C2C12 cells as shown by immunofluorescence staining. Scale bar: 100 μm.

[0015] Figure 4 The graph shows the effect of strong loquat extract intervention on TNF-α-induced myotube atrophy in Example 3. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. Figure 4 In the diagram, 'a' represents a schematic representation of a TNF-α-induced C2C12 myoblast atrophy model. Figure 4 In the figure, b represents the relative quantitative statistics of myosin heavy chain (MyHC) protein expression; Figure 4 In the figure, c represents the expression of MyHC (green) in C2C12 cells as detected by immunofluorescence. Scale bar: 100μm.

[0016] Figure 5 The graph shows the effects of potent loquat extract on the motor function and muscle fiber structure of *C. elegans* in Example 4. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Figure 5 In the diagram, 'a' represents the experimental design and dosing regimen. Figure 5 In the figure, b is a statistical graph showing the effects of different treatments on body movement and pharyngeal pump in young and old nematodes; Figure 5 c in the image represents a representative image showing the fluorescence intensity of muscle fibers. Scale bar: 100 μm. Figure 5 In the image, d represents a representative fluorescence image of muscle fiber integrity. Scale bar: 5 μm. Detailed Implementation

[0017] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0018] 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 invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0019] This invention provides the application of a potent loquat syrup in the preparation of a drug for improving and / or treating muscle atrophy. The potent loquat syrup comprises loquat leaf, poppy husk, stemona root, cynanchum paniculatum, mulberry bark, platycodon grandiflorus, and menthol, in a mass ratio of loquat leaf:poppy husk:stemon root:cynanchum paniculatum:mulberry bark:platycodon grandiflorus:menthol = 69:50:15:9:6:6:0.15.

[0020] In some embodiments, muscle atrophy includes age-related muscle atrophy or inflammatory factor-induced muscle atrophy.

[0021] In some embodiments, the medicament may also include pharmaceutically acceptable excipients.

[0022] In some embodiments, the excipients are one or more of diluents, buffers, binders, flavoring agents, and fillers.

[0023] In some embodiments, the drug is formulated into a clinically acceptable dosage form.

[0024] In some embodiments, the dosage form of the drug is an oral liquid.

[0025] Experimental materials and equipment: 1. The reagents, consumables and their sources are shown in Table 1.

[0026] Table 1. Reagents, Consumables, and Their Sources

[0027] 2. Reagent formulation: (1) Nematode synchronization lysis buffer: 750 μL of 5 mol / L NaOH + 2 mL of [unclear text - likely a solution or solution] +1 mL of NaClO.

[0028] (2) NGM solid culture medium: Weigh 2g peptone, 16g agar powder, 2.4g NaCl, and 0.8mL of 5mg / mL cholesterol, then use... Bring the volume to 800 mL, autoclave, and then add 800 μL of 1 mol / L solution to the laminar flow hood. 800 μL of 1 mol / L Add 20 mL of 1 mol / L PK buffer, shake well, pour the culture plate, and after the bacterial culture plate solidifies, wrap it with plastic wrap and store it at 4℃ for later use.

[0029] (3) PK buffer: Weigh 59.5g of PK buffer solution. 14.09g Then use Bring the volume to 500 mL, autoclave, and store at room temperature.

[0030] (4) M9 buffer: Weigh 3g of 15.12g 5g of NaCl, then use Bring the volume to 1L, autoclave, and then add 1mL of 1mol / L solution to the clean bench. Store at room temperature for later use.

[0031] (5) 5mg / mL cholesterol: Weigh 5mg cholesterol and dissolve it in 1mL of anhydrous ethanol. Store at 4℃ for later use.

[0032] (6) LB liquid medium: Weigh 10g NaCl, 10g Tryptone, and 5g yeast extract, then use... Bring the volume to 1L, sterilize under high temperature and high pressure, and store at 4℃ for later use.

[0033] (7) 10×PBS buffer: Weigh 80g of NaCl, 2g of KCl, and 36.3g of... 2.4g Then use Bring the volume to 1L, autoclave, and store at room temperature.

