Use of a traditional Chinese medicine composition in the preparation of a medicament for preventing and treating sarcopenia

Abstract

The application discloses application of a traditional Chinese medicine composition in preparation of a medicine for preventing and treating muscle loss, and the traditional Chinese medicine composition is prepared from raw medicinal materials in the following proportions by weight: Semen Euryae 150-350, Fructus Lycii 100-200, Schisandra chinensis 30-60, Cnidium 20-50, Chamaerops 20-50, Lepidium 20-50, Dipsacus 20-50, Cistanche 20-50, Rehmannia 30-60, Cyathula 20-50, Herba Epimedii 40-100, Rubus 20-50, Ginseng 15-35, Deer Horn 10-25, Sea Horse 15-35 and Melia 15-35. Animal experiments prove that the traditional Chinese medicine composition can improve the endurance and balance ability of a mouse with muscle loss, and can be used for preventing and treating muscle loss.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating sarcopenia. Background Technology

[0002] Sarcopenia, first proposed in the 1990s, refers to an age-related decrease in overall muscle mass and / or strength, or a decline in muscle function, leading to reduced physical activity. Clinically, it is also known as "skeletal muscle aging" or "sarcopenia." Skeletal muscle is the power source of the human musculoskeletal system; muscle aging and atrophy are significant markers of aging and greatly increase the risk of fractures and joint injuries. Elderly individuals with sarcopenia experience difficulty standing, slow gait, and are prone to falls and fractures. Sarcopenia can also affect organ function, potentially leading to heart and lung failure, and even death.

[0003] Sarcopenia mainly includes two types: physiological sarcopenia, which is primary sarcopenia caused solely by aging, and pathological sarcopenia, which is secondary sarcopenia caused by one or more factors related to chronic diseases, nutrition, and physical activity. Primary sarcopenia is more common and is one of the important causes and manifestations of the gradual decline in physiological function in the elderly.

[0004] Sarcopenia has long been considered to be related to aging, but some studies now suggest that its onset age is earlier and that many contributing factors beyond age-related increases exist. Current research indicates that sarcopenia results from the combined effects of multiple factors during aging, including decreased sex hormone secretion, muscle cell apoptosis, mitochondrial dysfunction, and neurodegenerative diseases. Nutritional deficiencies or malabsorption are also important contributing factors.

[0005] According to the latest report from EWGSOP, the global prevalence of sarcopenia is 6-12% (approximately 50 million people currently affected), with a prevalence of 14-33% in those aged 65 and older. This means that one in three people aged 65 and older has sarcopenia. Further statistics show that the prevalence among disabled and hospitalized patients is as high as 78%. It is projected that by 2050, the global population aged 60 and older will exceed 2.1 billion, and the number of people with sarcopenia will reach 500 million, becoming a global public health problem. In 2019, the Asian Sarcopenia Working Group (AWGS) indicated that the prevalence among the elderly population in Asia is approximately 5.5-25.7%.

[0006] Western medicine primarily treats sarcopenia through physical exercise and nutritional intervention, with protein and / or amino acid supplementation considered the most effective method. While these methods do have some effect in preventing sarcopenia, for those already experiencing symptoms, especially severe ones, the effects of exercise and nutritional supplementation are slow and minimal.

[0007] Traditional Chinese medicine does not have a specific disease name for "sarcopenia." Some scholars, based on its clinical manifestations and pathogenesis, classify it under the category of "atrophy syndrome." Traditional Chinese medicine treatment focuses on tonifying the kidneys and replenishing essence to nourish the innate constitution, strengthening the spleen and stomach to benefit qi and blood, and dispelling damp heat, resolving phlegm, and eliminating blood stasis. It can be further subdivided into: lung heat and fluid depletion syndrome, treated with modified Qingzao Jiufeng Decoction; qi stagnation and blood stasis syndrome, treated with modified Chaihu Shugan Powder or Shengyu Decoction combined with Xuefu Zhuyu Decoction; spleen and stomach weakness syndrome, treated with modified Buzhong Yiqi Decoction combined with Shenling Baizhu Powder; and liver and kidney deficiency syndrome, treated with modified Huqian Pill.

[0008] In his article "Legs Age First, Kidney Deficiency Causes Cold Legs," Zi Bu mentions that the main pathological changes in aging of the lower back and legs are muscle loss and bone weakening. In Traditional Chinese Medicine (TCM), the root cause of this aging is a severe deficiency of kidney essence, leading to muscle loss, bone weakening, and a series of other changes. Deer antler, an animal-derived medicine, is a representative drug with the most significant effect in tonifying the kidneys and replenishing essence, and is one of the main ingredients in the kidney-tonifying TCM formula, Bazibu Shen Capsules. Summary of the Invention

[0009] The purpose of this invention is to provide an application of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating sarcopenia.

[0010] To achieve the above objectives, the inventors have provided the following technical solutions.

[0011] The application of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating sarcopenia, the traditional Chinese medicine composition being made from the following raw materials in parts by weight: Cuscuta chinensis 150-350, Lycium barbarum 100-200, Schisandra chinensis 30-60, Cnidium monnieri 20-50, Rosa laevigata 20-50, Allium tuberosum 20-50, Morinda officinalis 20-50, Cistanche deserticola 20-50, Rehmannia glutinosa 30-60, Achyranthes bidentata 20-50, Epimedium brevicornu 40-100, Rubus idaeus 20-50, Panax ginseng 15-35, Cervus nippon antler 10-25, Hippocampus 15-35, and Melia toosendan 15-35.

[0012] The traditional Chinese medicine composition used to prepare the drug for preventing and treating sarcopenia is preferably made from the following raw materials in parts by weight: Cuscuta chinensis 150, Lycium barbarum 200, Schisandra chinensis 30, Cnidium monnieri 50, Rosa laevigata 20, Allium tuberosum 20, Morinda officinalis 50, Cistanche deserticola 20, Rehmannia glutinosa 30, Achyranthes bidentata 50, Epimedium brevicornu 40, Rubus idaeus 50, Panax ginseng 15, Cervus nippon antler 25, Hippocampus 15, and Melia toosendan 35.

[0013] The above-mentioned traditional Chinese medicine composition for preparing drugs to prevent and treat sarcopenia can preferably be made from the following raw materials in parts by weight: Cuscuta chinensis 350, Lycium barbarum 100, Schisandra chinensis 60, Cnidium monnieri 20, Rosa laevigata 50, Allium tuberosum 50, Morinda officinalis 20, Cistanche deserticola 50, Rehmannia glutinosa 60, Achyranthes bidentata 20, Epimedium brevicornu 100, Rubus idaeus 20, Panax ginseng 35, Cervus nippon antler 10, Hippocampus 35, and Melia toosendan 15.

[0014] The above-mentioned traditional Chinese medicine composition for preparing drugs to prevent and treat sarcopenia can also preferably be made from the following raw materials in parts by weight: Cuscuta chinensis 250, Lycium barbarum 138, Schisandra chinensis 46, Cnidium monnieri 35, Rosa laevigata 35, Allium tuberosum 35, Morinda officinalis 35, Cistanche deserticola 35, Rehmannia glutinosa 46, Achyranthes bidentata 35, Epimedium brevicornu 70, Rubus idaeus 35, Panax ginseng 25, Cervus nippon antler 16, Hippocampus 21, and Melia toosendan 23.

[0015] The above-mentioned traditional Chinese medicine composition for preparing drugs to prevent and treat sarcopenia can preferably be made from the following raw materials in parts by weight: Cuscuta chinensis 250, Lycium barbarum 150, Schisandra chinensis 45, Cnidium monnieri 30, Rosa laevigata 30, Allium tuberosum 30, Morinda officinalis 30, Cistanche deserticola 30, Rehmannia glutinosa 45, Achyranthes bidentata 30, Epimedium brevicornu 70, Rubus idaeus 30, Panax ginseng 20, Cervus nippon antler 19, Hippocampus 20, and Melia toosendan 20.

