Traditional Chinese medicine composition for enhancing functions of mature skeletal muscle cells and application

Abstract

The invention discloses a traditional Chinese medicine composition for enhancing functions of mature skeletal muscle cells and application of the traditional Chinese medicine composition. The composition is prepared from ginseng, astragalus membranaceus, pericarpium citri reticulatae, dogwood and rhizoma polygonati according to a specific ratio. The invention also provides an establishment method based on the quality standard of various active components (ginsenoside Rg1, hesperidin and the like) in the composition. The traditional Chinese medicine composition disclosed by the invention does not have a remarkable activity promoting effect on undifferentiated C2C12 myoblasts in a proliferation period, but can specifically enhance the cell activity of differentiated mature C2C12 myotubular cells. The action characteristics of the cell state dependence show that the composition does not simply stimulate cell proliferation, but is targeted to improve the functional state of mature muscle cells, so that a high-precision solution is provided for treating diseases (such as sarcopenia) characterized by mature muscle fiber function decline.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a traditional Chinese medicine composition for enhancing the function of mature skeletal muscle cells and its application; in particular, it relates to a new use of a traditional Chinese medicine composition in the preparation of products for specifically enhancing the function of differentiated mature skeletal muscle cells (myotubes), which is particularly suitable for improving diseases related to muscle function decline. Background Technology

[0002] Skeletal muscle is a crucial component of the human musculoskeletal system, and its functional state directly impacts physical ability and quality of life. With age or due to certain medical conditions, a decline in skeletal muscle mass and function can occur, a condition known as sarcopenia, leading to weakness, increased risk of falls, and a decreased quality of life. Currently, Western medical treatments for sarcopenia are limited, primarily focusing on nutritional support and exercise rehabilitation, with a lack of specific medications.

[0003] Traditional Chinese medicine theory holds that "the spleen governs the muscles and limbs" and "the kidneys are the organ of strength, from which skill arises," indicating that muscle strength is closely related to the functions of the spleen and kidneys. Spleen deficiency leads to insufficient production of qi and blood, resulting in malnourishment of the muscles; kidney deficiency leads to insufficient essence and marrow, resulting in weak bones and muscles. Therefore, tonifying qi, strengthening the spleen, and nourishing the kidneys are fundamental principles for treating muscle weakness. Currently, some research attempts to find substances that can promote muscle cell growth. However, many substances (such as certain growth factors) may pose potential risks while promoting myoblast proliferation, such as interfering with normal differentiation processes or causing uncontrollable stimulation of non-target cells. Ideally, drugs for treating muscle dysfunction (such as sarcopenia) should not simply and indiscriminately promote the proliferation of all muscle precursor cells, but should precisely improve the survival rate and functional state of differentiated, mature muscle fibers that perform contractile functions. In the current technology, drugs or natural product compositions that can clearly distinguish between undifferentiated and differentiated cells and specifically enhance the function of the latter are extremely rare. Summary of the Invention

[0004] The purpose of this invention is to provide a traditional Chinese medicine composition and its application for enhancing the function of mature skeletal muscle cells; the composition can specifically enhance the activity of differentiated mature skeletal muscle cells, while having little or no effect on undifferentiated myoblasts or even inhibiting them, thereby improving muscle function more safely and precisely.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a traditional Chinese medicine composition for enhancing the function of mature skeletal muscle cells, wherein the raw materials of the traditional Chinese medicine composition are ginseng, astragalus, tangerine peel, cornus officinalis, and polygonatum; the mass ratio of ginseng, astragalus, tangerine peel, cornus officinalis, and polygonatum is 4:2.5-3.5:2.5-3.5:2.5-3.5:2.5-3.5. Preferably, it is 4:3:3:3:3.

[0006] This traditional Chinese medicine composition has no significant promoting effect on the activity of undifferentiated skeletal muscle myoblasts or even has an inhibitory effect. Specifically, it has no significant promoting effect on the activity of undifferentiated C2C12 myoblasts in the proliferative phase, but it can specifically enhance the cell activity of differentiated mature C2C12 myotube cells. This cell state-dependent characteristic of action indicates that the composition does not simply stimulate cell proliferation, but rather targets the improvement of the functional state of mature muscle cells. This provides a highly precise solution for the treatment of diseases characterized by the decline of mature muscle fiber function, such as sarcopenia.

[0007] In one embodiment, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 12 parts ginseng, 8-10 parts astragalus, 8-10 parts dried tangerine peel, 8-10 parts cornus officinalis, and 8-10 parts polygonatum sibiricum. In some embodiments, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 12 parts ginseng, 9 parts astragalus, 9 parts dried tangerine peel, 9 parts cornus officinalis, and 9 parts polygonatum sibiricum.