[0034] (8) 1×PBST buffer: Measure 1L of 1×PBS buffer, add 1mL of Tween-20, shake well and store at room temperature for later use.

[0035] (9) Membrane regeneration solution: 15g glycine + 1g SDS; dissolve in deionized water to 1L, adjust pH to 2.2 and then add 10mL Tween-20.

[0036] (10) 10× Electrophoresis Buffer: Weigh 10g SDS, 144g glycine, and 30.2g Tris-base, then use... Bring the volume to 1L and store at room temperature.

[0037] (11) 10× transfer buffer: Weigh 144g glycine and 30.3g Tris-base, then use 800mL of... Dissolve and then use. Bring the volume to 1L and store at room temperature. When needed, follow the 10× transfer buffer formula. Dilute with anhydrous methanol at a ratio of 2:7:1 to prepare 1× transfer buffer.

[0038] (12) 30% Acrylamide / 0.8% Methylenebisacrylamide (30%AB): Weigh 145g of acrylamide and 5g of methylenebisacrylamide, then use... Make up to 500 mL, and after complete dissolution, filter through filter paper and store at 4°C protected from light.

[0039] (13) 4×Tris-HCl / SDS (pH=6.8): Weigh 30.25g of Tris-base, then use 300mL of... Dissolve the contents, adjust the pH to 6.8 with concentrated hydrochloric acid, then bring the volume to 500 mL, add 0.4 g of SDS, and store at room temperature.

[0040] (14) 4×Tris-HCl / SDS (pH=8.8): Weigh 91g of Tris-base, then use 300mL of... Dissolve the contents, adjust the pH to 8.8 with concentrated hydrochloric acid, then bring the volume to 500 mL, add 1 g of SDS, and store at room temperature.

[0041] (15) 10% ammonium persulfate (10% AP): Weigh 1g of ammonium persulfate, then use 10mL of... Dissolve, aliquot into 1.5mL centrifuge tubes, and store at -20℃.

[0042] (16) 6× protein denaturation loading buffer: Weigh 0.93g dithiothreitol (DTT), 1.2mg bromophenol blue, 3mL glycerol, 7mL 4×Tris-HCl / SDS, and finally weigh 1g SDS until fully dissolved. Aliquot into 1.5mL centrifuge tubes and store at -20℃ for later use.

[0043] (17) 5% skim milk powder blocking solution: Weigh 2.5g of skim milk and dissolve it in 50mL of 1×PBST buffer. Note that it should be prepared and used immediately.

[0044] (18) 5% BSA blocking solution: Weigh 2.5g of bovine serum albumin and dissolve it in 50mL of 1×PBST buffer. Note that it should be prepared and used immediately.

[0045] 3. The experimental equipment and their manufacturers are shown in Table 2.

[0046] Table 2 Experimental Equipment and Companies

[0047] Example 1: Non-targeted metabolomics identification of the chemical composition of a potent loquat extract Qiangli Pipa Syrup (PPS): A traditional Chinese medicine compound preparation, mainly composed of loquat leaves and other Chinese medicinal materials. Clinically, it is often used to relieve cough and respiratory inflammation-related symptoms, and has pharmacological effects such as antitussive, expectorant, and anti-inflammatory properties. The Qiangli Pipa Syrup in this embodiment includes loquat leaves, poppy husks, Stemona japonica, Cynanchum paniculatum, mulberry bark, Platycodon grandiflorus, and menthol. By weight ratio, loquat leaves: poppy husks: Stemona japonica: Cynanchum paniculatum: mulberry bark: Platycodon grandiflorus: menthol = 69:50:15:9:6:6:0.15, which is the prescription in the *Pharmacopoeia of the People's Republic of China*, specifically: 69g loquat leaves, 50g poppy husks, 15g Stemona japonica, 9g Cynanchum paniculatum, 6g mulberry bark, 6g Platycodon grandiflorus, and menthol. 0.15g of the above seven ingredients, except for menthol, the other six ingredients including loquat leaf are decocted twice with water, 2 hours each time. The decoctions are combined, filtered, and the filtrate is concentrated to an appropriate amount. 2.5g of sodium benzoate is added and stirred to dissolve. 600g of sucrose is added and heated to boiling. The mixture is kept at boiling point for 20 minutes, allowed to stand, filtered, and then an appropriate amount of citric acid, an appropriate amount of flavoring dissolved in ethanol, and menthol are added. The mixture is stirred, mixed well, allowed to stand, filtered, and water is added to 1000mL. The mixture is then mixed well to obtain the final product.