[0016] The dosage form of the above-mentioned traditional Chinese medicine composition used to prepare drugs for the prevention and treatment of sarcopenia can be decoction, capsule, tablet, granule, powder or pill.

[0017] In the above application, sarcopenia refers to senile sarcopenia.

[0018] In the above application, sarcopenia is referred to as sarcopenia-osteoporosis.

[0019] In the above applications, sarcopenia refers to muscle atrophy caused by reduced physical activity.

[0020] Osteoporosis (OP) is a systemic skeletal disease characterized by decreased bone strength, increased bone fragility, and a higher risk of fractures. Muscles and bones are anatomically adjacent, share common paracrine and endocrine regulation, similar molecular signaling pathways, and common therapeutic targets and drugs. The combined effects of sarcopenia and osteoporosis lead to decreased balance, increased risk of falls, and increased fragility fractures, ultimately resulting in a reduced quality of life for older adults. Binkley et al., combining research on the correlation between muscles and bones with the incidence of sarcopenia and osteoporosis, proposed the concept of "sarcopenia-osteoporosis," which refers to the presence of a clinical or bone mineral density diagnosis of osteoporosis accompanied by a decline in muscle mass and / or function.

[0021] To achieve the objectives of this invention, theoretically, the traditional Chinese medicine composition of this invention can be formulated into any dosage form according to general pharmaceutical methods, preferably as a decoction, capsule, tablet, granule, powder, or pill. To enable the above dosage forms, appropriate pharmaceutical excipients can be added during the preparation of these dosage forms, such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrices. Fillers include, but are not limited to, starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, or combinations thereof; disintegrants include, but are not limited to, starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, sodium croscarmellose, or combinations thereof. Lubricants include: magnesium stearate, sodium dodecyl sulfate, talc, silica, etc.; suspending agents include, but are not limited to, polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, or combinations thereof; binders include, but are not limited to, starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, or combinations thereof; sweeteners include, but are not limited to, sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, or combinations thereof; flavoring agents include, but are not limited to, sweeteners and various flavorings; preservatives include, but are not limited to, parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, or combinations thereof; and matrices include, but are not limited to, PEG6000, PEG4000, insect wax, or combinations thereof.

[0022] The present invention also provides a method for preparing capsules of the above-mentioned traditional Chinese medicine composition for treating sarcopenia, comprising the following steps:

[0023] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 6-10 times the amount of 70% ethanol, reflux extract 1-3 times, 1-3 hours each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0024] b. Combine the medicinal residue after alcohol extraction in step a with the appropriate amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 7-12 times the amount of water and decoct 1-3 times, 1-3 hours each time. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.25-1.30 when measured at 60℃ to obtain the extract.

[0025] c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder, mix them evenly with the extract obtained in step b, dry at 60-70℃, grind, sieve and fill into capsules.

[0026] The present invention has been demonstrated through tail suspension experiments in mice. The herbal composition of this invention can significantly increase the suspension time of tail-suspended mice, reaching normal levels. Furthermore, the herbal composition can significantly increase the total rotarod time of tail-suspended mice, reaching normal levels. This indicates that the herbal composition of this invention can improve the endurance and balance of sarcopenic mice, enhance muscle strength, and can be used for the prevention and treatment of sarcopenia, especially age-related sarcopenia.

[0027] Behavioral experiments using a D-galactose-induced aging mouse model confirmed that the traditional Chinese medicine composition of this invention has a significant effect on age-related sarcopenia.

[0028] The present invention also constructed a mouse model of sarcopenia using the hind limb suspension method, demonstrating that the traditional Chinese medicine composition of the present invention has a significant effect on sarcopenia caused by reduced exercise. Attached Figure Description

[0029] Figure 1 This is a diagram of the box used to test the suspension duration in Example 7.

[0030] Figure 2 This is a box diagram showing the test results of the total rotor duration in Example 7.

[0031] Figure 3 This is a diagram of HE staining of the extensor digitorum longus muscle in the tailless group in Example 7.

[0032] Figure 4 This is a diagram of HE staining of the extensor digitorum longus muscle in the group consisting of the suspended tail and the herbal composition of the present invention in Example 7.

[0033] Figure 5 This is a diagram of HE staining of the extensor digitorum longus muscle in the tail suspension + resveratrol group in Example 7.

[0034] Figure 6 This is a diagram of HE staining of the extensor digitorum longus muscle in the normal control group in Example 7.

[0035] Figure 7 This is a box plot comparing the size of the extensor digitorum longus muscle in the four experimental groups in Example 7.

[0036] Figure 8 The images are HE-stained electron micrographs (20x) of the cross-sectional area of ​​the gastrocnemius muscle fibers of mice in each experimental group in Example 9.

[0037] Figure 9 The image shows Sirius red staining of mice in each experimental group in Example 10 (10x).

[0038] Figure 10 , 11 12 represents the results of satellite cell activation, proliferation, and differentiation related indicators in each experimental group of mice in Example 10. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments.

[0040] Example 1: Preparation of Capsules

[0041] Formula: Cuscuta chinensis 250g, Lycium barbarum 138g, Schisandra chinensis 46g, Cnidium monnieri 35g, Rosa laevigata 35g, Allium tuberosum 35g, Morinda officinalis 35g, Cistanche deserticola 35g, Rehmannia glutinosa 46g, Achyranthes bidentata 35g, Epimedium brevicornu 70g, Rubus idaeus 35g, Panax ginseng 25g, Cervus nippon antler 16g, Hippocampus 21g, Melia toosendan 23g.

[0042] Preparation method:

[0043] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 3208 mL of 70% ethanol, reflux and extract 3 times, 2 hours each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0044] b. Combine the medicinal residue after alcohol extraction in step a with the prescribed amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 6732ml of water and decoct twice, 2 hours each time. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.30 when measured at 60℃ to obtain the extract.

[0045] c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder, mix them evenly with the extract obtained in step b, dry at 60-70℃, grind, sieve, fill into capsules, and obtain capsules.

[0046] Example 2: Preparation of Tablets

[0047] Formula: Cuscuta chinensis 250g, Lycium barbarum 150g, Schisandra chinensis 45g, Cnidium monnieri 30g, Rosa laevigata 30g, Allium tuberosum 30g, Morinda officinalis 30g, Cistanche deserticola 30g, Rehmannia glutinosa 45g, Achyranthes bidentata 30g, Epimedium brevicornu 70g, Rubus idaeus 30g, Panax ginseng 20g, Cervus nippon antler 19g, Hippocampus 20g, Melia toosendan 20g.

[0048] Preparation method:

[0049] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 2765mL of 70% ethanol, reflux and extract 3 times, 1 hour each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0050] b. Combine the medicinal residue after alcohol extraction in step a with the prescribed amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 5110ml of water and decoct once for 3 hours. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.25 when measured at 60℃ to obtain the extract.

[0051] c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder and mix them evenly with the extract obtained in step b, dry at 60-70℃, grind, sieve, add 1% magnesium stearate, compress into tablets to obtain tablets.

[0052] Example 3: Preparation of pills

[0053] Formula: Cuscuta chinensis 350g, Lycium barbarum 100g, Schisandra chinensis 60g, Cnidium monnieri 20g, Rosa laevigata 50g, Allium tuberosum 50g, Morinda officinalis 20g, Cistanche deserticola 50g, Rehmannia glutinosa 60g, Achyranthes bidentata 20g, Epimedium brevicornu 100g, Rubus idaeus 20g, Panax ginseng 35g, Cervus nippon antler 10g, Hippocampus 35g, Melia toosendan 15g.