[0008] As one implementation scheme, the traditional Chinese medicine composition is prepared by the following method: Ginseng, Astragalus, Tangerine Peel, Cornus officinalis, and Polygonatum sibiricum are weighed and soaked in water for 0.5-1.5 hours. Then, 9-11 times the amount of water is added, and the mixture is decocted for 0.4-0.6 hours and filtered. Then, 7-9 times the amount of water is added again, and the mixture is decocted for 0.4-0.6 hours and filtered. The two filtrates are combined, and the combined filtrate is collected. The filtrate is concentrated to a volume of 1 / 3 to 1 / 2 of the original combined volume of the medicinal liquid to obtain a concentrated solution. The concentrated solution is then purged with nitrogen until a solid product is obtained. In some implementation examples, 12, 9, 9, 9 g each of ginseng, astragalus, tangerine peel, cornus officinalis, and polygonatum were accurately weighed. The herbs were soaked in water for 1 hour, then 10 times the amount of water was added, and the mixture was decocted for 0.5 hours. The mixture was filtered, and then 8 times the amount of water was added again and decocted for 0.5 hours. The mixture was then filtered again. The two filtrates were combined and concentrated to obtain the Qi-tonifying, spleen-strengthening, and kidney-nourishing herbal composition.

[0009] As one implementation, the traditional Chinese medicine composition is prepared into tablets, pills, powders, hard capsules, soft capsules, granules, or oral liquids.

[0010] Secondly, the present invention provides the use of the aforementioned traditional Chinese medicine composition in the preparation of formulations for the treatment or prevention of diseases characterized by decreased or atrophied function of mature muscle fibers. Based on the fact that the compound / composition can specifically enhance the activity of differentiated myotube cells, it indicates that it has the potential to promote muscle protein synthesis and / or inhibit muscle degradation, and therefore can be used to prepare formulations for the prevention or treatment of diseases characterized by decreased or atrophied function of mature muscle fibers, such as sarcopenia. The traditional Chinese medicine composition, in a concentration range of 100 μg / mL to 1000 μg / mL, enhances the activity of differentiated skeletal muscle cells more effectively than undifferentiated skeletal muscle myoblasts. The differentiated skeletal muscle cells are myotube cells formed through induced differentiation. The traditional Chinese medicine composition of the present invention exhibits the most significant enhancing effect on the activity of differentiated skeletal muscle cells at a concentration of approximately 300 μg / mL.

[0011] Thirdly, the present invention also provides a drug, functional food, or health food for enhancing the activity of differentiated skeletal muscle cells, wherein the active ingredient is a traditional Chinese medicine composition in the aforementioned weight proportions. Specifically, it is a traditional Chinese medicine composition prepared from 12 parts ginseng, 8-10 parts astragalus, 8-10 parts dried tangerine peel, 8-10 parts cornus officinalis, and 8-10 parts polygonatum sibiricum. This drug, functional food, or health food is used to specifically enhance the activity of differentiated skeletal muscle cells and is suitable for treating sarcopenia.

[0012] Fourthly, the present invention provides a method for establishing the quality standard of the aforementioned traditional Chinese medicine composition, the method comprising the following steps: S1. Ginsenoside Rg1, hesperidin, loganin, rutin, and verbascoside were used as standard references. The concentrations of the series of standard reference solutions were determined by high performance liquid chromatography (HPLC). The peak area of ​​each standard reference was measured. The linear regression equation was calculated by plotting the peak area of ​​each standard reference as the ordinate and the injection mass of each standard reference as the abscissa. S2. The test solution of the traditional Chinese medicine composition is subjected to high performance liquid chromatography (HPLC) in the same step as S1 to determine the peak areas of ginsenoside Rg1, hesperidin, loganin, rutin, and verbascoside in the test solution. Based on the linear regression equation of the standard references of ginsenoside Rg1, hesperidin, loganin, rutin, and verbascoside, the mass of ginsenoside Rg1, hesperidin, loganin, rutin, and verbascoside in the test solution is calculated. The content of ginsenoside Rg1, hesperidin, loganin, rutin, and verbascoside in the test solution is then calculated.

[0013] As one implementation plan, the linear regression equations for each standard reference are as follows: The standard curve equation for ginsenoside Rg1 is: y = 678.05x + 14.623, with a correlation coefficient R. 2 =0.9975; The standard curve equation for hesperidin is: y = 1971.3x + 3.699, with a correlation coefficient R0. 2 =0.9988; The standard curve equation for loganin is: y = 1073.9x + 5.9191, with a correlation coefficient R0. 2 =0.999; The standard curve equation for rutin is: y = 1898x + 10.345, and the correlation coefficient R0 is... 2 =0.9996; The standard curve equation for hesperidin is: y = 3809.5x + 4.3465, with a correlation coefficient R0. 2 =0.9993.