[0048] 1. Sample preparation: (1) Accurately measure 100 μL of Strong Loquat Syrup (National Drug Approval Number Z36021533, Quality Standard: Pharmacopoeia of the People's Republic of China 2025 Edition, Part I, Batch No. 2402035J) and place it in a 1.5 mL EP tube.

[0049] (2) Add 400 μL of pre-cooled 80% methanol aqueous solution (V / V, dissolved in mass spectrometry grade water), vortex for 5 minutes to ensure thorough mixing.

[0050] (3) Place the mixed solution in an ice bath and let it stand for 5 minutes, then centrifuge it at a relative centrifugal force of 15000g for 20 minutes in a 4°C low-temperature centrifuge.

[0051] (4) Carefully aspirate 200 μL of supernatant and transfer it to a new EP tube. Add an appropriate amount of mass spectrometry grade water to accurately dilute the final concentration of methanol to 53% (V / V).

[0052] (5) The diluted solution was centrifuged again at 4°C and 15000g for 20 minutes. After centrifugation, all supernatant was carefully collected and analyzed by liquid chromatography-mass spectrometry. Metabolite separation was performed using... System, mass spectrometry analysis in The procedure was performed on a mass spectrometer using an electrospray ionization (ESI) source.

[0053] 2. Compound identification results:

[0054] The chemical composition of the strong loquat syrup was identified by liquid chromatography-mass spectrometry analysis as follows: Figure 1As shown, the results revealed 3244 compound characteristics in positive ion (POS) mode and 1891 compound characteristics in negative ion (NEG) mode. Comparison with secondary mass spectra further confirmed several key active ingredients, including ursolic acid, oleanolic acid, papaverine, stemona alkaloid, morinone A, menthol, platycodon saponin D, and corosolic acid. This chemical characterization spectrum comprehensively reveals the complex composition of the potent loquat syrup.

[0055] Example 2: Determination of C2C12 cell proliferation and differentiation 1. Cell resuscitation: (1) Take out the C2C12 cell (mouse myoblast cell line) cryopreservation tube from the liquid nitrogen tank and immediately place it in a 37°C constant temperature water bath. Gently shake it to thaw it completely within 1 minute.

[0056] (2) Wipe the outer wall of the cryopreservation tube thoroughly with 75% ethanol. In a clean bench, transfer all the cell suspension to a 15mL centrifuge tube containing 5mL of pre-warmed complete culture medium (high glucose DMEM + 10% fetal bovine serum).

[0057] (3) Centrifuge at 1000 rpm for 5 minutes and carefully discard the supernatant. Add 1 mL of complete culture medium to the cell pellet, gently pipette to resuspend, transfer the cell suspension to a 10 cm cell culture dish, and add 9 mL of complete culture medium.

[0058] (4) Gently shake the culture flask in a cross shape to distribute the cells evenly, and place it in a 37°C, 5% carbon dioxide incubator for 24 hours. After that, discard the old culture medium and replace it with 10 mL of fresh complete culture medium and continue culturing.

[0059] 2. Cell passage: (1) Observe under an inverted microscope daily. When the cell density reaches 80%-90%, passage the cells.

[0060] (2) Discard the old culture medium, add 3 mL of pre-warmed PBS buffer, gently shake to wash the cell surface and then discard.

[0061] (3) Add 4 mL of trypsin solution, gently shake to cover the bottom of the bottle, and place in a 37°C incubator for 3 minutes to digest.

