Composition and application thereof in preparation of medicine for treating osteoporosis-sarcopenia-obesity triple symptoms

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a composition and application thereof in preparation of a medicine for treating osteoporosis-sarcopenia-obesity triple symptoms. The invention provides a composition. The composition contains sanggenone C, morusin, sanggenone D, phellinus linteus ketone G and morusin. The composition can synchronously regulate and control bone, muscle and fat tissues; osteoclast differentiation is inhibited, and osteogenesis is promoted; the muscle strength and endurance are enhanced, and the quality of gastrocnemius muscle and soleus muscle is improved; visceral fat is reduced, white fat browning is promoted, and fat reduction without muscle reduction is achieved. Compared with an existing single-target medicine, the composition has the advantages that the muscle function and fat metabolism are remarkably improved while the bone mineral density is improved, and the limitation of single treatment of a single disease is overcome. The composition is derived from plants, is high in safety, has a synergistic effect when being combined with glucosamine chondroitin calcium tablets, and has good clinical transformation and application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine. More specifically, it relates to a composition and its use in the preparation of a medicament for treating osteoporosis-sarcopenia-obesity syndrome. BACKGROUND

[0002] With the accelerating process of global aging, osteoporosis (OP), sarcopenia (SP) and obesity in the elderly population show a high tendency of comorbidity. These three diseases are not isolated from each other, but are intertwined through various mechanisms such as chronic inflammation, metabolic disorders, hormonal disorders and mechanical imbalance, and together form a complex clinical syndrome known as "osteosarcopenic obesity" (OSO). Epidemiological studies have shown that OSO patients not only face higher risks of fractures, falls and disability, but also have significantly higher all-cause mortality than individuals with only one of the three diseases, making it a major public health problem that needs to be addressed urgently.

[0003] The pathological core of OSO lies in the vicious cycle formed between bone, muscle and fat. On the one hand, excessive accumulation of visceral fat caused by obesity leads to the secretion of a large number of pro-inflammatory factors (such as TNF-α, IL-6), activation of inflammatory signaling pathways such as NF-κB, inhibition of muscle protein synthesis and promotion of ubiquitin-proteasome system-mediated muscle breakdown, and stimulation of osteoclast activity, exacerbating bone resorption. At the same time, obesity is often accompanied by insulin resistance, which weakens the positive regulation of insulin on muscle synthesis and bone formation. On the other hand, sarcopenia leads to a decrease in basal metabolic rate and a decrease in physical activity, further promoting fat accumulation; and the decrease in mechanical stimulation of muscle on bone accelerates bone loss. At the same time, chronic pain caused by osteoporosis and fear of falling limit physical activity, thereby exacerbating muscle atrophy and weight gain. The three are causally related, forming a pathological closed loop that is difficult to break.

[0004] Currently, there is no specific drug for OSO in clinical practice, and existing treatment strategies mostly follow the "single disease single treatment" model, which has obvious limitations. For example, anti-osteoporosis drugs may indirectly exacerbate sarcopenia and obesity by causing reduced activity in patients; there is no approved targeted drug for sarcopenia, and its efficacy is highly dependent on patient compliance and overall physical condition; and the new type of weight loss drugs (such as GLP-1 receptor agonists) widely used in recent years have shown significant effects in weight loss, but clinical studies have confirmed that they can cause more than 10% muscle loss while reducing fat, significantly worsening sarcopenia, and may adversely affect bone density due to rapid weight loss, thereby exacerbating the overall risk of OSO.

[0005] The deeper contradiction is that the therapeutic goal of OSO itself has inherent contradictions: weight loss helps to improve metabolic status, but may accelerate the loss of muscle and bone mass; and promoting muscle and bone mass growth requires sufficient nutrient intake and weight-bearing stimulation, which conflicts with the traditional "low-calorie" weight loss concept. Therefore, it is urgent to develop a multi-target intervention strategy that can simultaneously regulate bone formation and absorption, muscle synthesis and decomposition, and fat distribution and metabolism. SUMMARY

[0006] The present application aims to overcome the lack of drugs for osteoporosis-sarcopenia-obesity syndrome in the prior art, and the defects and deficiencies of existing drugs (such as anti-osteoporosis drugs or new weight loss drugs) that may exacerbate other comorbidities (such as muscle loss caused by weight loss, indirect exacerbation of obesity by anti-osteoporosis drugs, etc.) during treatment, and provides a composition that can simultaneously intervene in the functions of bone, muscle, and fat, and achieve the treatment of osteoporosis-sarcopenia-obesity syndrome (OSO).

[0007] The first object of the present application is to provide a composition.

[0008] The second object of the present application is to provide the use of the above-mentioned composition.

[0009] The third object of the present application is to provide a drug for treating osteoporosis-sarcopenia-obesity syndrome.

[0010] The above objects of the present application are achieved by the following technical solutions: The present application provides a composition containing mulberroside C, morusin, mulberroside D, morusinol, and moracin G; the mass ratio of mulberroside C, morusin, mulberroside D, morusinol, and moracin G is (1-2):(1-2):(1-2):(1-2):(1-2).

[0011] Preferably, the mass ratio of mulberroside C, morusin, mulberroside D, morusinol, and moracin G is (1-2):1:(1-2):(1-2):1.

[0012] Alternatively, the mass ratio of mulberroside C, morusin, mulberroside D, morusinol, and moracin G is 1:1:1:1:1.