[0014] As one implementation scheme, the high-performance liquid chromatography (HPLC) conditions are as follows: Chromatographic conditions for ginsenoside Rg1: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: V (acetonitrile):V (0.1% phosphoric acid) = 15:85; flow rate: 1.0 mL / min; UV detection wavelength: 203 nm; injection volume: 10 μL; column temperature: 30℃. Chromatographic conditions for hesperidin: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: V(acetonitrile):V(water) = 22:78; flow rate: 1.0 mL / min; UV detection wavelength: 283 nm; injection volume: 10 μL; column temperature: 30℃. Chromatographic conditions for loganin: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: V (acetonitrile):V (0.3% phosphoric acid water) = 12:88; flow rate: 1.0 mL / min; UV detection wavelength: 240 nm; injection volume: 10 μL; column temperature: 35℃. Chromatographic conditions for rutin: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: V (acetonitrile):V (0.4% phosphoric acid water (adjusted to pH 3.0 with triethylamine)) = 4:6; flow rate: 1.0 mL / min; UV detection wavelength: 254 nm; injection volume: 10 μL; column temperature: 30℃; Chromatographic conditions for verbascoside isoflavone glucoside: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase acetonitrile (A)-0.2% formic acid water (B), gradient elution, 0–20 min, 20% A–40% A; 20–30 min, 40% A; flow rate: 1.0 mL / min; UV detection wavelength: 240 nm; injection volume: 10 μL; column temperature: 35℃.

[0015] As one implementation scheme, the quality control standard is as follows: the content of ginsenoside Rg1 in the test solution of the traditional Chinese medicine composition is 0.328±0.053 mg / g, the content of hesperidin is 1.713±0.056 mg / g, the content of loganin is 2.458±0.091 mg / g, the content of rutin is 0.0415 mg / g (n=2, relative deviation≈1.1%), and the content of verbascoside isoflavone glucoside is 0.0583±0.0027 mg / g.

[0016] As one implementation plan, the preparation of each standard reference solution is as follows: take the standard reference powder, add chromatographic methanol to dissolve it, and prepare the standard reference solution.

[0017] As one implementation plan, the preparation of each standard reference solution is as follows: Accurately weigh ginsenoside Rg1 reference standard, add chromatographic methanol, and prepare solutions containing 80 μg, 64 μg, 40 μg, 20 μg, and 10 μg of ginsenoside Rg1 per 1 mL; Accurately weigh the hesperidin reference standard, add chromatographic methanol, and prepare solutions containing 0.76 μg, 1.53 μg, 3.05 μg, 6.11 μg, 12.21 μg, and 24.42 μg of hesperidin per mL; Accurately weigh loganin reference standard, add chromatographic methanol, and prepare solutions containing 1.95 μg, 3.90 μg, 7.81 μg, 19.52 μg, and 39.03 μg of loganin per mL; Accurately weigh rutin reference standard, add chromatographic methanol, and prepare solutions containing 0.76 μg, 1.53 μg, 3.05 μg, 6.11 μg, and 12.21 μg loganin per 1 mL; accurately weigh verbenafil isoflavone glucoside reference standard, add chromatographic methanol, and prepare solutions containing 0.56 μg, 1.12 μg, 2.23 μg, 4.46 μg, and 8.93 μg verbenafil isoflavone glucoside per 1 mL.

[0018] As one implementation scheme, the test solution is prepared as follows: accurately weigh 0.1 g of the traditional Chinese medicine composition, dilute with 0.5 mL of 70% methanol, sonicate for 30 minutes, accurately measure 0.25 mL, dilute with 70% methanol 20 times, shake well, filter, take the filtrate, and filter with a 0.45 μm microporous membrane to obtain the test solution.

[0019] The present invention has the following beneficial effects: 1) This invention uses ginseng and astragalus as the principal herbs to replenish vital energy, strengthen the spleen and benefit the lungs; polygonatum and cornus officinalis as the assistant herbs to nourish yin and tonify the kidneys, replenish essence and generate marrow; and tangerine peel is added to regulate qi and strengthen the spleen, preventing the tonifying effect from becoming cloying. The whole formula works together to replenish qi, strengthen the spleen and tonify the kidneys, addressing the root cause of muscle weakness caused by "deficiency of both spleen and kidney".

[0020] 2) This invention uses modern detection techniques (such as HPLC) to determine the content of various active ingredients in the formula, including ginsenoside Rg1, hesperidin, loganin, rutin, and verbascoside, and establishes reliable quality standards to ensure the stability and consistency of the product.