[0054] Preparation method:

[0055] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis, and Epimedium according to the proportion, add 5300mL of 70% ethanol, reflux and extract once for 3 hours, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0056] b. Combine the medicinal residue after alcohol extraction in step a with the prescribed amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 10140mL of water and decoct three times, one hour each time. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.26 when measured at 60℃ to obtain the extract.

[0057] c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder, mix them evenly with the extract obtained in step b, dry at 60-70℃, grind, sieve and make into pills.

[0058] Example 4: Preparation of Granules

[0059] Formula: Cuscuta chinensis 150g, Lycium barbarum 200g, Schisandra chinensis 30g, Cnidium monnieri 50g, Rosa laevigata 20g, Allium tuberosum 20g, Morinda officinalis 50g, Cistanche deserticola 20g, Rehmannia glutinosa 30g, Achyranthes bidentata 50g, Epimedium brevicornu 40g, Rubus idaeus 50g, Panax ginseng 15g, Cervus nippon antler 25g, Hippocampus 15g, Melia toosendan 35g.

[0060] Preparation method:

[0061] a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 2430mL of 70% ethanol, reflux extract twice, 1.5 hours each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract.

[0062] b. Combine the medicinal residue after alcohol extraction in step a with the remaining medicinal materials in the prescription amount, add 8800mL of water and decoct 3 times, 1 hour each time, filter, combine the filtrates and mix with the alcohol extract obtained in step a, concentrate under reduced pressure until the relative density is 1.28 when measured at 60℃, to obtain the extract.

[0063] c. Mix the extract obtained in step b with 300g of dextrin and 200g of sucrose powder evenly, dry at 60-70℃, pulverize, sieve, and granulate to obtain granules.

[0064] Example 5 Preparation of Powder

[0065] Formula: Cuscuta chinensis 250g, Lycium barbarum 150g, Schisandra chinensis 45g, Cnidium monnieri 30g, Rosa laevigata 30g, Allium tuberosum 30g, Morinda officinalis 30g, Cistanche deserticola 30g, Rehmannia glutinosa 45g, Achyranthes bidentata 30g, Epimedium brevicornu 70g, Rubus idaeus 30g, Panax ginseng 20g, Cervus nippon antler 19g, Hippocampus 20g, Melia toosendan 20g.

[0066] Preparation method: Weigh the Chinese medicinal materials in the formula according to the proportion, mix and grind them into fine powder to obtain 823g of powder.

[0067] Example 6: Preparation of the decoction

[0068] Formula: Cuscuta chinensis 250g, Lycium barbarum 150g, Schisandra chinensis 45g, Cnidium monnieri 30g, Rosa laevigata 30g, Allium tuberosum 30g, Morinda officinalis 30g, Cistanche deserticola 30g, Rehmannia glutinosa 45g, Achyranthes bidentata 30g, Epimedium brevicornu 70g, Rubus idaeus 30g, Panax ginseng 20g, Cervus nippon antler 19g, Hippocampus 20g, Melia toosendan 20g.

[0069] Preparation method: Weigh the Chinese medicinal materials according to the proportion, add water to cover the medicinal materials, soak for 15 minutes, heat to boiling, continue to simmer for 30 minutes, filter, add water to the dregs again to cover the medicinal materials, simmer for 30 minutes again, filter, combine the filtrates to obtain the decoction.

[0070] Example 7

[0071] This invention investigated the therapeutic effect of the traditional Chinese medicine composition of this invention on sarcopenia using a mouse tail suspension experiment. The specific experimental details are as follows:

[0072] 1. Experimental Materials

[0073] 1.1 Test sample

[0074] The test samples used in this experiment were prepared according to the prescription and method provided in Example 1, and were the contents of capsules.

[0075] 1.2 Positive drugs, main reagents and materials

[0076] 1.2.1 Positive drugs

[0077] Resveratrol: A non-flavonoid polyphenol compound, manufacturer: Shanghai Aladdin Biochemical Technology Co., Ltd., product number: R107315.

[0078] 1.2.2 Tool drugs, main reagents and materials

[0079] Sodium carboxymethyl cellulose (CMC-Na): Tianjin Damao Chemical Reagent Factory, batch number: 20181006, expiration date: December 30, 2021.

[0080] 1.3 Experimental system

[0081] 1.3.1 Animal strain: C57BL / 6J mice.

[0082] 1.3.2 Animal level: SPF level.

[0083] 1.3.3 Animal gender and quantity: 60 were purchased, all 60 were male, and 60 (60 / male) were selected for this experiment. 1.3.4 Animal age at the start of dosing: 13 months old.

[0084] 1.3.5 Animal weight at the start of dosing: Actually 20 - 40 g.

[0085] 1.3.6 Animal source: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0086] 1.3.7 Animal certificate number, issuing unit, and receiving date: Certificate number 1100112011043405, license number SCXK (Beijing) 2016 - 0006, the issuing unit is Beijing Vital River Laboratory Animal Technology Co., Ltd., and the receiving date is July 15, 2020.

[0087] 1.3.8 Feeding conditions: Mice were caged and raised in the New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd. The light was 12 hours per day, the temperature was 20 - 26 °C, and the relative humidity was 40 - 70%. The experimental animal use license number: SYXK (Hebei) 2020 - 003.

[0088] 1.3.9 Quarantine process: The animals were quarantined for 3 days and adaptively raised for 2 days. During this period, the animals' drinking water, food intake, and health status were observed, as well as whether there were signs of disease and death.

[0089] 1.3.10 Feed: SPF rat and mouse maintenance diet, provided by Beijing Keao Xieli Feed Co., Ltd. Feed production license number: Jingsizheng (2018) 06073, feed batch number: 20103213, 20113213.

[0090] 1.3.11 Drinking water: Clean water prepared by the ROA50 laboratory animal drinking system (instrument number YL-LE-A01) was filled into drinking bottles for free drinking. The water quality was tested and met the "Standards for Drinking Water Quality".

[0091] 1.3.12 Bedding material: Ordinary grade corn cob bedding material, which is sterilized by high temperature and high pressure before use. It is provided by Beijing Keao Xieli Feed Co., Ltd., bedding material batch number: 20079811.

[0092] 1.3.13 Identification: Ear tag markings for laboratory animals.

[0093] 2 Experimental Methods

[0094] 2.1 Experimental Design Basis

[0095] 2.1.1 Design basis: Published literature.

[0096] 2.1.2 Experimental System Selection Instructions

[0097] Aged C57BL / 6J mice are used in research applications including immunology, cancer, longevity intervention, and biomarker research. In 13-month-old C57 mice, the balance and stability of stem cells are disrupted, leading to rapid aging. Since a 13-month-old mouse is approximately equivalent to a 52-year-old human, and a 15-month-old mouse is approximately equivalent to a 60-year-old human, 13-month-old C57BL / 6J mice can be used to simulate the natural aging process. A sarcopenic muscular dystrophy model can be created through a two-week tail suspension assay.

[0098] 2.2 Dosage and Grouping

[0099] Sixty C57 mice were randomly divided into four groups of 15 mice each according to their body weight: a normal control group, a tail suspension group, a tail suspension plus the invented traditional Chinese medicine composition group, and a tail suspension plus resveratrol group. Each group was given a different drug.

[0100] ① Normal control group (CON): No medication was given, and the animals were fed normally.

[0101] ②Suspended Tail Group (TS): An equal volume of distilled water was given.

[0102] ③Suspended tail + the traditional Chinese medicine composition of the present invention (TS+BZ): Administer 2g / kg / d of the contents of the capsule of the composition of the present invention.