[0062] (4) Observe under a microscope. When the intercellular spaces increase and the shape becomes round, immediately add 1 mL of complete culture medium containing 10% FBS (fetal bovine serum) to stop digestion.

[0063] (5) Gently and repeatedly blow the bottom of the dish with a pipette until all cells are detached and a uniform cell suspension is formed.

[0064] (6) Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add an appropriate amount of complete culture medium to resuspend the cells.

[0065] (7) Subculture in a ratio of 1:2 to 1:4, add complete culture medium to a total volume of 10 mL, shake to mix well, and then put back into the incubator to continue culturing.

[0066] 3. Cell proliferation assay (MTS method): (1) After digesting C2C12 cells in the logarithmic growth phase with trypsin, the digestion was terminated with complete medium containing 10% FBS and the cells were resuspended. The cell density was adjusted to the target concentration using a hemocytometer. .

[0067] (2) Dispense the cell suspension at 100 μL per well (i.e., Precisely seed the cells into the central 60 wells of a 96-well cell culture plate, and add 100 μL of 10% FBS to the outer 36 wells to reduce edge effects.

[0068] (3) Place the inoculated culture plate in a constant temperature incubator at 37°C, 5% carbon dioxide and saturated humidity for 8 hours to allow the cells to adhere fully.

[0069] (4) Carefully aspirate the original culture medium in each well. Add fresh complete culture medium containing a series of concentration gradients of strong loquat syrup (0, 0.03, 0.3, 3.0, 30 μg / mL) to the experimental group respectively. Set up 6 technical replicate wells for each concentration. Add an equal volume of cell-free complete culture medium to the blank control group.

[0070] (5) Return the culture plate to the incubator and continue culturing for 24, 48, 72, 96 and 120 hours respectively. At each detection time point, add 100 μL of MTS detection reagent (DMEM:MTS=5:1) to each well.

[0071] (6) Wrap the culture plate with aluminum foil to protect it from light and continue incubation at 37°C for 1.5 hours.

[0072] (7) Use a multi-functional microplate reader to measure the absorbance of each well at a wavelength of 490 nm.

[0073] The absorbance test results are as follows Figure 2 As shown, the results indicated that there were no significant differences in the concentrations of Qiangli Pipa Lu (a type of loquat extract) treated with concentrations ranging from 0.03 to 30 μg / mL compared with the control group, indicating that Qiangli Pipa Lu has no obvious toxicity in the low concentration range. Similarly, within this concentration range, Qiangli Pipa Lu had no significant effect on cell proliferation.

[0074] 4. Myotube differentiation and drug intervention: (1) After digestion and resuspension, C2C12 cells in the logarithmic growth phase were seeded into six-well cell culture plates, and 2 mL of complete culture medium containing 10% FBS was added to each well.

[0075] (2) Place the culture plate in a 37°C, 5% carbon dioxide incubator and observe the cell growth status under an inverted microscope every 24 hours.

[0076] (3) When the cell density reaches 70-80%, discard the original complete culture medium and gently rinse the cells twice with pre-warmed PBS to completely remove serum residue.

[0077] (4) The experimental group was replaced with high-glucose DMEM differentiation medium containing 2% horse serum, and strong loquat syrup was added to the medium at final concentrations of 0.3 and 3 μg / mL respectively; the control group used an equal volume of differentiation medium without drugs (i.e., the final concentration of strong loquat syrup was 0); the volume of each well was 2 mL.

[0078] (5) Return the cells to the incubator and continue culturing, taking this day as day 0 of differentiation; from this point onward, regularly discard the old culture medium daily and replace it with the appropriate fresh differentiation medium containing or without the drug, continuing to induce differentiation for 6 days. The C2C12 cell culture process is as follows: Figure 3 As shown in 'a'.

[0079] (6) During differentiation, the morphology and length of myotubes were observed and recorded daily using an inverted microscope.

[0080] (7) After 6 days of differentiation, the culture medium was discarded and washed with PBS. Then, 200 μL of 3× protein loading buffer was added directly to each well. Cells were scraped with a cell scraper and the resulting suspension was transferred to a centrifuge tube. The protein was denatured by heating in a 95°C metal bath for 10 minutes. The resulting sample was stored at -80°C for Western blotting analysis.