[0021] 3) This invention demonstrates the effective effect of the traditional Chinese medicine composition in improving skeletal muscle function through in vitro cell experiments (C2C12 mouse skeletal muscle myoblast model). Attached Figure Description

[0022] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 Chromatogram of ginsenoside Rg1 reference standard; Figure 2 Chromatogram of hesperidin reference standard; Figure 3 Chromatogram of strychnine reference standard; Figure 4 Chromatogram of rutin reference standard; Figure 5 Chromatogram of verbascoside isoflavone glucoside reference standard; Figure 6 The effect of the Qi-tonifying, spleen-strengthening, and kidney-tonifying formula in Example 1 on the activity of undifferentiated and differentiated C2C12 cells; Figure 7 The effect of the Qi-tonifying, spleen-strengthening, and kidney-tonifying formula in Comparative Example 1 on the activity of undifferentiated and differentiated C2C12 cells. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments should be understood as illustrative only and not as limiting the scope of protection of the present invention. After reading the description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0024] Medicinal materials, main reagents and experimental instruments (1) Medicinal materials Ginseng, astragalus, tangerine peel, cornus officinalis, and polygonatum were all purchased from Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine; all the above medicinal materials were identified as conforming to the relevant provisions of the 2020 edition of the Pharmacopoeia of the People's Republic of China. Ginsenoside Rg1, loganin, rutin, and verbascoside glucose reference standards were purchased from Chengdu Mansite Biotechnology Co., Ltd., and hesperidin reference standard was purchased from Shanghai Shidander Biotechnology Co., Ltd.

[0025] (2) Main reagents Acetonitrile (analytical grade) (SIGMA), methanol (analytical grade) (MACKLIN), phosphoric acid (MAKLIN).

[0026] (3) Experimental instruments Induction cooker, Agilent Infinity 2 high-performance liquid chromatograph (HPLC), Agilent Poroshell 120 EC-C18 HPLC with 4.6 mm × 150 mm column, ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.), rotary evaporator (Shanghai Xiande Experimental Instrument Co., Ltd.), electronic analytical balance (Sartorius Scientific Instruments).

[0027] Example 1 This embodiment relates to a traditional Chinese medicine composition (Qi-tonifying, spleen-strengthening, and kidney-tonifying formula) for enhancing the function of mature skeletal muscle cells and its preparation: The traditional Chinese medicine composition of this embodiment uses 12 g of ginseng, 9 g of astragalus, 9 g of dried tangerine peel, 9 g of cornus officinalis, and 9 g of polygonatum as raw materials. These are soaked in water for 1 hour, then decocted for 0.5 hours with 10 times the amount of water, filtered, and then decocted again with 8 times the amount of water for 0.5 hours, filtered again. The two filtrates are combined and concentrated to obtain the qi-tonifying, spleen-strengthening, and kidney-tonifying traditional Chinese medicine composition.

[0028] Example 2 This embodiment relates to the determination of ginsenoside Rg1 content in a traditional Chinese medicine composition (Yiqi Jianpi Bushen Fang) used to enhance the function of mature skeletal muscle cells.

[0029] Chromatographic conditions: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: V (acetonitrile): V (0.1% phosphoric acid) = 15:85; flow rate: 1.0 mL / min; UV detection wavelength: 203 nm; injection volume: 10 μL; column temperature: 30℃.

[0030] Preparation of reference solutions: Accurately weigh ginsenoside Rg1 reference standard, add chromatographic methanol, and prepare solutions containing 80 μg, 64 μg, 40 μg, 20 μg, and 10 μg of ginsenoside Rg1 per mL.

[0031] Preparation of the test solution: Weigh 0.1 g of the traditional Chinese medicine composition of the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula in Example 1, dilute with 0.5 mL of 70% methanol, sonicate for 30 minutes, accurately measure 0.25 mL, dilute with 70% methanol 4 times, shake well, filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0032] Linearity assessment: A series of reference solutions were determined under the chromatographic conditions for ginsenoside Rg1. The chromatograms of the reference solutions are shown below. Figure 1 Peak areas were recorded, and a plot was constructed using ginsenoside Rg1 concentration and peak area. 10 μL of each peak area was injected. The results showed that the injection amount of ginsenoside Rg1 in the range of 0.1–0.8 μg exhibited a good linear relationship with the peak area. Linear regression was performed with the concentration of the standard (x, μg) as the abscissa and the corresponding peak area (y) as the ordinate, yielding the following standard curve regression equation: The coefficient of determination (R²) for y = 678.05x + 14.623 is... 2 The value is 0.9975.

[0033] Test sample determination: The test sample solution was determined according to the chromatographic conditions of ginsenoside Rg1, the peak area was recorded, and 10 μL was injected. The results showed that the total content of ginsenoside Rg1 in the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula was 0.328±0.053 mg / g.

[0034] Example 3 This embodiment relates to the determination of hesperidin content in a traditional Chinese medicine composition (Yiqi Jianpi Bushen Fang) used to enhance the function of mature skeletal muscle cells: Chromatographic conditions: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: V(acetonitrile):V(water) = 22:78; flow rate: 1.0 mL / min; UV detection wavelength: 283 nm; injection volume: 10 μL; column temperature: 30℃.