[0103] ④ Tail suspension + resveratrol group (TS+R): Resveratrol 150mg / kg / d was administered.

[0104] 2.3 Method of administering test sample

[0105] Gavage.

[0106] 2.4 Preparation and storage of test samples

[0107] Based on the number and weight of the experimental animals, weigh appropriate amounts of the contents of the capsules containing the traditional Chinese medicine composition of this invention, add solvent, grind and suspend, and dilute to volume to prepare the test solution. The solvent is 0.5% CMC-Na, used at a volume of 10 ml / kg. Weigh out CMC-Na, spread it evenly on the surface of pure water to fully swell, dilute to volume, and store at 2-8℃ until use. The test solution should be prepared fresh on the day of use. Specific preparation data are shown in Table 1.

[0108]

[0109] 2.5 Administration of the test sample

[0110] The test sample was administered using a syringe at a volume of 10 ml / kg. The groups were: tail suspension group, tail suspension + traditional Chinese medicine composition of the present invention group, and tail suspension + resveratrol group. The mice were given the corresponding volume of distilled water or drug solution according to their body weight.

[0111] 2.6 Model Making

[0112] For two consecutive weeks, mice were suspended by their tails using clips to prevent their hind limbs from touching the ground. Adhesive tape was attached to the mice's tails, and a cushioning pad was added before the suspension, adjusting its thickness to prevent tail ischemia. This tail suspension experiment was used to create a sarcopenia model.

[0113] 2.7 Detection Indicators

[0114] 2.7.1 Suspension Duration

[0115] Two months after drug administration, a hind limb suspension experiment was conducted. The forelimbs of mice were bound with rubber bands and placed on the suspension device. After the mice stabilized, the suspension device was flipped upside down so that only the hind limbs could be used for suspension. A timer was used to time the event until the mice fell and stopped. The total duration was recorded. After three training sessions, a formal test was conducted. A total of three tests were conducted, with a 10-minute interval between each test. The results were recorded and the average value was calculated.

[0116] 2.7.2 Total Rotation Time

[0117] Two months after drug administration, a rotarod test was conducted. The rotarod parameters were set as follows: Run limit: 300, Lines: 5, Start Rpm: 4, Top Rpm: 40, Ramp time: 30. Mice were placed on the rotarod, and the time from start to fall was recorded. Each mouse was measured three times, with a 30-minute interval between each measurement as fatigue recovery time, and the average value was calculated. A formal test was conducted after three training cycles.

[0118] 2.7.3 HE staining

[0119] After the last administration, skeletal muscle samples were sent for pathological examination and HE staining to observe the morphological changes of the extensor digitorum longus muscle fibers.

[0120] 2.8 Instrument System

[0121] Mettler ME104E analytical balance (YL-LE-B07, B08), METTLER TOLEDO.

[0122] DT2000 electronic balance (YL-LE-B01, B05), Changshu Shuangjie Test Instrument Factory.

[0123] IITC755 Rotary Bar Fatigue Tester (YL-LE-A051), IITC Biosciences.

[0124] 2.9 Statistical Methods

[0125] Experimental data were analyzed using SPSS 20.0 statistical software, with significance levels of 0.05 and 0.01. Results are expressed as mean ± standard deviation. The method involves first performing a normality test. For data that conforms to a normal distribution, one-way ANOVA is used to compare the means. If the variances are homogeneous, the least significant difference (LSD) test is used for pairwise comparisons. If the variances are not homogeneous, Dunnett's T3 test is used for pairwise comparisons. If the data does not conform to a normal distribution, nonparametric tests are used. For count data, the chi-square test or rank-sum test is used.

[0126] 3 Results

[0127] 3.1 Suspension time

[0128] Appendix Figure 1 A diagram of the test chamber is provided. Table 2 shows the test results of the suspension time experiment. ).

[0129]

[0130] As shown in Table 2, compared with the suspended tail group, the suspension time of the suspended tail + traditional Chinese medicine composition of the present invention group was significantly increased (P<0.01), and the suspension time of the suspended tail + resveratrol group was significantly increased (P<0.05). Compared with the normal control group, the suspension time of the suspended tail + resveratrol group was significantly decreased (P<0.05), while there was no statistically significant difference in the suspension tail + traditional Chinese medicine composition of the present invention group.

[0131] 3.2 Total duration of the rotating rod

[0132] Appendix Figure 2 A box diagram showing the test results of the rotating bar experiment is provided. Table 3 presents the test results of the rotating bar experiment. ).

[0133]

[0134] As shown in Table 3, compared with the suspended tail group, the total rotator time of the suspended tail + traditional Chinese medicine composition of the present invention group was significantly increased (P<0.01), and the suspension time of the suspended tail + resveratrol group was significantly increased (P<0.05). Compared with the normal control group, the total rotator time of the suspended tail + resveratrol group was significantly decreased (P<0.05), and there was no statistically significant difference in the suspended tail + traditional Chinese medicine composition of the present invention group.

[0135] 3.3 Pathological Results

[0136] Appendix Figure 3 Appendix Figure 4 Appendix Figure 5 Appendix Figure 6 HE staining images (20×) of the extensor digitorum longus muscle of the following groups are presented: the group consisting of the suspended tail, the group consisting of the suspended tail plus the traditional Chinese medicine composition of this invention, the group consisting of the suspended tail plus resveratrol, and the normal control group.

[0137] Appendix Figure 7 A box plot comparing the size of the extensor digitorum longus muscle fibers in the four experimental groups is provided.

[0138] From the appendix Figure 3-6 It can be seen that the number of degenerated muscle fibers in the suspended tail group is greater, the cells are lightly stained, and the cross-sectional area of ​​the muscle tissue in the suspended tail group is smaller than that in the normal control group.

[0139] The muscle fiber outer membrane of the group consisting of *Suspension Tail* and the herbal composition of this invention was clear, and the cell nuclei were small and regular. Compared with the *Suspension Tail* group, the cross-sectional area of ​​the muscle in the group consisting of *Suspension Tail* and the herbal composition of this invention was increased.

[0140] In the tail suspension + resveratrol group, the outer membrane of muscle fibers was slightly wrinkled, some muscle cell nuclei were swollen, and a small number of inflammatory cells with dark blue staining and small volume were visible. Compared with the tail suspension group, the cross-sectional area of ​​muscle in the tail suspension + resveratrol group increased.

[0141] In the normal control group, the outer membrane of the muscle fibers was clear, and the cell nuclei were small and regular.

[0142] Pathological results indicate that the herbal composition of this invention can increase muscle strength and muscle mass.

[0143] In summary, the traditional Chinese medicine composition of this invention can improve the endurance and balance of sarcopenic mice, enhance muscle strength, and can be used for the prevention and treatment of sarcopenia.

[0144] Example 8

[0145] The present invention also conducted behavioral experiments using a D-galactose-induced aging mouse model to confirm the efficacy of the traditional Chinese medicine composition of the present invention in treating age-related sarcopenia.

[0146] I. Experimental Materials

[0147] Test sample: Capsules of traditional Chinese medicine composition prepared in accordance with Example 1 of the present invention, and the contents were taken.

[0148] Positive control: Nicotinic acid mononucleotide, abbreviated as NMN.

[0149] Experimental animals: Seventy-five male SPF-grade C57BL / 6J mice, aged 7-8 weeks and weighing approximately 18-22g, were purchased from Vital River Company (Animal Certificate No.: 1100112011036225). All mice were housed in SPF-grade animal barriers with alternating 12-hour light and dark cycles, a temperature of 20-26℃, and a relative humidity of 40%-70%. Clean feeding and drinking equipment was provided. During the experiment, the animals had free access to regular feed and sterile water. Cages and bedding were changed regularly. Mice were housed in separate stainless steel cages, with 3-4 mice per cage.

