Application of icariin in preparation of product with lipid-lowering and muscle protection functions
By applying icariin in cell and animal models, the problem of muscle damage caused by existing lipid-lowering drugs has been solved, achieving safe and effective lipid-lowering and muscle protection effects, and expanding the applicable population of statins.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GANSU UNIV OF CHINESE MEDICINE
- Filing Date
- 2026-05-11
- Publication Date
- 2026-06-05
Smart Images
Figure CN122140741A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to the application of icariin in the preparation of products with both lipid-lowering and muscle-protecting functions. Background Technology
[0002] Lipid metabolism disorders are the common pathological basis for a series of major chronic diseases, including obesity, non-alcoholic fatty liver disease, hyperlipidemia, and metabolic syndrome. Their core characteristic is the abnormal accumulation of lipid components such as triglycerides (TG) and total cholesterol (TC) in the body. These diseases are interconnected, significantly increasing the risk of cardiovascular and cerebrovascular events, seriously threatening patients' lives and quality of life. Currently, lipid-lowering therapy, represented by statins, is the first-line clinical intervention for lipid metabolism disorders. These drugs effectively reduce total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels by inhibiting HMG-CoA reductase, a key enzyme in cholesterol synthesis, while also increasing high-density lipoprotein cholesterol. They are widely used in lipid-lowering and cardiovascular protection. However, statins also have significant limitations, with a series of adverse reactions, including muscle-related toxicity (myalgia, myositis, rhabdomyolysis), liver damage, and diabetes, which to some extent limits their clinical applicability. Therefore, developing a novel treatment strategy that can effectively regulate lipid metabolism while maintaining muscle safety has become a critical issue that urgently needs to be addressed in this field.
[0003] Icariin is a natural flavonoid compound extracted from the traditional Chinese medicine Epimedium, possessing various pharmacological activities such as anti-inflammatory, antioxidant, and antitumor effects. In recent years, increasing research has demonstrated that icariin can regulate fatty acid synthesis and oxidative decomposition, effectively inhibiting fat deposition and exhibiting significant lipid-lowering potential. Furthermore, Epimedium extract has shown positive effects in improving muscle mass and preventing sarcopenia. However, no research has yet revealed whether icariin can effectively lower lipids while mitigating the risk of muscle damage and protecting muscle tissue from injury, nor has its specific application strategy in this area been disclosed. Therefore, this study investigates the effects of icariin on muscle function during lipid-lowering, aiming to provide a theoretical basis for developing drugs that combine lipid-lowering and muscle safety, thus addressing the shortcomings of statin therapy. Summary of the Invention
[0004] In existing technologies, mainstream lipid-lowering drugs (such as statins) pose a risk of inducing muscle damage with long-term use, which limits their safety in clinical application. Therefore, the purpose of this invention is to provide a novel use for an active ingredient that combines highly effective lipid-lowering and muscle-protective effects. This invention establishes a free fatty acid (FFA)-induced cellular hyperlipidemia model and a high glucose-induced nematode hyperlipidemia model, providing an important theoretical basis for developing novel drugs with both lipid-lowering and muscle-protective functions, thereby overcoming the deficiency of existing drugs in avoiding muscle toxicity during long-term lipid-lowering treatment.
[0005] To solve the above problems, the present invention includes:
[0006] Application of icariin in the preparation of drugs with both lipid-lowering and muscle-protecting functions.
[0007] The aforementioned medications are used to prevent and treat lipid metabolism disorders associated with the risk of muscle damage.
[0008] The above-mentioned lipid metabolism disorders associated with the risk of muscle damage are selected from any one of hyperlipidemia, obesity, non-alcoholic fatty liver disease, or metabolic syndrome.
[0009] The aforementioned muscle protection functions include maintaining the structural integrity of muscle cells and improving muscle function.
[0010] The above-mentioned drugs can reduce the levels of triglycerides and total cholesterol in cells.
[0011] The aforementioned drugs can reduce the triglyceride content in Caenorhabditis elegans.
[0012] The above application is achieved through at least one of the following methods:
[0013] (a) Reduces intracellular triglyceride and total cholesterol levels in a free fatty acid-induced hyperlipidemic cell model;
[0014] (b) In a high-sugar-induced high-lipid Caenorhabditis elegans model, the triglyceride content in the nematodes was reduced, while the integrity of their body wall muscle fiber structure was maintained.
[0015] In (a), the concentration of icariin was 2.5–10 μM; in (b), the concentration of icariin was 50–150 μM.
[0016] It also includes a pharmaceutical composition comprising a therapeutically effective amount of icariin or a pharmaceutically acceptable salt, derivative or prodrug thereof, and one or more pharmaceutically acceptable carriers or excipients, said pharmaceutical composition for improving lipid metabolism disorders while protecting muscle tissue.
[0017] The administration methods of the drugs in the above applications include oral, injection or local administration, and the dosage forms are one or more combinations of tablets, capsules, granules, powders, oral liquids, injections, and liposomes.
[0018] Furthermore, the drug is used to prevent and / or treat lipid metabolism disorders associated with the risk of muscle damage, and is suitable for patient populations who are intolerant to statins or have myopathy.
[0019] The experimental results of this invention show that
[0020] (1) At the cellular level, in the free fatty acid (FFA) induced hyperlipidemia model of HepG2 cells, icariin can dose-dependently and significantly reduce the accumulation of intracellular triglycerides (TG) and total cholesterol (TC), confirming its direct lipid-lowering effect on hepatocytes.
[0021] (2) At the whole animal level, in the high glucose-induced Caenorhabditis elegans model, icariin intervention not only effectively reduced the triglyceride level in the nematodes (by about 30.27% compared with the model control group), but also significantly maintained the integrity of the muscle fiber structure of the body wall. Fluorescence microscopy showed that the muscle fibers were arranged regularly without obvious breakage, which indicates that icariin has the advantage of muscle protection while exerting lipid-lowering function.