[0035] Preparation of reference solutions: Accurately weigh hesperidin reference standard, add chromatographic methanol, and prepare solutions containing 0.76 μg, 1.53 μg, 3.05 μg, 6.11 μg, 12.21 μg, and 24.42 μg of hesperidin per mL.

[0036] Preparation of the test solution: Weigh 0.1 g of the traditional Chinese medicine composition of the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula in Example 1, dilute with 0.5 mL of 70% methanol, sonicate for 30 minutes, accurately measure 0.25 mL, dilute with 70% methanol 20 times, shake well, filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0037] Linearity assessment: A series of reference solutions were determined according to the chromatographic conditions for hesperidin. The chromatograms of the reference solutions are as follows: Figure 2 Peak areas were recorded, and a plot was constructed using hesperidin concentration and peak area. 10 μL of each sample was injected. The results showed that the injection amount of hesperidin showed a good linear relationship with the peak area within the range of 0.007–0.244 μg. Linear regression was performed with the concentration of the standard (x, μg) as the abscissa and the corresponding peak area (y) as the ordinate, yielding the following standard curve regression equation: The coefficient of determination (R²) for y = 1971.3x + 3.699 is... 2 The value is 0.9988.

[0038] Test sample determination: The test sample solution was determined according to the chromatographic conditions of hesperidin, the peak area was recorded, and 10 μL was injected. The results showed that the total content of hesperidin in the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula was 1.713±0.056 mg / g.

[0039] Example 4 This embodiment relates to the determination of loganin content in a traditional Chinese medicine composition (Yiqi Jianpi Bushen Fang) used to enhance the function of mature skeletal muscle cells: Chromatographic conditions: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: V (acetonitrile):V (0.3% phosphoric acid water) = 12:88; flow rate: 1.0 mL / min; UV detection wavelength: 240 nm; injection volume: 10 μL; column temperature: 35℃.

[0040] Preparation of reference solutions: Accurately weigh loganin reference standard, add chromatographic methanol, and prepare solutions containing 1.95 μg, 3.90 μg, 7.81 μg, 19.52 μg, and 39.03 μg of loganin per mL.

[0041] Preparation of the test solution: Weigh 0.1 g of the traditional Chinese medicine composition of the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula in Example 1, dilute with 0.5 mL of 70% methanol, sonicate for 30 minutes, accurately measure 0.25 mL, dilute with 70% methanol 20 times, shake well, filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0042] Linearity investigation: A series of reference solutions were determined according to the chromatographic conditions for loganin. The chromatograms of the reference solutions are as follows: Figure 3 Peak areas were recorded, and a plot was constructed using loganin concentration and peak area. 10 μL of each peak area was injected. The results showed that the injection amount of loganin showed a good linear relationship with the peak area within the range of 0.019–0.390 μg. A linear regression was performed with the concentration of the standard (x, μg) on ​​the x-axis and the corresponding peak area (y) on the y-axis, yielding the following standard curve regression equation: The coefficient of determination (R²) for y = 1073.9x + 5.9191 is... 2 The value is 0.999.

[0043] Test sample determination: The test sample solution was determined according to the chromatographic conditions of loganin, the peak area was recorded, and 10 μL was injected. The results showed that the total content of loganin in the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula was 2.458±0.091 mg / g.

[0044] Example 5 This embodiment relates to the determination of rutin content in a traditional Chinese medicine composition (Yi Qi Jian Pi Bu Shen Fang) used to enhance the function of mature skeletal muscle cells: Chromatographic conditions: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: V (acetonitrile):V (0.4% phosphoric acid water (adjusted to pH 3.0 with triethylamine)) = 4:6; flow rate: 1.0 mL / min; UV detection wavelength: 254 nm; injection volume: 10 μL; column temperature: 30℃.

[0045] Preparation of reference solutions: Accurately weigh rutin reference standard, add chromatographic methanol, and prepare solutions containing 0.76 μg, 1.53 μg, 3.05 μg, 6.11 μg, and 12.21 μg loganin per mL.

[0046] Preparation of the test solution: Weigh 0.1 g of the traditional Chinese medicine composition of the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula in Example 1, dilute with 0.5 mL of 70% methanol, sonicate for 30 minutes, accurately measure 0.25 mL, dilute with 70% methanol 4 times, shake well, filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0047] Linearity investigation: A series of reference solutions were determined according to the chromatographic conditions for rutin. The chromatograms of the reference solutions are as follows: Figure 4 Peak areas were recorded, and a plot was created using rutin concentration and peak area. 10 μL of rutin was injected at each peak. The results showed a good linear relationship between the rutin injection amount and peak area within the range of 0.007–0.122 μg. Linear regression was performed with the standard concentration (x, μg) as the abscissa and the corresponding peak area (y) as the ordinate, yielding the standard curve regression equation: The coefficient of determination (R²) for the equation y = 1898x + 10.345 is... 2 The value is 0.9996.