[0081] 5. Western blot analysis of proteins: (1) Take the denatured protein sample prepared above out of the -80℃ ultra-low temperature freezer, thaw it naturally at room temperature and then centrifuge it briefly; place the sample in a metal bath and heat it again at 95℃ for 5 minutes, then immediately transfer it to ice to cool, and vortex mix it after a brief centrifugation for later use.

[0082] (2) Accurately pipette 10 μL of sample volume and slowly add it to the loading well of the SDS-PAGE gel, and add 10 μL of pre-stained protein molecular weight standard to the adjacent well as a reference.

[0083] (3) In 1×Tris-glycine electrophoresis buffer, electrophoresis was initially performed at a constant voltage of 80V. After the bromophenol blue indicator entered the separating gel, the voltage was increased to 120V and electrophoresis was continued until the bromophenol blue front reached the bottom edge of the gel to terminate the electrophoresis.

[0084] (4) After electrophoresis, carefully remove the gel and equilibrate it with the PVDF membrane that has been activated with methanol for 30 seconds in pre-cooled 1× transfer buffer for 15 minutes.

[0085] (5) Use wet transfer method, assemble the transfer clamp in the order of cathode-filter paper-gel-PVDF membrane-filter paper-anode to ensure that there are no air bubbles between each layer; place the transfer device in an ice bath environment and transfer at a constant voltage of 100V for 60 minutes.

[0086] (6) After the transfer is completed, take out the PVDF membrane and place it in PBST blocking solution containing 5% skim milk. Block it at room temperature for 2 hours on a horizontal shaker.

[0087] (7) After blocking, rinse the membrane three times with PBST solution for 5 minutes each time. Then transfer the membrane into the corresponding primary antibody working solution [anti-MyHC antibody (MF20, 1:100), anti-MyoD antibody (1:500), anti-Myogenin antibody (1:500), anti-GAPDH antibody (1:20000)] and incubate slowly on a shaker overnight (12-14 hours) at 4°C.

[0088] (8) The primary antibody was recovered the next day, and the membrane was washed three times with PBST solution for 10 minutes each time. Then the membrane was incubated with the corresponding HRP-labeled secondary antibody (1:5000) on a shaker at room temperature for 1 hour.

[0089] (9) After the secondary antibody incubation, wash the membrane three times with PBST solution for 10 minutes each time. Mix ECL chemiluminescence reagent A and B in a 1:1 ratio, and then add the mixture evenly to the protein surface of the PVDF membrane. Acquire the signal in the chemiluminescence imaging system.

[0090] (10) The gray values ​​of the target protein band and the internal control GAPDH band were quantitatively analyzed using ImageJ image analysis software. The ratio of the gray values ​​of the target band to the internal control band was used as the relative expression level of the protein. All experiments were independently repeated 3 times.

[0091] Results of Western blot analysis of proteins as follows Figure 3 As shown, the results indicated that, compared with the control group, the protein expression levels of myosin heavy chain (MyHC), myogenic determinant (MyoD), and myogenin were significantly upregulated after intervention with Qiangli Pipa Lu (a traditional Chinese medicine formula). This confirms that Qiangli Pipa Lu can effectively promote the myogenic differentiation process of C2C12 cells.

[0092] 6. Immunofluorescence staining: (1) Cell fixation and permeabilization: Discard the culture medium and rinse the cells three times with pre-cooled PBS; add 4% paraformaldehyde (PFA) and fix at room temperature for 15 minutes; discard the PFA and wash three times with PBS (5 minutes each time); add 0.02% Triton X-100 PBS solution and permeabilize at room temperature for 15 minutes, then wash with PBS for 10 minutes.

[0093] (2) Blocking and primary antibody incubation: Discard PBS, add 0.5% BSA PBS blocking solution and block at room temperature for 2 hours; discard blocking solution, add MYH2 primary antibody working solution diluted with PBS at 1:300, and incubate overnight in a humidified chamber at 4°C.