[0150] II. Experimental Methods

[0151] 1. Experimental drugs

[0152] 0.5% Sodium Carboxymethyl Cellulose (CMC-Na) Solution: Weigh 5g of solid powder, mix it with a stir bar and 900ml of double-distilled water in a beaker, dissolve it thoroughly with a magnetic stirrer, and then add double-distilled water to bring the volume to 1000ml. Store the prepared 0.5% CMC-Na solution at 4℃ for later use.

[0153] Test sample gavage storage solution: Weigh 20g of the contents powder of the capsule prepared in Example 1, place it in a mortar, add a small amount of CMC-Na solution to completely cover the powder and grind it vigorously, then measure CMC-Na solution to make up to 200ml, stir and sonicate for 1h to fully dissolve, and prepare a test sample gavage storage solution with a final concentration of 0.1g / ml. Store the prepared suspension in a refrigerator at 4℃ for later use.

[0154] NMN oral storage solution: Take 1000mg of NMN capsule contents powder, grind it into a very fine state, mix it with 100ml of CMC-Na solution, continue grinding for 3min, make up to 200ml, sonicate for 60min to obtain NMN oral storage solution with a final concentration of 5mg / ml, and store the prepared suspension in a 4℃ refrigerator for later use.

[0155] D-galactose (D-gal) solution: Dissolve 300 mg of D-galactose powder in 25 mL of sterile water for injection in a centrifuge tube, then bring the volume to 30 mL and gently shake to mix. Filter with a microporous membrane to obtain a D-galactose solution with a concentration of 10 g / mL.

[0156] 2. Grouping of experimental animals

[0157] Seventy-five male C57BL / 6J mice were acclimatized for one week and then randomly divided into five groups according to their body weight: normal group, model group, low-dose test sample group (LDBZ group), high-dose test sample group (HDBZ group), and nicotinamide mononucleotide capsule group (NMN group), with 15 mice in each group. All mice were fed normal diet, provided with sterile drinking water, and underwent a 12-hour light / dark cycle daily.

[0158] 3. Modeling and drug administration

[0159] The clinical equivalent dose of the traditional Chinese medicine composition capsules prepared in Example 1 is 0.5 g / kg / d. The test samples selected in this application were based on this recommended dosage. The dosage of the low-dose group of the test samples was twice the clinical equivalent dose, i.e., 1 g / kg / d, and the dosage of the high-dose group of the test samples was 2 g / kg / d.

[0160] The Normal group received a subcutaneous injection of sterile water (0.1 ml / 10 g) into the neck and back of mice, while the other four groups received a subcutaneous injection of D-gal solution (100 mg / kg) into the neck and back of mice. This administration was repeated for 12 weeks. Starting from week 7, mice in the LDBZ and HDBZ groups were administered the test sample solution by gavage daily at 1 g / kg and 2 g / kg, respectively. Mice in the NMN group were administered 100 mg / kg NMN solution by gavage daily, while mice in the Normal and Model groups were administered the corresponding volume of 0.5% CMC-Na solution by gavage. This continued for 6 weeks.

[0161] The D-gal aging model is the most commonly used subacute aging animal model in China. Compared to naturally occurring aging animal models, this model is quicker to prepare, less expensive, easier to establish, and can last for more than 30 days, facilitating subsequent experiments, making it an ideal endogenous aging model. Studies have shown that nicotinamide mononucleotide (NMN) can increase the content of nicotinamide adenine dinucleotide (NAD+) to delay aging and effectively intervene in the progression of age-related diseases; therefore, NMN capsules were selected as a positive control drug. After administration, observations showed that the Model group mice exhibited dry skin, fine lines, reduced elasticity, dullness, and significantly decreased activity, indicating successful D-gal model establishment.

[0162] 4. General observation

[0163] During the experiment, we observed the mice's general condition daily, including their eating, drinking, urination and defecation, fur color, mental state, and activity.

[0164] 5. Mouse pulling force measurement experiment

[0165] After drug administration, a tensile strength measurement experiment was conducted to evaluate changes in mouse muscle strength. The DFE2-002 grip strength meter was placed horizontally on the operating table. The experimental mouse was gently placed on the grip grid, and after the mouse crawled across the grid until its forepaws and hind paws were within the grid area, the mouse's tail was grasped with one hand and pulled horizontally and evenly backward until all four limbs completely released the grid. At this point, the data acquisition device displayed the mouse's maximum grip strength for this experiment. Each mouse underwent three measurements, and the average value was taken, with each measurement spaced 10 minutes apart. Before the formal experiment, the mice underwent acclimatization training for three consecutive days.

[0166] 6. Mouse fatigue rotarod test

[0167] After drug administration, a rotarod test was performed on mice to determine the rotarod latency time, which was used to evaluate the mice's balance ability. Five mice were placed on the rotarod simultaneously each time. The test duration was set to 300 seconds, the initial speed was 5 r / min, the maximum speed was 30 r / min, the acceleration time was 30 seconds (i.e., it takes 30 seconds to accelerate to the maximum speed), and the rotation was clockwise. The instrument (IITC rat and mouse rotarod fatigue tester) was started, and the mice began to run. When a mouse became fatigued and fell off the rotarod, touching the sensor plate, the control panel displayed the mouse's movement time. Mice underwent adaptive training at a lower rotation speed for 3 consecutive days before the formal experiment. On the 4th day, each mouse underwent 3 repeated measurements, and the average results were taken. A 20-minute rest interval was maintained between each measurement.

[0168] 7. Mouse suspension test

[0169] After drug administration, a suspension test was conducted to evaluate the mice's grasping and motor coordination abilities. A homemade suspension test apparatus was used: a circular sieve with a diameter of 45 cm was placed between two platforms, 50 cm above the clean bedding at the bottom, ensuring stability. During the experiment, the mice were placed on the circular sieve, and once they had a firm grip with their four paws, the sieve was inverted, suspending them. The time it took for them to fall freely was recorded using a timer. Three tests were conducted consecutively, with 20-minute intervals between each test, and the average value was calculated. Mice underwent acclimatization training for three consecutive days prior to the formal experiment.

[0170] 8. Data Statistical Analysis

[0171] All experimental measurement data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 25.0 software. One-way ANOVA was used to compare means between groups, with tests for homogeneity of variance. If variances were homogeneous, the LSD test was used for pairwise comparisons between groups; if variances were unequal, Dunnett's T3 test was used for pairwise comparisons between groups. Data were considered statistically significant with p < 0.05.

[0172] III. Experimental Results

[0173] 1. Appearance of fur in each group of mice

[0174] After the drug administration was completed, the changes in the fur of mice in each group were observed by taking photos. Mice in the Normal group had smooth, shiny black fur, normal activity, and a docile temperament. Mice in the Model group had sparse, dull fur, with some showing signs of whitening. Some mice also showed localized hair loss, reduced activity, and poor mental state. Mice in the LDBZ and HDBZ groups showed significant improvement compared to the Model group. Their fur was thick, smooth, and close to the skin, with a bright color. No hair loss or whitening was observed. They were in good mental state, active, and highly adaptable. Mice in the NMN group had smoother, brighter fur than the Model group. No obvious hair loss or whitening was observed. Their activity was normal.

[0175] 2. Results of mouse behavioral experiments

[0176] Table 4 presents the results of the mouse behavioral experiments of this invention. The results show that, compared with the Normal group, the Model group had significantly reduced suspension time, grip strength, and rotarod dwell time (P < 0.01). Compared with the Model group: the suspension time of both the LDBZ and HDBZ groups was significantly increased (P < 0.01); the grip strength of the LDBZ group was significantly increased (P < 0.05), and the grip strength of the HDBZ group was significantly increased (P < 0.01); the rotarod dwell time of mice in the HDBZ group was significantly increased (P < 0.01), while there was no statistically significant difference in rotarod dwell time in the LDBZ group (P > 0.05); the NMN group showed only a significant increase in grip strength (P < 0.01), with no statistically significant difference in suspension time and rotarod dwell time (P > 0.05).