[0048] Test sample determination: The test sample solution was analyzed according to the chromatographic conditions for rutin, and the peak area was recorded. 10 μL of sample was injected. Due to sample size limitations, the rutin content determination was performed in only two parallel experiments (n=2), and the precision was assessed using relative deviation. The results showed that the total rutin content in the Qi-tonifying, spleen-strengthening, and kidney-tonifying formula was 0.0415 mg / g (n=2, relative deviation ≈ 1.1%).

[0049] Example 6 This embodiment relates to the determination of verbascoside content in a traditional Chinese medicine composition (Yiqi Jianpi Bushen Fang) used to enhance the function of mature skeletal muscle cells: Chromatographic conditions: Agilent Poroshell 120 EC-C18 (4.6 mm × 150 mm, 4 μm); mobile phase: acetonitrile as mobile phase A and 0.2% formic acid water as mobile phase B, with gradient elution as specified in the table below; flow rate: 1.0 mL / min; UV detection wavelength: 240 nm; injection volume: 10 μL; column temperature: 35℃.

[0050] Table 1: Gradient Elution Table of Mobile Phase

[0051] Preparation of reference solutions: Accurately weigh verbenafil isoflavone glucoside reference standard, add chromatographic methanol, and prepare solutions containing 0.56 μg, 1.12 μg, 2.23 μg, 4.46 μg, and 8.93 μg verbenafil isoflavone glucoside per 1 mL.

[0052] Preparation of the test solution: Weigh 0.1 g of the traditional Chinese medicine composition of the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula in Example 1, dilute with 0.5 mL of 70% methanol, sonicate for 30 minutes, accurately measure 0.25 mL, dilute with 70% methanol 10 times, shake well, filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the test solution.

[0053] Linearity assessment: A series of reference solutions were determined under the chromatographic conditions for verbascoside glucoside. The chromatograms of the reference solutions are shown below. Figure 5 Peak areas were recorded, and a plot was constructed using the concentration of vernixone glucoside and peak area. 10 μL of each sample was injected. The results showed that the injection amount of vernixone glucoside in the range of 0.005–0.089 μg had a good linear relationship with the peak area. A linear regression was performed with the concentration of the standard (x, μg) on ​​the x-axis and the corresponding peak area (y) on the y-axis, yielding the following standard curve regression equation: The coefficient of determination (R²) for y = 3809.5x + 4.3465 is... 2 The value is 0.9993.

[0054] Test sample determination: The test sample solution was determined according to the chromatographic conditions of verbascoside, the peak area was recorded, and 10 μL was injected. The results showed that the total content of verbascoside in the Qi-invigorating, spleen-strengthening, and kidney-tonifying formula was 0.0583±0.0027 mg / g.

[0055] Example 7 This embodiment relates to the effect of a traditional Chinese medicine composition (Yi Qi Jian Pi Bu Shen Fang) for enhancing the function of mature skeletal muscle cells on the activity of C2C12 cells. C2C12 cell suspension was administered at 100 μL per well, with a final concentration of 1 × 10⁻⁶. 5 ·L -1 The mixture was added to a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. The old culture medium was then removed. The herbal composition of the Qi-tonifying, spleen-strengthening, and kidney-tonifying formula from Example 1 was diluted with 0.5% DMEM culture medium to prepare concentrations of 10, 30, 100, 300, and 1000 μg·L⁻¹. -1 Add 100 μL of the solution to each well of a 96-well plate, with four replicates for each concentration. Four replicates containing only 0.5% DMEM culture medium were also included as a blank control group. After culturing for 24 h, the old culture medium was discarded, and 100 μL of 1% MTT was added to each well. After culturing for 4 h, the MTT solution was discarded, and 100 μL of DMSO was added to each well. The absorbance (OD) value was then measured using a microplate reader.

[0056] Conclusion: In undifferentiated C2C12 cells, cell viability was lower than that in all drug treatment groups (10-1000 μg / mL) than in the control group. In differentiated C2C12 myotube cells: cell viability was higher than that in the control group at a concentration of 300 μg / mL; while at other concentrations, the viability was not significantly different from or slightly lower than that in the control group. Figure 6 Therefore, this traditional Chinese medicine composition specifically enhances the activity of differentiated mature skeletal muscle cells, with the optimal effect at around 300 μg / mL.

[0057] Comparative Example 1 This comparative example relates to a traditional Chinese medicine composition for invigorating qi, strengthening the spleen, and tonifying the kidneys. The composition consists of 12 g of ginseng, 9 g of astragalus, 9 g of dried tangerine peel, and 18 g of polygonatum. The ingredients are soaked in water for 1 hour, then decocted with 10 times the amount of water for 0.5 hours, filtered, and then decocted again with 8 times the amount of water for 0.5 hours, and filtered again. The two filtrates are combined and concentrated to obtain the qi-invigorating, spleen-strengthening, and kidney-tonifying formula (without cornus officinalis).