[0094] (3) Secondary antibody incubation and nuclear staining: The primary antibody was recovered and washed three times with PBST (5 minutes each time); FITC-labeled secondary antibody diluted with PBST at 1:300 was added and incubated at room temperature in the dark for 2 hours; the secondary antibody was discarded and washed three times with PBST; DAPI staining solution diluted with PBS at 1:5000 was added and incubated at room temperature in the dark for 10 minutes.

[0095] (4) Image acquisition: After washing three times with PBS, 500 μL of PBS was added to each well to cover the cells. Using a high-content imaging system, MYH2 (green fluorescence) and DAPI (blue fluorescence) images were acquired under a 20x objective lens.

[0096] Immunofluorescence staining results as follows Figure 3 As shown in Figure e, the results indicate that the diameter of the myotubes in the group treated with the strong loquat syrup was significantly increased compared to the control group. Quantitative analysis showed that the average myotube size increased in a concentration-dependent manner after administration of the strong loquat syrup, increasing from 23.56±1.063 μm in the control group to 25.82±1.096 μm and 27.17±1.445 μm, respectively.

[0097] The results of immunofluorescence staining and Western blot analysis showed that MyHC protein expression was upregulated, indicating that Qiangli Pipa Lu can effectively promote myogenic differentiation and myotube formation of C2C12 cells.

[0098] Example 3: Establishment of a TNF-α-induced myotube atrophy model and determination of drug effects 1. Construction of a myotube atrophy model and drug intervention: (1) The mature C2C12 myotubes differentiated in Example 2 were randomly divided into a model group and a drug administration group.

[0099] (2) The model group was replaced with fresh differentiation medium containing 20 ng / mL TNF-α; the drug treatment group was replaced with fresh differentiation medium containing 20 ng / mL TNF-α and different concentrations of strong loquat syrup (0.3, 3 μg / mL); a blank control group (without TNF-α and drugs) was set up.

[0100] (3) Treat each group of cells for 48 hours, keeping the culture conditions constant during the period.

[0101] 2. Protein expression analysis: Following the Western blot analysis method described in Example 2, the protein expression level of myosin heavy chain (MyHC) in each group of cells was extracted and analyzed.

[0102] Results of Western blot analysis of proteins as follows Figure 4 As shown in b, the results indicated that compared with the blank control group, the expression level of myosin heavy chain (MyHC) protein in the TNF-α model group was significantly reduced (p<0.05), indicating that the myotube atrophy model was successfully established. Compared with the model group, the expression of MyHC protein was upregulated in all concentrations of the strong loquat extract treatment group. Among them, the intervention effect was significant when the concentration of strong loquat extract was 3 μg / mL (p<0.05), indicating that strong loquat extract can effectively antagonize the degradation of TNF-α-induced myotube atrophy-related proteins.

[0103] 3. Immunofluorescence staining analysis: Following the immunofluorescence staining method in Example 2, cells in each group were treated and MYH2 staining was performed. Subsequently, myotube morphology was observed and myotube diameter was statistically analyzed.

[0104] Immunofluorescence staining results as follows Figure 4 As shown in c, the results showed that TNF-α treatment significantly reduced the myotube diameter to 23.46±3.620 μm, while the myotube diameter in the treatment groups (treated with different concentrations of strong loquat syrup) increased to 25.59±4.014 μm and 26.80±2.790 μm, respectively. This indicates that strong loquat syrup effectively prevented TNF-α-induced myotube morphological atrophy.

[0105] In summary, the results of Western blot analysis and immunofluorescence staining, from both the protein expression and cell morphology perspectives, jointly confirmed that Qiangli Pipa Lu (a traditional Chinese medicine) has a clear ameliorative effect on TNF-α-induced myotube atrophy.

[0106] Example 4: A method for evaluating motor function based on the Caenorhabditis elegans model 1. Nematode culture: (1) Under aseptic conditions, spread 100 μL of Escherichia coli OP50 bacterial solution evenly in the center of a 60 mm nematode growth medium (NGM) plate to form an inoculation area with a diameter of about 2 cm.