[0177]

[0178] IV. Conclusion

[0179] The D-gal aging model leads to aging of many organs, including the immune system, heart, and brain, and the aging process is accompanied by a decline in learning and memory abilities, motor skills, and immune function. This study can improve the motor abilities of aging mice. In this experiment, compared with the Normal group, the Model group mice showed significantly reduced suspension time, grip strength, and rotarod dwell time (P<0.01). Compared with the Model group, the test sample group mice showed significantly improved muscle strength, balance, grip, and motor coordination, indicating that the herbal composition of this invention can improve the motor abilities of aging mice and has a significant therapeutic effect on age-related sarcopenia.

[0180] Example 9

[0181] This invention also used 11-month-old BALB / c mice as research subjects, and constructed a sarcopenia model by hindlimb suspension method to investigate the effect of the traditional Chinese medicine composition of this invention in preventing and treating sarcopenia.

[0182] 1. Experimental Materials

[0183] 1.1 Test sample

[0184] Take the contents of the capsule prepared according to Example 1.

[0185] 1.2 Positive Drug

[0186] none.

[0187] 1.3 Experimental System

[0188] 1.3.1 Animal strain: BALB / c mouse.

[0189] 1.3.2 Animal grade: SPF grade.

[0190] 1.3.3 Animal gender and quantity: 50 animals, with an equal number of males and females.

[0191] 1.3.4 Animal age at the start of dosing: 11 months old.

[0192] 1.3.5 Animal weight at the start of dosing: actually 20 - 30 g.

[0193] 1.3.6 Animal source: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0194] 1.3.7 Animal certificate number, issuing unit, and receiving date:

[0195] Certificate number: 110322211103085363, License number: SCXK(Beijing)2019 - 0008, Issuing unit: Beijing Huafukang Biotechnology Co., Ltd., Receiving date: November 22, 2021.

[0196] Certificate number: 110322210102868962, License number: SCXK(Beijing)2019 - 0008, Issuing unit: Beijing Huafukang Biotechnology Co., Ltd., Receiving date: November 22, 2021.

[0197] 1.3.8 Rearing conditions: Mice are caged, with 12 - hour light per day, temperature 20 - 26 °C, relative humidity 40 - 70%, Experimental Animal Use License number: SYXK(Hebei)2020 - 003.

[0198] 1.3.9 Quarantine process: Animals are quarantined for 3 days and adaptively reared for 2 days. During this period, the drinking water, food intake, and health status of the animals are observed, as well as the presence of signs of disease and death.

[0199] 1.3.10 Feed: SPF mouse and rat maintenance feed, provided by Beijing Keao Xieli Feed Co., Ltd. Feed production license number: Beijing Feed License(2018)06073, Feed batch numbers: 20103213, 20113213.

[0200] 1.3.11 Drinking water: Drink clean water prepared by the ROA50 experimental animal drinking water system (instrument number YL - LE - A01). The drinking water bottles are filled for free drinking, and the water quality is tested to meet the "Hygienic Standard for Drinking Water".

[0201] 1.3.12 Litter: Ordinary - grade corncob litter. The litter is used after being sterilized by high - temperature and high - pressure, provided by Beijing Keao Xieli Feed Co., Ltd., Litter batch number: 20079811.

[0202] 1.3.13 Identification: Marked with ear tags for experimental animals.

[0203] 2 Experimental methods

[0204] 2.1 Experimental Design

[0205] 2.1.1 Design basis: relevant literature and materials.

[0206] 2.1.2 Experimental System Selection Instructions

[0207] BALB / c mice: BALB / c mice are an inbred strain, bred from siblings, resulting in smaller individual differences, purer genetic material, and better overall health. They are widely used in animal experiments for immunology and physiology. Therefore, BALB / c mice are used to create a model for research.

[0208] 2.2 Dosage and Grouping

[0209] The subjects were divided into a normal group (equal volume of distilled water), a model group (equal volume of distilled water), a high-dose test sample group (4 g / kg / d), a medium-dose test sample group (2 g / kg / d), and a low-dose test sample group (1 g / kg / d), and were given drug intervention.

[0210] The recommended daily dosage of the test drug is 2.4 g of crude drug per person (based on a body weight of 60 kg). For experimental mice, the low-dose, medium-dose, and high-dose groups were set at 1 g crude drug / kg, 2 g crude drug / kg, and 4 g crude drug / kg, respectively. Fifty BALB / c mice were randomly divided into five groups based on their body weight: normal group, model group, high-dose group, medium-dose group, and low-dose group. See Table 5 for details.

[0211]

[0212] 2.3 Method of administering test sample

[0213] Gavage, consistent with the intended route.

[0214] 2.4 Preparation and storage of test samples

[0215] High-dose test sample group: 2.4g capsule contents + 12ml CMC-Na, prepared and used immediately on the same day.

[0216] Medium-dose group of test sample: 1.2g capsule contents + 12ml CMC-Na, prepared and used immediately on the same day.

[0217] Low-dose test sample group: 0.6g capsule contents + 12ml CMC-Na, prepared and used immediately on the same day.

[0218] Based on the dosage of 10 ml / kg and the dosage settings in Table 5, calculate the concentration, weigh the contents of the capsules according to the number of animals, add solvent, grind and suspend, and make up to volume. The test sample should be prepared fresh before use. The solvent is 0.5% CMC-Na, which is evenly spread on the surface of pure water to fully swell, made up to volume, and stored at 2-8℃ for later use.

[0219] 2.5 Administration of the test sample

[0220] Animals were administered the corresponding volume of solvent or drug solution via gavage using a syringe at a dose of 10 ml / kg. The model group, high-dose group, medium-dose group, and low-dose group were given the appropriate volume of solvent or drug solution. The solution was changed every 48 hours.

[0221] 2.6 Model Making

[0222] A sarcopenia model was established using a hindlimb suspension method. Specifically, the mouse's hind limbs were raised, causing its head to tilt downwards at a 30° angle, resulting in fluid displacement of the head and preventing weight-bearing on the hind limbs. The mouse model of sarcopenia was induced by hind limb suspension for two weeks.

[0223] 2.7 Detection Indicators

[0224] 2.7.1 Hind limb grip strength

[0225] Two weeks after drug administration, the gripping force was tested. The gripping force meter was set to peak tension mode, and the unit was kilogram-force. During the test, one hand held the mouse's head and forelimbs, tilting it at a 45° angle to the ground, while the other hand held its tail so that its hind paws gripped the metal wire. Then, the mouse's tail was gently pulled, and the gripping force value was read from the gripping force meter screen and recorded. Each mouse was tested three times, and the average value was taken as the gripping force.

[0226] 2.7.2 Cross-sectional area of ​​muscle fibers

[0227] Muscle tissue was fixed in 10% neutral formalin solution, routinely embedded in paraffin, and prepared into 4 μm thick sections. HE staining was used to assess the degree of muscle fiber atrophy, and the tissue was observed and photographed under a microscope.

[0228] 2.8 Instrument System

[0229] Mettler ME104E analytical balance.

[0230] DT2000 electronic balance, manufactured by Changshu Shuangjie Test Instruments Factory.

[0231] DFE2-002 grip strength tester, Chatilon.

[0232] Paraffin slicer, LEICA, Germany.

[0233] Tissue section staining machine, Sakura, Japan.