[0058] The following relates to the effect of Comparative Example 1, the Qi-tonifying, spleen-strengthening, and kidney-nourishing formula (without Cornus officinalis), on the activity of C2C12 cells. C2C12 cell suspension was administered at 100 μL per well, with a final concentration of 1 × 10⁻⁶. 5 ·L -1 Add the mixture to a 96-well plate and incubate at 37°C with 5% CO2 for 24 h. Then, remove the old culture medium. Dilute the Qi-tonifying, spleen-strengthening, and kidney-tonifying formula (without Cornus officinalis) composition of Comparative Example 1 with 0.5% DMEM culture medium to prepare concentrations of 10, 30, 100, 300, and 1000 μg·L⁻¹. -1 Add 100 μL of the solution to each well of a 96-well plate, with four replicates for each concentration. Four replicates containing only 0.5% DMEM culture medium were also included as a blank control group. After culturing for 24 h, the old culture medium was discarded, and 100 μL of 1% MTT was added to each well. After culturing for 4 h, the MTT solution was discarded, and 100 μL of DMSO was added to each well. The absorbance (OD) value was then measured using a microplate reader.

[0059] Conclusion: In undifferentiated C2C12 cells, all drug concentrations (10-1000 μg / mL) showed no significant difference or slightly higher activity compared to the control group. In differentiated C2C12 myotube cells: at a concentration of 1000 μg / mL, cell viability was lower than the control group; while at other concentrations, activity was not significantly different from the control group or slightly higher. Figure 7 Therefore, a 1000 μg / mL Chinese herbal composition without Cornus officinalis exhibits strong toxicity in differentiated mature skeletal muscle cells.

[0060] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A traditional Chinese medicine composition for enhancing mature skeletal muscle cell function, characterized in that, The raw materials of the traditional Chinese medicine composition are ginseng, astragalus, dried tangerine or orange peel, cornus officinalis and polygonatum; the mass ratio of the ginseng, astragalus, dried tangerine or orange peel, cornus officinalis and polygonatum is 4: 2.5-3.5 : 2.5-3.5 : 2.5-3.5 : 2.5-3.

5.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 12 parts of ginseng, 8-10 parts of astragalus, 8-10 parts of dried tangerine or orange peel, 8-10 parts of cornus officinalis and 8-10 parts of polygonatum.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is prepared by the following method: ginseng, astragalus, dried tangerine or orange peel, cornus officinalis and polygonatum are weighed, the medicinal materials are soaked in water for 0.5-1.5 hours, 9-11 times the amount of water is added, and decoction is performed for 0.4-0.6 hours, and then filtration is performed; 7-9 times the amount of water is added again for decoction for 0.4-0.6 hours, and then filtration is performed; the filtrates obtained in the two steps are combined, and the combined filtrate is collected; the filtrate is concentrated to a volume of 1 / 3 to 1 / 2 of the total volume of the original combined filtrate to obtain a concentrated solution; then the concentrated solution is subjected to nitrogen blowing until a solid product is obtained.

4. Use of the traditional Chinese medicine composition according to any one of claims 1-3 in the preparation of a preparation for treating or preventing a disease characterized by reduced or atrophied mature muscle fiber function.

5. A medicine, functional food or health food for enhancing the activity of differentiated skeletal muscle cells, the active ingredient of which is the traditional Chinese medicine composition according to any one of claims 1-3.

6. A method for establishing a quality standard of the traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, The method comprises the following steps: S1, ginsenoside Rg1, hesperidin, loganin, rutin and calycosin-7-glucoside are used as standard control substances; a series of standard control substance solutions with different concentrations are determined by high performance liquid chromatography, the peak areas of the standard control substances are measured, the peak areas of the standard control substances obtained are used as the ordinate, and the injection mass of each standard control substance is used as the abscissa, and a linear regression equation is calculated; S2, the test sample solution of the traditional Chinese medicine composition is determined by high performance liquid chromatography in the same step S1, and the peak areas of ginsenoside Rg1, hesperidin, loganin, rutin and calycosin-7-glucoside in the test sample solution are obtained, the mass of ginsenoside Rg1, hesperidin, loganin, rutin and calycosin-7-glucoside in the test sample solution is calculated according to the linear regression equation of ginsenoside Rg1, hesperidin, loganin, rutin and calycosin-7-glucoside standard control substances, and the content of ginsenoside Rg1, hesperidin, loganin, rutin and calycosin-7-glucoside in the test sample solution is further calculated.