[0013] Alternatively, the mass ratio of mulberroside C, morusin, mulberroside D, morusinol, and moracin G is 2:1:2:2:1.

[0014] This invention demonstrates that the composition can significantly inhibit osteoclast differentiation while promoting osteogenic differentiation of bone marrow mesenchymal stem cells, effectively improving bone microstructure and bone density. In terms of muscle, it significantly enhances grip strength, rotarod endurance, and relative wet weight of the gastrocnemius / soleus muscles in mice, improving sarcopenia. Regarding fat metabolism, it significantly reduces visceral fat accumulation and promotes the conversion of white adipose tissue to brown adipose tissue (upregulation of Ucp-1 and Pgc1α expression), enhancing energy expenditure and achieving the ideal intervention effect of "fat reduction without muscle loss." Therefore, this invention claims protection for the following applications: The use of the above composition in the preparation of a medicament for the prevention or treatment of osteoporosis-sarcopenia-obesity triad.

[0015] The use of the above composition in the preparation of drugs for the prevention or treatment of osteoporosis.

[0016] The use of the above composition in the preparation of drugs for the prevention or treatment of sarcopenia.

[0017] The use of the above composition in the preparation of drugs for the prevention or treatment of obesity.

[0018] Furthermore, the present invention provides a medicament for treating osteoporosis-sarcopenia-obesity triad, comprising the above-described composition.

[0019] Preferably, the above-mentioned drug further contains pharmaceutical excipients, including any one or a combination of at least two of phosphatidylcholine, PVP-K30, glyceryl monostearate, and stearic acid.

[0020] Preferably, in the above-mentioned drug, the mass ratio of the above-mentioned composition, phosphatidylcholine and PVP-K30 is 1:(2~3):(3~4).

[0021] As an alternative implementation, in the above-mentioned drug, the mass ratio of the above-mentioned composition, phosphatidylcholine and PVP K30 is 1:2:3.

[0022] Preferably, the above-mentioned drug also contains glucosamine chondroitin calcium tablets.

[0023] The present invention has the following beneficial effects: This invention provides a natural plant-derived composition composed of mulberry root ketone C, mulberry cinnamon, mulberry root ketone D, mulberry cinnamon ketone G, and mulberry cinnamon alcohol, which can be used to prevent or treat osteoporosis-sarcopenia-obesity triad (OSO). This composition has multi-target, synergistic regulatory effects on the bone-muscle-fat axis. 1. The composition of the present invention can significantly inhibit osteoclast differentiation (manifested as downregulation of TRAP activity and NFATc1 protein expression) and promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) (manifested as enhanced ALP activity, increased formation of mineralized nodules and upregulation of JNK expression), effectively improving bone microstructure and bone density. 2. In terms of muscles, it significantly improved the grip strength, rotarod endurance, and relative wet weight of the gastrocnemius / soleus muscles in mice, and improved sarcopenia; 3. In terms of fat metabolism, it significantly reduces visceral fat accumulation and promotes the conversion of white fat to brown fat (upregulation of Ucp-1 and Pgc1α expression), enhances energy consumption, and achieves the ideal intervention effect of "fat reduction without muscle loss".

[0024] Compared with alendronate sodium, a commonly used anti-osteoporosis drug in clinical practice, the composition of this invention not only has a comparable effect in improving bone density, but also shows significant advantages in improving muscle quality and function and promoting fat browning. It overcomes the limitations of the traditional "single disease, single treatment" strategy and, in particular, avoids the risk of muscle loss caused by weight loss drugs.

[0025] The compositions of the present invention are derived from natural sources, have good biocompatibility and safety, and are suitable for long-term use. The combination of the compositions of the present invention with glucosamine chondroitin calcium tablets can further enhance the effect and expand their application prospects in compound preparations or functional health products.

[0026] In summary, the composition of this invention achieves simultaneous intervention for the osteoporosis-sarcopenia-obesity triad for the first time, providing solid technical support for the development of innovative drugs or health products for the treatment of OSO syndrome, and has great application prospects and value. Attached Figure Description

[0027] Figure 1 The results show the effects of different treatment groups on the expression level of the JNK gene in bone marrow mesenchymal stem cells.

[0028] Figure 2 The effects of different treatment groups on the expression of NFATc1 protein, a marker transcription factor of osteoclasts, in bone marrow mesenchymal stem cells (Figure A shows the results of Western blot analysis; Figure B shows the results of Western blot grayscale analysis).

[0029] Figure 3 The effects of different treatment groups on the mRNA expression levels of Ucp-1 and Pgc-1a, key genes for white adipose tissue browning (Figure A shows the mRNA expression level of the Ucp-1 gene; Figure B shows the mRNA expression level of the Pgc-1a gene).

[0030] Figure 4 The results show the effects of different treatment groups on tartrate-resistant acid phosphatase in bone marrow macrophages (Figure A shows the staining results of tartrate-resistant acid phosphatase in cells of different treatment groups; Figure B shows the results of tartrate-resistant acid phosphatase content determination).

[0031] Figure 5The effects of different treatment groups on the formation of bone mineralization nodules in bone marrow mesenchymal stem cells (Figure A shows the results of Alizarin Red staining of cells in different treatment groups; Figure B shows the results of staining area analysis).

[0032] Figure 6 The results show the effects of different treatment groups on alkaline phosphatase activity in bone marrow mesenchymal stem cells.