[0107] (2) After the culture plate is coated with bacteria, it is dried in a clean bench for 15 minutes, and then transferred to a 37℃ constant temperature incubator and inverted for 12-16 hours.

[0108] (3) Place the NGM plates that have completed bacterial culture in a 20℃ constant temperature incubator for 2 hours in advance for equilibration.

[0109] (4) Use platinum wire to pick out healthy nematodes in the growth period and transfer them to the edge of the pretreated NGM plate OP50 mycelium.

[0110] (5) Place the culture plate inoculated with nematodes in a 20℃ constant temperature incubator and culture it in the dark. Replace the food source with fresh OP50 every 2-3 days until the nematodes enter the oviposition period.

[0111] 2. Nematode synchronization treatment: (1) Use M9 buffer to rinse the oviposition adults from the surface of the NGM culture plate into a 15 mL sterile centrifuge tube.

[0112] (2) Centrifuge the centrifuge tube at 1000 rpm for 1 minute, carefully discard the supernatant, and retain the nematode precipitate.

[0113] (3) Repeat the above washing steps three times to ensure that the bacteria residue attached to the nematode body surface is completely removed.

[0114] (4) Add 1 mL of freshly prepared lysis buffer to the precipitate and immediately place it on a vortex shaker and shake vigorously for about 3 minutes.

[0115] (5) After observing under a stereomicroscope to confirm that most of the adult insect body walls have ruptured and the eggs have been fully released, add 10 mL of M9 buffer to terminate the lysis reaction.

[0116] (6) Centrifuge the centrifuge tube at 3500 rpm for 1.5 minutes, discard the supernatant, and retain the larval precipitate at the bottom.

[0117] (7) Resuspend the precipitate with 5 mL of M9 buffer, centrifuge and discard the supernatant, and repeat the washing three times.

[0118] (8) Finally, resuspend the eggs in 2 mL of M9 buffer and place the centrifuge tube in a 20°C constant temperature incubator for 16-20 hours.

[0119] (9) After hatching, the hatching status of L1 stage larvae was confirmed under a stereomicroscope, and synchronized larvae were collected for subsequent experiments.

[0120] 3. Drug treatment: (1) The nematodes were cultured in a standardized environment at a constant temperature of 20°C throughout their entire life cycle. The young adult Caenorhabditis elegans, which had been cultured in a synchronized environment for 5 days, was transferred to a fresh NGM plate without drugs using platinum wire. The first motor function test was performed under a stereomicroscope, and the frequency of body bending and the number of pharyngeal pump movements were recorded as data for the young control group.

[0121] (2) Continue culturing until day 10, then randomly group the nematodes and transfer them to NGM plates containing different concentrations (0, 0.3, 0.6 mg / mL) of potent loquat extract using platinum wire for drug intervention. Each plate contains 30 nematodes, and each concentration has 3 biological replicates.

[0122] (3) After 48 hours of continuous intervention, the nematode's motor function was measured on the twelfth day.

[0123] 4. Motor function testing: (1) Transfer the nematodes to fresh NGM plates that do not contain OP50 bacterial growth and wash off any residual bacteria on the body surface with M9 buffer; (2) Body swaying measurement: Add 1 drop of M9 buffer to the center of the measurement plate, transfer the nematode into the droplet, observe and record the number of body flexions within 30 seconds under a stereomicroscope. Body flexion is defined as a complete sinusoidal motion along the long axis of the body. Body swing measurement, such as Figure 5 As shown, the results indicated that on the fifth day, the average body swing rate of young *C. elegans* was 51 times / min. Compared with when they were young, the musculoskeletal function of older nematodes was significantly reduced, with an average body swing rate of 21 times / min. However, the intervention of strong loquat extract significantly improved the age-dependent decline in musculoskeletal function, increasing it to 24 times / min and 26 times / min, respectively.