[0234] 2.9 Experimental Reagents

[0235] Hematoxylin staining solution Beijing Solarbio Technology Co., Ltd.

[0236] Eosin staining solution Beijing Solarbio Technology Co., Ltd.

[0237] Neutral resin mounting tablets Shanghai Beyotime Biotechnology Co., Ltd.

[0238] 2.10 Statistical Methods

[0239] Experimental data were analyzed using SPSS 22.0 statistical software, with significance levels of 0.05 and 0.01. Results are expressed as mean ± standard deviation. The statement indicates that a normality test is first performed. For data that conforms to a normal distribution and has homogeneous variances, one-way ANOVA is used to compare means. If the variances are not homogeneous or do not conform to a normal distribution, nonparametric tests are used. Chi-square test or rank-sum test is used for count data.

[0240] 3 Results

[0241] 3.1 Hind limb grip strength

[0242] Table 6 shows the results of the hindlimb grip strength test of mice in each experimental group.

[0243]

[0244] As shown in Table 6, compared with the normal group, the hind limb muscle strength of mice in the model group was significantly reduced (P<0.05); compared with the model group, the hind limb muscle strength of mice in the medium-dose group and the high-dose group of the test product was significantly increased (P<0.05).

[0245] 3.2 Cross-sectional area of ​​muscle fibers

[0246] Table 7 shows the results of the detection of the cross-sectional area of ​​gastrocnemius muscle fibers in mice in each experimental group. Compared with the normal group, the cross-sectional area of ​​muscle fibers in the model group mice was significantly reduced (P < 0.05), indicating successful model establishment. Compared with the model group, the cross-sectional area of ​​muscle fibers in the high-dose group of mice was significantly increased (P < 0.01), and the cross-sectional area of ​​muscle fibers in the medium-dose group of mice was significantly increased (P < 0.05).

[0247]

[0248] Appendix Figure 8 HE-stained electron micrographs (20x) of the cross-sectional area of ​​the gastrocnemius muscle fibers in mice from each experimental group are provided. Figure 8It can be seen that the gastrocnemius muscle fibers in the normal group mice are neatly and tightly arranged; while the muscle fibers in the model group are disordered and broken. The pathological structure of muscle fibers in the medium and high dose groups of the test sample was improved, and most of the muscle fibers were neatly arranged.

[0249] 4. Conclusion

[0250] The results of this experiment show that the traditional Chinese medicine composition described in this invention can significantly improve the limb strength of mice with sarcopenia caused by reduced exercise and improve the muscle atrophy in mice with sarcopenia.

[0251] Example 10

[0252] This invention further investigated the therapeutic effect of the traditional Chinese medicine composition of this invention on sarcopenia using a mouse model of sarcopenia. The specific experimental details are as follows:

[0253] 1. Experimental Materials

[0254] 1.1 Test sample

[0255] 1.1.1 Test sample: The contents (BZBS) of the capsule prepared according to Example 1 were taken.

[0256] 1.1.2 Route and dosage: Oral administration

[0257] 1.1.6 Source and batch number: Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch number is XB2103001.

[0258] Positive test drugs, main reagents and materials

[0259] none

[0260] 1.3 Experimental System

[0261] 1.3.1 Animal strain: BALB / c mouse.

[0262] 1.3.2 Animal grade: SPF grade.

[0263] 1.3.3 Animal sex and number: Purchase 250 animals, 50 males and 200 females. Select 50 animals (half male and half female) for this experiment.

[0264] 1.3.4 Animal age at the start of administration: 11 months.

[0265] 1.3.5 Animal body weight at the start of administration: 20-30g.

[0266] 1.3.6 Animal source: Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0267] 1.3.7 Animal Certificate Number, Issuing Authority, and Date of Acceptance:

[0268] Certificate number: 110322211103085363, License number: SCXK(Beijing)2019 - 0008, Issuing unit: Beijing Huafukang Biotechnology Co., Ltd., Receiving date: November 22, 2021.

[0269] Certificate number: 110322210102868962, License number: SCXK(Beijing)2019 - 0008, Issuing unit: Beijing Huafukang Biotechnology Co., Ltd., Receiving date: November 22, 2021.

[0270] 1.3.8 Feeding conditions: Mice are caged and raised in the New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd. The lighting is 12 hours per day, the temperature is 20 - 26°C, and the relative humidity is 40 - 70%. License number for the use of experimental animals: SYXK(Hebei)2020 - 003.

[0271] 1.3.9 Quarantine process: Animals are quarantined for 3 days and adaptively raised for 2 days. During this period, the drinking water, food intake, and health status of the animals are observed, as well as the signs of disease and death.

[0272] 1.310 Feed: SPF mouse and rat maintenance feed, provided by Beijing Keao Xieli Feed Co., Ltd. Feed production license number: Beijing Feed License(2018)06073, Feed batch numbers: 20103213, 20113213.

[0273] 1.3.11 Drinking water: Drink clean water prepared by the ROA50 experimental animal drinking water system (instrument number YL - LE - A01). The drinking water bottles are filled and provided for free drinking. The water quality has been tested and complies with the "Hygienic Standard for Drinking Water".

[0274] 1.3.12 Litter: Ordinary - grade corn cob litter. The litter is used after being sterilized by high - temperature and high - pressure, provided by Beijing Keao Xieli Feed Co., Ltd. Litter batch number: 20079811.

[0275] 1.3.13 Identification: Mark the ear tags of experimental animals.

[0276] 2 Test methods

[0277] 2.1 Basis for test design

[0278] 2.1.1 Design basis: Relevant literature materials.

[0279] 2.1.2 Explanation for the selection of experimental system:

[0280] BALB / c mice: BALB / c mice are an inbred strain, bred from siblings, resulting in smaller individual differences, purer genetic material, and better overall health. They are widely used in animal experiments for immunology and physiology. Therefore, BALB / c mice are used to create a model for research.

[0281] 2.2 Dosage and Grouping

[0282] The patients were divided into a normal group (equal volume of distilled water), a model group (equal volume of distilled water), a high-dose BZBS group (4 g / kg / d), a medium-dose BZBS group (2 g / kg / d), and a low-dose BZBS group (1 g / kg / d), and were given medication intervention.

[0283] The daily dosage of BZBS capsules for humans is 2.4g of crude drug (based on a human body weight of 60kg). For mice, the low, medium, and high doses of BZBS were set at 1g crude drug / kg, 2g crude drug / kg, and 4g crude drug / kg, respectively. Fifty BALB / c mice were randomly divided into five groups according to body weight: a control group, a model group, a high-dose BZBS group (BZ-high), a medium-dose BZBS group (BZ-middle), and a low-dose BZBS group (BZ-low) (see Table 8).

[0284]

[0285] 2.3 Method of administering test sample

[0286] Gavage, consistent with the intended route.

[0287] 2.4 Preparation and storage of test samples

[0288] High-dose BZBS: 2.4g BZBS ultrafine powder + 12ml CMC, prepare and use immediately on the same day.

[0289] Medium dose of BZBS: 1.2g BZBS ultrafine powder + 12ml CMC, prepare and use immediately on the same day.

[0290] BZBS low dose: 0.6g BZBS ultrafine powder + 12ml CMC, prepare and use immediately on the same day.

[0291] Based on the dosage of 10 ml / kg and the prescribed dosage, weigh out the BZBS according to the number of animals, add solvent, grind and suspend, and make up to volume. Prepare the test sample immediately before use. The solvent is 0.5% CMC-Na, which is evenly spread on the surface of pure water to fully swell, and then made up to volume. Store at 2-8℃ until use.

[0292] 2.5 Administration of the test sample

[0293] The test sample was administered using a syringe at a volume of 10 ml / kg. Animals in the model group, high-dose BZBS group, medium-dose BZBS group, and low-dose BZBS group were given the corresponding volume of solvent or drug solution, which was changed every 48 hours.