[0033] Figure 7 The effects of the composition on the grip strength of the four limbs, the grip strength of the forelimbs, and the rotation bar test in OSO model mice are shown in Figure A (the results of the grip strength measurement of the four limbs; the results of the grip strength measurement of the forelimbs; and the results of the rotation bar test).

[0034] Figure 8 The results show the effects of the composition on the relative wet weight of the gastrocnemius muscle and the relative wet weight of the soleus muscle in OSO model mice (Figure A shows the relative wet weight of the gastrocnemius muscle; Figure B shows the relative wet weight of the soleus muscle).

[0035] Figure 9 Figure A shows the effect of the composition on bone mineral density in mice; Figure B shows the bone mineral content measurement results; Figure C shows the muscle content measurement results; Compared with the control group, #P<0.05, ####P<0.0001; Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0036] Figure 10 The results show the effect of the composition on body fat percentage in mice; Figure A shows the results of measuring the perirenal fat / body weight ratio in mice; Figure B shows the results of measuring the epididymal fat / body weight ratio in mice; Figure C shows the results of measuring the (epididymal fat + perirenal fat) / body weight ratio in mice; Figure D shows the results of measuring the brown fat / body weight ratio in mice; Compared with the control group, #P<0.05, ####P<0.0001; Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] In the following examples, the monomeric compounds (including morinone C, morinol, morinone D, morinone G, morinone B, morinone, naringin, naringenin, morinol, and icariin) were dissolved using DMSO, and the final concentration of DMSO in the solution was strictly controlled within 0.1% (v / v); the corresponding control group, induction group, and model group were also added with the same amount of DMSO to ensure that the experimental conditions were consistent.

[0040] Sancinin, CAS: 62596-29-6, has the following structural formula:

[0041] Phellinus linteus, CAS: 62949-79-5, has the following structural formula:

[0042] Morus root ketone C, CAS: 80651-76-9, has the following structural formula:

[0043] Morus bark ketone B, CAS: 62949-78-4, has the following structural formula:

[0044] Morus root ketone D, CAS: 81422-93-7, has the following structural formula:

[0045] Phellinus linteus G, CAS: 75629-19-5, has the following structural formula:

[0046] Morphine, CAS: 62949-93-3, has the following structural formula:

[0047] Naringin, CAS No.: 4493-40-7, has the following structural formula:

[0048] Naringenin, CAS No.: 480-41-1, has the following structural formula:

[0049] Icariin, CAS No.: 489-32-7, has the following structural formula:

[0050] Grasp force test method: Weigh each mouse before the test and bring the animal into the testing room at least one hour before the test to acclimatize to the environment. Gently remove the animal from the test cage, gently grasp its tail, and allow the animal to grasp the digital force gauge while being gently pulled parallel to the bar by its tail. Record the reading on the force gauge when the maximum force is applied to the experimental mouse. Repeat the measurement multiple times to ensure the results.

[0051] Forelimb gripping force test method: Place the experimental mouse on the gripping force meter, allowing its forelimbs to grasp the probe. Gently pull the mouse's tail to induce a gripping force. Record the gripping force meter reading when the mouse exerts maximum force, and repeat the measurement multiple times. The hind paws and forelimbs will be tested by allowing all four paws to grasp the bar and gently pulling the tail until release. After each test, clean the instrument with alcohol. Control the light / dark cycle, temperature, and humidity of the environment. The gripping force meter measures the maximum peak force in Newtons. Calculation and statistical results: The maximum force measured for each mouse (in Newtons (N)) is divided by its body weight to obtain force / body weight (N / g).

[0052] Rotating rod test method: The adaptation phase of the experiment is divided into three stages with no time interval between the three stages: In stage T1, the rotating rod is accelerated uniformly from rest to a speed of 5 r / min at an acceleration of 5 r / min; in stage T2, the acceleration is changed to 10 r / min, and the rod is accelerated to 15 r / min before rotating at a constant speed for 2 minutes; in stage T3, the rod is accelerated again to 20 r / min at an acceleration of 10 r / min, and then rotated at a constant speed for 2 minutes. If a mouse falls off during the test, the time of fall is recorded. After 10 minutes, the experiment is restarted for the fallen mouse, and the duration of the mouse on the rotating rod is recorded. During the adaptation experiment, the mice's physical strength and coordination abilities are trained. In the formal experimental stage, the acceleration remains unchanged in stages T1 and T2, and the uniform speed reaches 5 r / min and 40 r / min respectively at the end. The duration of the mouse on the rotating rod (s) is recorded.