7. The method of claim 6, wherein the quality criterion is established based on a comparison of the first and second values of the quality metric. The linear regression equations of the standard control substances are as follows: The standard curve equation of ginsenoside Rg1 is: y=678.05x+14.623, and the correlation coefficient R=0.9975. 2 =0.9975; The standard curve equation of hesperidin is: y = 1971.3x + 3.699, and the correlation coefficient R = 0.9988. 2 =0.9988; The standard curve equation of strychnine is: y = 1073.9x + 5.9191, and the correlation coefficient R = 0.

999. 2 =0.999; The standard curve equation of rutin is: y = 1898x + 10.345, the correlation coefficient R = 0.9996. 2 =0.9996; The standard curve equation of hesperidin is: y=3809.5x+4.3465, and the correlation coefficient R=0.9993. 2 =0.9993.

8. The method of claim 6, wherein the quality criterion is established based on a result of a test performed on the product. The high performance liquid chromatography conditions are as follows: Ginsenoside Rg1 chromatography conditions: Agilent Poroshell 120 EC-C18 (4.6 mm x 150 mm, 4 μm); mobile phase: V(acetonitrile): V(0.1% phosphoric acid) = 15:85; flow rate: 1.0 mL / min; UV detection wavelength: 203 nm; injection volume: 10 μL; column temperature: 30℃; HPLC conditions of hesperidin: Agilent Poroshell 120 EC-C18 (4.6 mm x 150 mm, 4 μm); mobile phase: V(acetonitrile):V(water)=22:78; flow rate: 1.0 mL / min; UV detection wavelength: 283 nm; injection volume: 10 μL; column temperature: 30 °C; HPLC conditions of loganin: Agilent Poroshell 120 EC-C18 (4.6 mm x 150 mm, 4 μm); mobile phase: V(acetonitrile):V(0.3% phosphoric acid water)=12:88; flow rate: 1.0 mL / min; UV detection wavelength: 240 nm; injection volume: 10 μL; column temperature: 35 °C; HPLC conditions of rutin: Agilent Poroshell 120 EC-C18 (4.6 mm x 150 mm, 4 μm); mobile phase: V(acetonitrile):V(0.4% phosphoric acid water (pH adjusted to 3.0 with triethylamine))=4:6; flow rate: 1.0 mL / min; UV detection wavelength: 254 nm; injection volume: 10 μL; column temperature: 30 °C; HPLC conditions of calycosin-7-glucoside: Agilent Poroshell 120 EC-C18 (4.6 mm x 150 mm, 4 μm); mobile phase: acetonitrile (A)-0.2% formic acid water (B), gradient elution, 0-20 min, 20% A-40% A; 20-30 min, 40% A, flow rate: 1.0 mL / min; UV detection wavelength: 240 nm; injection volume: 10 μL; column temperature: 35 °C.

9. The method of claim 6, wherein the quality criterion is established based on a result of a test performed on the product. The quality control standard is that the content of ginsenoside Rg1 in the test solution of the traditional Chinese medicine composition is 0.328±0.053 mg / g, the content of hesperidin is 1.713±0.056 mg / g, the content of loganin is 2.458±0.091 mg / g, the content of rutin is 0.0415 mg / g (n=2, relative deviation ≈1.1%), and the content of calycosin-7-glucoside is 0.0583±0.0027 mg / g.

10. The method of claim 6, wherein the quality criterion is established based on a result of a test performed on the product. The preparation of each standard control solution is as follows: The ginsenoside Rg1 control sample was precisely weighed, and chromatographic methanol was added to prepare a solution containing 80 μg, 64 μg, 40 μg, 20 μg, and 10 μg of ginsenoside Rg1 per 1 mL; The hesperidin control sample was precisely weighed, and chromatographic methanol was added to prepare a solution containing 0.76 μg, 1.53 μg, 3.05 μg, 6.11 μg, 12.21 μg, and 24.42 μg of hesperidin per 1 mL; The loganin control sample was precisely weighed, and chromatographic methanol was added to prepare a solution containing 1.95 μg, 3.90 μg, 7.81 μg, 19.52 μg, and 39.03 μg of loganin per 1 mL; Accurately weigh rutin reference substance, add chromatographic methanol to prepare a solution containing 0.76 μg, 1.53 μg, 3.05 μg, 6.11 μg, 12.21 μg of loganin per 1 mL; accurately weigh calycosin-7-glucoside reference substance, add chromatographic methanol to prepare a solution containing 0.56 μg, 1.12 μg, 2.23 μg, 4.46 μg, 8.93 μg of calycosin-7-glucoside per 1 mL; and / or, The preparation of the test sample solution is as follows: accurately weigh 0.1 g of the traditional Chinese medicine composition, dilute with 70% methanol 0.5 mL, treat with ultrasonic for 30 minutes, accurately take 0.25 mL, dilute 20 times with 70% methanol, shake well, filter, take the filtrate, filter with 0.45 μm microporous filter membrane, and then obtain.

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