[0124] (3) Measurement of pharyngeal pump frequency: The same nematode was transferred to the edge of an NGM plate coated with OP50 bacterial motility. The pharynx was focused under a stereomicroscope, and the number of pharyngeal pump contractions within 30 seconds was observed and recorded. Pharyngeal pump contraction was defined as one complete relaxation-contraction cycle of the pharynx.

[0125] Pharyngeal pump frequency measurement, such as Figure 5 As shown, the results indicated that the average pharyngeal pump rate of young *C. elegans* (day 5) reached 95 times / minute; while the motor function of older nematodes showed a significant decline, with the average pharyngeal pump rate decreasing to 28 times / minute. However, after intervention with potent loquat extract, the age-related decline in motor function of nematodes was significantly improved, with the average pharyngeal pump rate in the intervention group increasing to 38 times / minute and 39 times / minute, respectively.

[0126] 5. Observation of muscle fiber structure: (1) The RW1596 transgenic strain (myo-3p::GFP) was treated with the same drug intervention protocol as the N2 wild-type nematodes. Nematodes were collected on the 5th day (young adult stage) and the 12th day (senescent stage), washed with M9 buffer, and anesthetized with 10 mM imidazole solution; (2) The anesthetized nematodes were transferred onto a glass slide with a 2% agarose pad, and GFP fluorescence signal images of their body wall muscles were observed and collected using a Zeiss upright fluorescence microscope (LSM800); (3) The collected images were quantitatively analyzed for fluorescence intensity using Image J software to measure the average fluorescence intensity in the myofiber region, evaluate the myosin expression level; and observe the continuity and morphological structure of the myofibril arrangement.

[0127] The results of the myofiber structure observation are as Figure 5 shown. The results showed that Qiangli pipa lu could improve the disorder of the myofiber structure in old nematodes, and at the same time increase the total level of myo3 protein in myofibers. And consistent with the previous results, Qiangli pipa lu at a concentration of 0.3 mg / mL showed the best intervention effect. This indicates that Qiangli pipa lu can relieve the muscle function decline in old nematodes in a dose-dependent manner.

[0128] According to Examples 1 - 4, the conclusion is drawn that Qiangli pipa lu can enhance motor function, improve the integrity of myofiber structure, promote myotube differentiation and inhibit inflammation-induced muscle atrophy. In the C2C12 myoblast model, treatment with Qiangli pipa lu can promote the expression of myosin heavy chain (MyHC) in a concentration-dependent manner, increase the myotube diameter, and specifically activate the MyoD-myogenin signaling axis; in the TNF-α-induced muscle atrophy model, Qiangli pipa lu effectively inhibits the degradation of MyHC protein and maintains the integrity of the myotube structure. In the Caenorhabditis elegans model, Qiangli pipa lu significantly improves the body swing frequency and pharyngeal pumping motor function of old nematodes, alleviates age-related myofiber structure disorder, and upregulates the expression of muscle-related genes. Qiangli pipa lu can provide a new precursor molecule for the development of muscle atrophy treatment drugs based on natural products by consistently showing anti-muscle atrophy activity in multi-species models.

[0129] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. The use of potent loquat syrup in the preparation of drugs for improving and / or treating muscle atrophy, characterized in that, Strong loquat syrup contains loquat leaves, poppy husks, stemona root, cynanchum paniculatum, mulberry bark, platycodon grandiflorum, and menthol. By mass ratio, loquat leaves: poppy husks: stemona root: cynanchum paniculatum: mulberry bark: platycodon grandiflorum: menthol = 69:50:15:9:6:6:0.

15.

2. The application as described in claim 1, characterized in that, The muscle atrophy includes age-related muscle atrophy or inflammatory factor-induced muscle atrophy.

3. The application as described in claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application as described in claim 3, characterized in that, The excipients are one or more of the following: diluents, buffers, adhesives, flavoring agents, and fillers.

5. The application as described in claim 4, characterized in that, The drug is formulated into a clinically acceptable dosage form.

6. The application as described in claim 5, characterized in that, The drug is in the form of an oral liquid.

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