[0053] Methods for establishing the osteoporosis-sarcopenia-obesity triad (OSO) model: Mice were fed a high-sugar, high-fat diet combined with a D-galactose mixed diet, and received intraperitoneal injections of dexamethasone (2.5 mg / kg) twice weekly for 6 weeks (1.5 months). After model establishment, the success of model construction was assessed using multidimensional indicators. Compared with the blank control group, the OSO model group exhibited the following significant pathological features, confirming the successful establishment of the osteoporosis-sarcopenia-obesity triad model: 1. Abnormal weight and glucose metabolism: The model group had a significant increase in weight and a significant increase in fasting blood glucose (Glu-G) level, indicating obesity and insulin resistance. 2. Bone metabolism disorder: Serum bone formation markers type I procollagen N-terminal propeptide (P1NP) and bone-specific alkaline phosphatase (BALP) were significantly decreased; bone resorption markers type I collagen C-terminal peptide (CTX-1) and tartrate-resistant acid phosphatase (TRAP) were significantly increased; at the same time, serum calcium (Ca) and serum phosphorus (P) levels decreased, consistent with the biochemical characteristics of osteoporosis. 3. Sarcopenia-related inflammatory state: The levels of myostatin (MSTN), C-reactive protein (CRP), and interleukin-6 (IL-6) are significantly elevated, reflecting the activation of chronic inflammation and the inhibition of muscle synthesis; 4. Abnormal lipid metabolism: Decreased levels of total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), and increased levels of low-density lipoprotein cholesterol (LDL-C), presenting typical obesity-related dyslipidemia; 5. Bone microstructure damage (Micro-CT analysis): In the model group, bone connectivity density (Conn.D.), bone volume fraction (BV / TV), number of trabeculae (Tb.N), and bone mineral density (BMD) were all significantly decreased; trabecular separation (Tb.Sp) was also reduced. 6. Reduced muscle function: The grid suspension test and grip strength test showed that the average grip strength of the model group mice was reduced by more than 35% compared with the blank group, indicating a significant reduction in muscle strength.

[0054] Example 1: Effects of different treatments on the MAPK / JNK pathway Within the MAPK / JNK pathway, JNK activation is a signal that promotes osteogenic differentiation. JNK is an important member of the MAPK signaling pathway, and it primarily responds to stress signaling factors such as inflammatory factors and oxidative stress.

[0055] I. Experimental Methods The mRNA expression of the JNK gene in bone marrow mesenchymal stem cells (BMSCs) under different treatments was detected by qRT-PCR. All drugs were added at the start of induction and continued until the end of induction (a total of 4 days of treatment). The final concentration of each active ingredient in groups A through L was 2 μM; the final concentration of the positive control drug, alendronate sodium, was also 10 μM.

[0056] After cell induction and drug administration, the supernatant was discarded, and the cells were washed twice with PBS. Cell RNA samples were used to synthesize cDNA according to the Evo M-MLV reverse transcription premix kit (brand: Aikerui Biotechnology, catalog number: AG11728), and then Real-time PCR amplification was performed according to the SYBR GreenPCR Master Mix (brand: ThermoFisher, catalog number: A25742) kit. Two... -ΔΔCtThe method calculates the relative gene expression level.

[0057] The processing groups are specifically grouped as follows: Control group: Uninduced bone marrow mesenchymal stem cells; Induction group: induced differentiated bone marrow mesenchymal stem cells; Alendronate sodium group: 10 μM alendronate sodium; Group A: Mori senna G, Mori root ketone C, Mori bark ketone B, Mori root ketone D, Icariin; Group B: Mori flavonoids G, Mori root ketone C, Mori bark ketone B, Mori root ketone D, Naringin; Group C: Mori senna G, Mori root ketone C, Mori cinnamic acid, Mori senna, Mori bark ketone B; Group D: Moriton G, Morigenin C, Morigenin, Moriton, Naringin; Group E: Morus root ketone D, Morus root ketone C, Morusin, Morus flavonoids, Morus bark ketone B; Group F: Morus root ketone D, Morus root ketone C, Morusin, Morus flavonoids, Icariin; Group G: Morus root ketone C, Morusin, Morus root ketone D, Morus flavonoid G, Morus bark ketone B; Group H: Morus root ketone C, Morusin, Morus root ketone D, Morus flavonoid G, Naringin; Group I: Mori root ketone C, Mori cinnamic acid, Mori root ketone D, Mori flavonoid G, Icariin; Group J: Morigenin C, Morigenin D, Morigenin G, Naringin; Group K: Morus root ketone C, Morus root ketone D, Morus root ketone G, Morus root ketone; Group L: Morus root ketone C, Morusin, Morus root ketone D, Morus huang ketone G, Morus huang ketone.

[0058] II. Experimental Results Test results as follows Figure 1 As shown, the results indicate that different treatment groups had significantly different regulatory effects on JNK gene mRNA expression after intervention with BMSCs, with group K showing the most significant effect, which was significantly better than other treatment groups. The decrease in JNK expression in the induced group suggests that simple induction may not be sufficient to maintain the sustained activation of the JNK pathway, requiring synergistic effects of exogenous regulatory factors. Most combinations exhibited moderate to strong activation effects.

[0059] Example 2: Effects of different treatments on the expression of osteoclast-related proteins in bone marrow macrophages of BMM patients. I. Experimental Methods Western blot analysis was used to detect the expression levels of the osteoclast marker transcription factor NFATc1 under different treatments. The treatment groups were the same as in Example 1, using primary mouse bone marrow macrophages (BMMs) induced to differentiate into mature osteoclasts by 100 ng / mL RANKL for 4 days. The drugs in each treatment group were added at the beginning of RANKL induction and continued until the end of induction (a total of 4 days of treatment). The final concentration of each active ingredient in groups A through L was 2 μM; the final concentration of the positive control drug, alendronate sodium, was also 10 μM.

[0060] The specific method is as follows: After different cell treatments, proteins were extracted using RIPA lysis buffer and quantified using the BCA method. Protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes, blocked in 5% skim milk for 1 h, and then incubated overnight at 4°C with the corresponding antibodies. Secondary antibodies were then added and incubated at room temperature for 1.5 h. The samples were then developed using a high-sensitivity ECL chemiluminescence assay kit, and the bands were quantitatively analyzed using ImageJ software.