[0294] 2.6 Model Making

[0295] A sarcopenia model was established using a hindlimb suspension method: the hind limbs of mice were raised, causing their heads to tilt downwards by 30°, resulting in fluid displacement of the head and avoiding weight-bearing on the hind limbs. The hind limb suspension was maintained for 2 weeks to induce and establish a sarcopenia mouse model.

[0296] 2.7 Detection Indicators

[0297] 2.7.1 Sirius Crimson Staining

[0298] Muscle tissue was fixed in 10% neutral formalin solution, routinely embedded in paraffin, and prepared into 4 μm thick sections. Intramuscular fiber deposition was assessed using Sirius red staining, and the tissue was observed and photographed under a microscope.

[0299] 2.7.2 Satellite cell activation, proliferation, and differentiation related indicators

[0300] qPCR detects PAX7, MYOD, and MYOG.

[0301] 2.8 Instrument System

[0302] Mettler ME104E analytical balance.

[0303] DT2000 electronic balance, manufactured by Changshu Shuangjie Test Instruments Factory.

[0304] DFE2-002 grip strength tester, Chatilon.

[0305] Paraffin slicer, LEICA, Germany.

[0306] Tissue section staining machine, Sakura, Japan.

[0307] 2.9 Experimental Reagents

[0308] Hematoxylin staining solution Beijing Solarbio Technology Co., Ltd.

[0309] Eosin staining solution Beijing Solarbio Technology Co., Ltd.

[0310] Neutral resin mounting tablets Shanghai Beyotime Biotechnology Co., Ltd.

[0311] 2.10 Statistical Methods

[0312] Experimental data were analyzed using SPSS 22.0 statistical software, with significance levels of 0.05 and 0.01. Results are expressed as mean ± standard deviation. The statement indicates that a normality test is first performed. For data that conforms to a normal distribution and has homogeneous variances, one-way ANOVA is used to compare means. If the variances are not homogeneous or do not conform to a normal distribution, nonparametric tests are used. Chi-square test or rank-sum test is used for count data.

[0313] 3 Results

[0314] 3.1 Sirius Crimson Staining

[0315] Appendix Figure 9 The deposition of intramuscular fibers in mice in each experimental group is presented.

[0316] 3.2 Satellite cell activation, proliferation, and differentiation related indicators

[0317] Appendix Figures 10-12 Results of satellite cell activation, proliferation and differentiation related indicators (PAX7, MYOD, MYOG) in mice of each experimental group are presented.

[0318] 4. Conclusion

[0319] The BZBS provided by this invention can improve intramuscular fiber deposition in sarcopenic mice and can restore the normal morphology of muscles in sarcopenic model mice and maintain their normal function by promoting the proliferation and differentiation of satellite cells (the main type of skeletal muscle stem cells) into mature muscle cells.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating sarcopenia, characterized in that, This traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 150-350, Lycium barbarum 100-200, Schisandra chinensis 30-60, Cnidium monnieri 20-50, Rosa laevigata 20-50, Allium tuberosum 20-50, Morinda officinalis 20-50, Cistanche deserticola 20-50, Rehmannia glutinosa 30-60, Achyranthes bidentata 20-50, Epimedium brevicornu 40-100, Rubus idaeus 20-50, Panax ginseng 15-35, Cervus nippon antler 10-25, Hippocampus 15-35, and Melia toosendan 15-35.

2. The application according to claim 1, characterized in that, This traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 150, Lycium barbarum 200, Schisandra chinensis 30, Cnidium monnieri 50, Rosa laevigata 20, Allium tuberosum 20, Morinda officinalis 50, Cistanche deserticola 20, Rehmannia glutinosa 30, Achyranthes bidentata 50, Epimedium brevicornu 40, Rubus idaeus 50, Panax ginseng 15, Cervus nippon antler 25, Hippocampus 15, and Melia toosendan 35.

3. The application according to claim 1, characterized in that, This traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 350, Lycium barbarum 100, Schisandra chinensis 60, Cnidium monnieri 20, Rosa laevigata 50, Allium tuberosum 50, Morinda officinalis 20, Cistanche deserticola 50, Rehmannia glutinosa 60, Achyranthes bidentata 20, Epimedium brevicornu 100, Rubus idaeus 20, Panax ginseng 35, Cervus nippon antler 10, Hippocampus 35, and Melia toosendan 15.

4. The application according to claim 1, characterized in that, This traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 250, Lycium barbarum 138, Schisandra chinensis 46, Cnidium monnieri 35, Rosa laevigata 35, Allium tuberosum 35, Morinda officinalis 35, Cistanche deserticola 35, Rehmannia glutinosa 46, Achyranthes bidentata 35, Epimedium brevicornu 70, Rubus idaeus 35, Panax ginseng 25, Cervus nippon antler 16, Hippocampus 21, and Melia toosendan 23.

5. The application according to claim 1, characterized in that, This traditional Chinese medicine composition is made from the following raw materials in parts by weight: Cuscuta chinensis 250, Lycium barbarum 150, Schisandra chinensis 45, Cnidium monnieri 30, Rosa laevigata 30, Allium tuberosum 30, Morinda officinalis 30, Cistanche deserticola 30, Rehmannia glutinosa 45, Achyranthes bidentata 30, Epimedium brevicornu 70, Rubus idaeus 30, Panax ginseng 20, Cervus nippon antler 19, Hippocampus 20, and Melia toosendan 20.

6. The application according to any one of claims 1-5, characterized in that, The dosage form of the drug is decoction, capsule, tablet, granule, powder or pill.

7. The method for preparing the capsules as described in claim 6, characterized in that, The preparation method includes the following steps: a. Weigh out Cnidium monnieri, Cuscuta chinensis, Schisandra chinensis and Epimedium according to the proportion, add 6-10 times the amount of 70% ethanol, reflux extract 1-3 times, 1-3 hours each time, filter, combine the filtrates, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the alcohol extract. b. Combine the medicinal residue after alcohol extraction in step a with the appropriate amounts of wolfberry, rehmannia root, raspberry, rosehip, leek seed, morinda root, and Sichuan pepper, add 7-12 times the amount of water and decoct 1-3 times, 1-3 hours each time. Filter, combine the filtrates, mix with the alcohol extract obtained in step a, and concentrate under reduced pressure until the relative density is 1.25-1.30 when measured at 60℃ to obtain the extract. c. Weigh out ginseng, deer antler, seahorse, cistanche and achyranthes according to the proportion, grind them into fine powder, mix them evenly with the extract obtained in step b, dry at 60-70℃, grind, sieve and fill into capsules.

8. The application according to any one of claims 1-5, characterized in that, The sarcopenia mentioned is senile sarcopenia.

9. The application according to any one of claims 1-5, characterized in that, The aforementioned sarcopenia is known as sarcopenia-osteoporosis.

10. The application according to any one of claims 1-5, characterized in that, The traditional Chinese medicine composition achieves the effect of preventing and treating sarcopenia by improving the body's endurance and balance.

11. The application according to any one of claims 1-5, characterized in that, The herbal composition described herein achieves the effect of preventing and treating sarcopenia by enhancing muscle strength and increasing muscle mass.

12. The application according to any one of claims 1-5, characterized in that, Sarcopenia is muscle atrophy caused by reduced physical activity.

13. The application according to any one of claims 1-5, characterized in that, The application of the traditional Chinese medicine composition in drugs for improving intermuscular fiber deposition.

14. The application according to any one of claims 1-5, characterized in that, The application of the traditional Chinese medicine composition in drugs that promote the proliferation and differentiation of satellite cells into mature muscle cells.

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