[0061] II. Experimental Results Experimental results are as follows Figure 2 As shown, the results indicate that RANKL induction significantly upregulated NFATc1 protein expression, demonstrating the successful establishment of the osteoclast differentiation model. Compared with the induction group, all drug treatment groups inhibited NFATc1 expression to varying degrees. The positive control drug alendronate sodium significantly inhibited NFATc1 expression, and the effect of group K was more significant than that of the positive control group, suggesting that the combination of morinone C, morinol, morinone D, morinone G, and morinol may have a synergistic inhibitory effect on osteoclastogenesis.

[0062] Example 3: Effects of different treatments on Ucp-1 and Pgc-1a in a 3T3-L1 adipocyte adipogenesis induction model I. Experimental Methods In the 3T3-L1 adipogenic induction model, the expression of key genes Ucp-1 and Pgc-1a for the browning of white adipose tissue under different treatments was detected by qRT-PCR. The treatment groups were the same as in Example 1, except that the positive control group was Lipitor (10 μM), and the qRT-PCR detection method was the same as in Example 1.

[0063] II. Experimental Results Experimental results are as follows Figure 3As shown, the results indicate that group K had the most significant effect compared to other treatment groups, significantly upregulating Ucp-1 mRNA expression. Although group K did not have the highest upregulation of Pgc-1α, considering its Ucp-1 induction ability, it is speculated that group K did not simply upregulate Pgc-1α transcription, but effectively and synergistically activated the "Pgc-1α→Ucp-1" functional axis. In summary, these results suggest that the combination of morula root ketone C, morula root ketone D, morula root ketone G, and morula root ketone can promote the conversion of white adipose tissue to brown adipose tissue and enhance energy expenditure.

[0064] Example 4: Effects of different treatments on osteoclast differentiation I. Experimental Methods Primary mouse bone marrow macrophages (BMMs) were induced to differentiate into mature osteoclasts by inducing them with 100 ng / mL RANKL for 4 days.

[0065] All drugs were added at the start of RANKL induction and continued to be administered until the end of induction (a total of 4 days of treatment). The final concentration of each active ingredient in groups A through F was 2 μM; the final concentration of the positive control drug, alendronate sodium, was also 10 μM.

[0066] The treatment groups are specifically divided as follows (Group A is Group K of Example 1): Control group: Uninduced primary bone marrow macrophages from mice; Induction group: osteoclasts induced by RANKL (100 ng / mL) for 4 days; Alendronate sodium group: 10 μM alendronate sodium; Group A: Morus root ketone C, Morusin, Morus root ketone D, Morus flavonoid G, Morusin alcohol (i.e. Group K of Example 1); Group B: Morigenin C, Morigenin D, Morigenin G, Naringin; Group C: Mori senna G, Mori root ketone C, Mori cinnamic acid, Mori senna, Mori bark ketone B; Group D: Morus root ketone C, Morusin, Morus root ketone D, Morus fruit ketone G, Morus bark ketone B; Group E: Morus root ketone D, Morus root ketone C, Morusin, Morus flavonoids, Morus bark ketone B; Group F: Moriton G, Morigenin C, Morigenin, Moriton, Naringin.

[0067] The intracellular tartrate-resistant acid phosphatase (TRAP) content was detected using a Solarbio anti-tartrate acid phosphatase staining kit (product number: G1492) according to its instructions.

[0068] II. Experimental Results Experimental results are as follows Figure 4As shown, the results indicate that the TRAP content in the induction group was significantly higher than that in the control group, indicating successful induction of osteoclast differentiation. The TRAP content in all drug treatment groups was lower than that in the induction group, suggesting inhibition of osteoclast formation.

[0069] Example 5: Effects of different treatments on bone mineralization nodules in BMSCs (promoting bone formation) The formation of bone mineralization nodules in different treatment groups was detected by alizarin red staining.

[0070] All drugs were added at the start of induction and continued to be administered until the end of induction (a total of 4 days of treatment). The final concentration of each active ingredient in groups A through F was 2 μM; the final concentration of the positive control drug, alendronate sodium, was also 10 μM.

[0071] The processing groups are specifically grouped as follows: Control group: Uninduced primary bone marrow macrophages from mice; Induction group: induced differentiated bone marrow mesenchymal stem cells (BMSCs); Alendronate sodium group: 10 μM alendronate sodium; Group A: Morus root ketone C, Morus root ketone D, Morus root ketone G, Morus root ketone; Group B: Morigenin C, Morigenin D, Morigenin G, Naringin; Group C: Mori senna G, Mori root ketone C, Mori cinnamic acid, Mori senna, Mori bark ketone B; Group D: Morus root ketone C, Morusin, Morus root ketone D, Morus fruit ketone G, Morus bark ketone B; Group E: Morus root ketone D, Morus root ketone C, Morusin, Morus flavonoids, Morus bark ketone B; Group F: Moriton G, Morigenin C, Morigenin, Moriton, Naringin.

[0072] I. Experimental Methods 1. Main reagents and consumables Alizarin Red S staining quantitative detection kit (brand: Solarbio, catalog number: G3283), phosphate buffered saline (PBS), 4% paraformaldehyde (PFA) fixative, and ultrapure water.

[0073] 2. Experimental Procedure (1) Extraction of bone marrow mesenchymal stem cells (BMSCs) ① Prepare surgical instruments in advance, including ophthalmic scissors, scalpels, forceps, etc., and sterilize them with high-pressure steam for later use; ② Each time BMSCs were extracted, one 1-month-old SPF-grade male C57 mouse (approximately 15 g) was purchased from the Animal Center of Guangzhou University of Chinese Medicine. After being anesthetized with CO2, the mouse was euthanized by cervical dislocation and disinfected by soaking in 75% alcohol. ③ Immediately separate the bilateral femurs and tibias completely in a clean bench, rinse the bone surface with PBS, cut open both ends of the femur to expose the medullary cavity, draw 2 mL of 10% complete culture medium with a 1 mL syringe, rinse the medullary cavity, and then rinse the medullary cavity with the sterile cell suspension in a 6-well plate 2-3 times. ④ Use a 200-mesh filter to repeatedly filter the flushed cell suspension to remove bone fragments and other debris.

[0074] ⑤ Use a pipette to transfer the filtered cell suspension into a cell culture flask, gently shake to mix, and place in a cell culture incubator (37℃, 5% CO2) to culture until the cells adhere and grow.

[0075] (2) After bone marrow mesenchymal stem cells (BMSCs) are induced to differentiate, the culture supernatant is discarded and the cells are gently washed three times with pre-cooled PBS to remove residual culture medium; (3) Add 4% PFA fixative and fix at room temperature for 30 min; (4) Discard the fixative and wash three times with ultrapure water to thoroughly remove residual PFA; (5) Add an appropriate amount of Alizarin Red S staining solution to each well (enough to cover the cell layer), and stain at room temperature in the dark for 3-5 minutes; (6) Discard the staining solution and rinse repeatedly with ultrapure water 5-6 times; (7) Observe the morphology of mineralized nodules under an inverted optical microscope and take pictures to record them.

[0076] II. Experimental Results Results of cellular bone mineralization nodules, such as Figure 5 As shown, the results indicated that the number of mineralized nodules in the induction group was significantly higher than that in the control group, confirming the effectiveness of osteogenic induction. Among the drug administration groups, group A (Morus alba root ketone C, Morus alba root ketone D, Morus alba root ketone G and Morus alba root ketone) had the most prominent mineralization-promoting effect, and its relative area of ​​ARS staining was significantly higher than that of the positive drug alendronate sodium group and other combination treatments, indicating that it has a strong osteogenic activity.

[0077] Example 6: Effects of different treatments on ALP activity in BMSCs cells Bone marrow mesenchymal stem cells (BMSCs) were used, and the treatment group was the same as in Example 5. The activity of ALP, an early marker of osteogenic differentiation, was assessed using ALP staining.

[0078] I. Experimental Methods 1. Main reagents and consumables BCIP / NBT alkaline phosphatase colorimetric kit (brand: Beyotime, catalog number: C3206), phosphate buffered saline (PBS), 4% paraformaldehyde (PFA) fixative, and ultrapure water.

[0079] 2. Experimental Procedure (1) Wash with PBS 3 times, 3-5 minutes each time.

[0080] (2) After removing the PBS, add 4% paraformaldehyde fixative and fix for 30 min. (3) After proper fixation, wash with distilled water 3-5 times, each time for 3-5 minutes.

[0081] (4) After the final wash, remove the washing solution and add an appropriate amount of BCIP / NBT staining working solution to ensure that the sample is fully covered. (5) Incubate at room temperature in the dark for 5-30 minutes or longer (up to 24 hours) until the color develops to the desired depth. (6) Remove the BCIP / NBT staining working solution and wash with distilled water 1-2 times to terminate the color development reaction.

[0082] II. Experimental Results ALP staining results of different treatment groups are as follows Figure 6 As shown, the results indicate that ALP activity was significantly increased in the induction group compared with the control group, indicating the initiation of early osteogenic differentiation. ALP activity was further enhanced in all drug treatment groups, with group A showing the largest increase, significantly higher than the induction group and the positive control group, suggesting that it exerts a strong promoting effect in the early stage of osteogenic differentiation.

[0083] Example 7: Effect of the composition on improving muscle function in mice with osteoporosis-sarcopenia-obesity triad (OSO). I. Experimental Methods Based on the cell experiments in Examples 1-6, the K group (Morus alba root ketone C, Morus alba root ketone D, Morus alba root ketone G, Morus alba root ketone) composition was selected for animal pharmacodynamic evaluation. An OSO model was established using a high-sugar, high-fat diet combined with dexamethasone, and the mice were administered the medication continuously for 8 weeks. After administration, grip strength, forelimb grip strength, and rotating rod tests were performed on the mice in each group. The treatment groups were set as follows: Control group: administered an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage; Model group: administered an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage; Alendronate sodium group: Alendronate sodium (20 mg / kg); Composition A: Morus root ketone C, Morusin, Morus root ketone D, Morus flavonoid G, Morusinol, in a ratio of 1:1:1:1:1; each component is 4 mg / kg.

[0084] Composition B: Morus root ketone C, morula syringin, morus root ketone D, morula syringin G, and morula syringol in a ratio of 2:1:2:2:1; morus root ketone C / D / G each 5 mg / kg, morula syringin / morula syringol each 2.5 mg / kg.

[0085] II. Experimental Results The grip strength test represents the "strength" of the muscle, while the dwell time on the rotating bar represents the "flexibility-endurance-strength" of the muscle. The combination of muscle strength and endurance provides objective data to verify the effectiveness of a drug.

[0086] Experimental results are as follows Figure 7 , Figure 8 As shown in Tables 1 and 2, the results indicate that compared with the control group, the claw grip strength of the four limbs (0.050±0.004 N / g vs. 0.111±0.014 N / g), the claw grip strength of the forelimbs (0.026±0.003 N / g vs. 0.041±0.003 N / g), and the rotarod time (307±25 s vs. 480±116 s) of the model group mice were significantly reduced (P<0.001), indicating that the sarcopenia model was successfully established.

[0087] Both compositions A and B significantly improved grip strength in the limbs and forelimbs (P<0.001) and significantly prolonged rotarod time (Composition A: 442±106 s; Composition B: 449±86 s, P<0.05 vs. model group). The compositions were superior to the positive control drug alendronate sodium in improving muscle strength (grip strength) and endurance (rotarod time), confirming their multidimensional therapeutic effects on sarcopenia.

[0088] Further anatomical analysis showed that, compared with the control group, the relative wet weight of the gastrocnemius muscle and the relative wet weight of the soleus muscle in the model group were significantly reduced, indicating that sarcopenia was successfully established.

[0089] The relative wet weight of the gastrocnemius and soleus muscles in the model group was significantly reduced, while the addition of the composition restored the wet weight of the gastrocnemius muscle to over 0.50% (P<0.01), and the soleus muscle also showed significant improvement (P<0.05). In conclusion, the composition can effectively improve muscle quality and function, and has a significant therapeutic effect on sarcopenia in OSO.

[0090] Table 1. Data from claw gripping force test and rotating rod fatigue test ( ± s, n=8)

[0091] Note: Different lowercase letters in the table indicate significant differences (P<0.05, for comparison of model groups), the same applies below.

[0092] Table 2. Relative wet weight of gastrocnemius muscle and relative wet weight of soleus muscle ( ± s, n=8)

[0093] Example 8: Interventional effect of the composition on sarcopenic osteoporosis I. Experimental Methods Bone mineral density (BMD), bone mineral content (BMC), and muscle mass were measured in mice of different treatment groups using dual-energy X-ray absorptiometry (DXA). The animal dual-energy X-ray bone densitometer was a KUBTEC / PARAMETER 3D, manufactured by KUBTEC. The treatment groups were set up as in Example 7.

[0094] II. Experimental Results Dual-energy X-ray data results are as follows Figure 9 As shown in Table 3, the results indicate that the model group had significantly lower BMD, BMC, and muscle mass than the control group, confirming the successful construction of the OSO model. Compositions A and B significantly improved all three indicators, while alendronate sodium only improved bone parameters and had limited effect on muscle mass improvement.

[0095] This indicates that the composition can simultaneously improve bone and muscle, and can treat the osteoporosis-sarcopenia-obesity triad, achieving multi-target intervention for sarcopenic osteoporosis.

[0096] Table 3 Bone mineral density data and muscle mass ( ± s, n=8)

[0097] Example 9: Regulatory effect of the composition on sarcopenic obesity I. Experimental Methods This embodiment examines the effect of the composition on improving sarcopenic obesity in an OSO model. Perirhinal fat, epididymal fat, and brown fat were obtained by dissection, and their ratios to body weight were calculated to assess body fat distribution. The treatment group was set up the same as in Example 7.

[0098] II. Experimental Results The experimental results are shown in Table 4 and Figure 10 As shown, the results indicated that the model group had significant visceral fat accumulation and reduced brown fat, consistent with sarcopenic obesity. The combination therapy group significantly reduced visceral fat (P<0.05~0.001) and increased the proportion of brown fat (P<0.0001), demonstrating that it can promote energy consumption and fat remodeling, exhibiting a dual effect of "fat reduction and energy enhancement".

[0099] Table 4 Comparison of body fat percentage data of mice in each group ( ± s, n=8)

[0100] Examples 7-9 collectively demonstrate that the composition has a multi-system, multi-target, and synergistic therapeutic effect on the osteoporosis-sarcopenia-obesity triad. It not only improves individual tissue lesions but also achieves overall regulation of the bone-muscle-fat axis by modulating the three common pathological bases of inflammation, oxidative stress, and energy metabolism, providing a solid experimental basis for the development of innovative traditional Chinese medicine preparations for the treatment of OSO syndrome.

[0101] Example 10 Screening of Dissolution Systems I. Processing Group Settings Treatment group 1 (phospholipid complex): The composition and lecithin (mass ratio 1:3) were mixed with 5 L of anhydrous ethanol and stirred at a constant speed of 200 r / min in a 60 ℃ water bath until completely dissolved. A reflux reflux apparatus was connected, and the reaction was carried out with shaking for 1 h while maintaining the temperature. After the reaction was complete, the ethanol was recovered under reduced pressure, and the resulting product was vacuum dried at 40 ℃ for 24 h. The dried product was then pulverized and passed through a 120-mesh sieve to obtain the phospholipid complex sample.

[0102] Treatment group 2 (PEG 6000 / poloxam 188 system): Weigh out each component according to the composition: PEG 6000: Poloxamer 188 = 1:2:2 (mass ratio). Melt PEG 6000 and Poloxamer 188 in a 70-80°C water bath, stirring until homogeneous. Then slowly add the composition to the molten carrier, stirring vigorously to ensure thorough dispersion and form a homogeneous mixture. Quickly pour the mixture into a pre-cooled stainless steel pan, spread it into a thin layer, and allow it to cool and solidify naturally. Crush, dry, and pulverize the resulting solid, passing it through a 60-80 mesh sieve to obtain the solid dispersion particles.

[0103] Treatment group 3 (PVP K30 solid dispersion): Weigh the raw materials according to the composition: polyvinylpyrrolidone K30 (PVP K30) = 1:4 (mass ratio). Select an appropriate amount of ethanol as a solvent (preferably just enough to completely dissolve both), mix the two and then treat with magnetic stirring or sonication to form a clear and transparent solution. Transfer the solution to a glass petri dish and place it on a water bath at 50-60 ℃. Stir and evaporate the solvent under aeration to remove the solvent (a rotary evaporator can also be used). After the solvent has completely evaporated, a transparent thin film-like solid is obtained. Place the solid in a desiccator and let it stand overnight, then grind it and pass it through an 80-100 mesh sieve to obtain a solid dispersion powder with uniform particle size. The obtained powder can be used directly for hard capsule filling, pellet preparation, granule forming, or mixed with 0.5% magnesium stearate for tableting.

[0104] Treatment group 4 (lipid matrix solid dispersion): Weigh out each component according to the composition: glyceryl monostearate (GMS): stearic acid (SA) = 1:2:3 (mass ratio). Melt GMS and SA in a water bath at 70–75 °C and stir until homogeneous; then add the composition to the molten lipid and stir under homogenization conditions to form a homogeneous suspension. Keep the system at 40–45 °C to maintain the molten state for later use.

[0105] Treatment group 5 (phospholipid-PVP K30 complex system): Weigh the raw materials according to the composition: phosphatidylcholine (LPC): PVP K30 = 1:2:3 (mass ratio). Add an appropriate amount of ethanol to adjust the final concentration of the composition to 50~100 mg / mL, and stir thoroughly in a 55 ℃ water bath for about 2 h to dissolve. Then remove the ethanol by rotary evaporation under reduced pressure at 50 ℃, and thoroughly dry the residue in a 40 ℃ oven (spray drying or belt drying can be used for industrial production). After grinding, pass the dried product through a 100-mesh sieve to obtain the target sample.

[0106] II. Solubility Test Method The solubility of the powders prepared in treatment groups 1-5 was compared. The determination of solubility was based on the process of establishing a dissolution equilibrium. An excess of the powders prepared in treatment groups 1-5 was dissolved in water and the solution was sonicated (40 kHz) at room temperature for 30 min to ensure that the composition and the solution reached a dynamic equilibrium (i.e., the dissolution rate and the precipitation rate were equal).

[0107] III. Test Results The results are shown in Table 5. The results show that the solubility of the composition powder is 0.1 mg / mL, and the solubility of the sample prepared in treatment group 5 is 6 mg / mL.

[0108] Table 5. Solubility test results for different formulation processes

[0109] Example 11: Health food formulated with the composition I. Experimental Methods An OSO model was established using a high-sugar, high-fat diet combined with dexamethasone, and the mice were administered the medication continuously for 8 weeks. After administration, bone mineral density, bone mineral content, muscle mass, and rotarod test time were measured in each group of mice.

[0110] Configure the processing group as follows: Control group: administered 0.5% sodium carboxymethyl cellulose solution by gavage; Model group: administered 0.5% sodium carboxymethyl cellulose solution by gavage; Glucosamine chondroitin calcium tablet group: Glucosamine chondroitin calcium tablets 300 mg / kg / day; Composition group: Morus root ketone C, Morusin, Morus root ketone D, Morus flavonoid G, Morusin alcohol (ratio 1:1:1:1:1); total dose 20 mg / kg / day; The combination composition of glucosamine chondroitin calcium tablets: glucosamine chondroitin calcium tablets 300 mg / kg / d + composition (Morus alba root ketone C, Morus alba root ketone D, Morus alba root ketone G ...

[0111] II. Experimental Results The experimental results are shown in Table 6. The results show that the composition alone can significantly improve bone mineral density and exercise endurance; when used in combination with glucosamine chondroitin calcium tablets, all indicators are further improved, especially showing a synergistic effect in terms of rotator time and bone mineral content.

[0112] Table 6. Effects of different intervention groups on bone metabolism and exercise capacity

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A composition, characterized in that, It contains morinone C, morinone, morinone D, morinone G and morinol; the mass ratio of morinone C, morinone, morinone D, morinone G and morinol is (1~2): (1~2): (1~2): (1~2): (1~2): (1~2).

2. The composition according to claim 1, characterized in that, The mass ratio of mulberry root ketone C, mulberry root ketone D, mulberry root ketone G and mulberry root ketone alcohol is (1~2):1:(1~2):(1~2):

1.

3. Use of the composition of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of osteoporosis-sarcopenia-obesity triad.

4. Use of the composition of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of osteoporosis.

5. Use of the composition of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of sarcopenia.

6. Use of the composition of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of obesity.

7. A drug for treating osteoporosis-sarcopenia-obesity triad, characterized in that, Contains the composition according to claim 1 or 2.

8. The drug according to claim 7, characterized in that, It also contains pharmaceutical excipients, including any one or a combination of at least two of phosphatidylcholine, PVP-K30, glyceryl monostearate, and stearic acid.

9. The drug according to claim 8, characterized in that, The mass ratio of the composition of claim 1 or 2, phosphatidylcholine and PVP-K30 is 1:(2~3):(3~4).

10. The drug according to any one of claims 7 to 9, characterized in that, It also contains glucosamine, chondroitin, and calcium tablets.

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