[0022] The beneficial effects of this invention are as follows:
[0023] (1) This study first clearly demonstrates the novel use of icariin in protecting the integrity of muscle tissue while lowering lipids. Compared with traditional statins, icariin does not cause muscle damage side effects such as rhabdomyolysis while exerting its lipid-lowering effect, providing a new treatment strategy that lowers lipids without damaging muscles and offering new ideas for the development of drugs that combine lipid-lowering and muscle safety.
[0024] (2) The lipid-lowering effect of icariin on hepatocytes was confirmed by an in vitro cell model (HepG2) to simultaneously reduce intracellular TG and TC levels and decrease lipid droplet deposition. The beneficial effects of icariin on reducing in vivo TG levels while effectively maintaining muscle structure and improving related motor functions were verified by an in vivo animal model (Caenorhabditis elegans).
[0025] (3) Outstanding clinical application value: The present invention is particularly suitable for people who are intolerant to statins, which can expand the applicable population for lipid-lowering treatment, reduce the risk of muscle damage from long-term lipid-lowering treatment, and has good industrialization prospects. Attached Figure Description
[0026] Figure 1 The effect of icariin on the survival rate of HepG2 cells;
[0027] Figure 2 The effect of icariin on triglyceride and total cholesterol levels induced by free fatty acids in HepG2 cells was investigated. A: triglycerides; B: cholesterol. Compared with the control group, #### p < 0.0001; Compared with the model group, **p < 0.01, ***p < 0.001, ****p < 0.0001;
[0028] Figure 3The effect of icariin on free fatty acid-induced lipid accumulation in HepG2 cells was investigated. A: Representative micrographs stained with Oil Red O (scale bar 50 μm); B: Quantitative analysis results of Oil Red O staining; compared with the control group... #### p < 0.0001; compared with the model group, p < 0.0001;
[0029] Figure 4 The effect of icariin on free fatty acid-induced accumulation of sarcoplasmic lipid droplets in HepG2 cells is shown in Figure A: Representative images of Bodipy 493 / 503 fluorescence staining (scale bar 50 μm); Figure B: Quantitative analysis results of fluorescence area; compared with the control group. #### p < 0.0001; compared with the model group, p < 0.0001. ns p>0.05;
[0030] Figure 5 The effect of icariin on the lifespan of Caenorhabditis elegans induced by high sugar;
[0031] Figure 6 The effect of icariin on high glucose-induced locomotion behavior in *C. elegans* was investigated, where A: body flexion frequency; B: head swing frequency; compared with the control group. #### p < 0.0001; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001. ns p>0.05;
[0032] Figure 7 The effect of icariin on the triglyceride content of Caenorhabditis elegans induced by high glucose was investigated, with comparisons made with the control group. #### p < 0.0001; Compared with the model group, **p < 0.01, ***p < 0.001, ****p < 0.0001;
[0033] Figure 8 The effect of icariin on lipid droplet content in *C. elegans* induced by high glucose. A: Representative micrographs stained with Oil Red O (scale bar 50 μm); B: Staining area analysis results; compared with the control group. #### p < 0.0001; compared with the model group, p < 0.0001;
[0034] Figure 9 Effects of icariin on high glucose-induced body wall muscle fibers of Caenorhabditis elegans (scale bar 50 μm). Detailed Implementation
[0035] To enable those skilled in the art to better understand the present invention, specific embodiments will now be described in further detail. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.
[0036] Example 1
[0037] Cell passage, culture, and establishment of a high-fat model
[0038] Cell resuscitation: Cells were frozen at -80 ℃, thawed in a 37 ℃ water bath, transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Then, 5 mL of serum-containing culture medium (containing a mixture of 10% FBS and 1% penicillin and streptomycin) was added and mixed well. The mixture was then transferred to cell culture flasks and incubated in a 37 ℃, 5% CO2 incubator. Cell growth was observed and the culture medium was replaced as needed.
[0039] Cell medium change: Observe the cell status after 24 h of resuscitation. When a large number of cells adhere to the culture vessel and there is a lot of floating matter, aspirate the original culture medium and wash twice with 2 mL PBS. During the washing process, shake the cell culture flask to ensure full contact with the cells. After washing, aspirate the PBS, add 5 mL of complete culture medium, and continue to culture in a constant temperature incubator at 37℃ and 5% CO2. When the cell density is close to 80%, perform passage treatment.
[0040] Cell passage: When the cell adhesion density reaches about 80%, wash twice with an appropriate amount of PBS, add 1 mL of trypsin, place in a constant temperature incubator, and digest for about 3 min. After observing the adhered cells detaching under a microscope, add 2 mL of complete culture medium to stop the digestion, pipette the cells to obtain a cell suspension, transfer the cell suspension to a 5 mL centrifuge tube, place in a centrifuge, centrifuge at 1000 rpm for 5 min, remove the supernatant, add complete culture medium, pipette the cell clumps to resuspend the cells, and inoculate the resuspended cell suspension into a new culture flask for continued passage and culture. After the cells adhere and grow, replace with fresh complete culture medium every 2-3 days.
[0041] Establishment of the high-fat model: Adjust the number of cells in the logarithmic growth phase to 1×10⁻⁶. 6Cells were seeded at a density of 1 / mL in 6-well plates. After complete cell adhesion, the cells were divided into groups: the model group and the drug treatment group were replaced with induction medium containing 0.75 mM free fatty acids (FFA, oleic acid OA to palmitic acid PA molar ratio of 2:1), while the blank control group continued to use complete medium. All groups were cultured for another 24 h to induce intracellular lipid accumulation. This embodiment aims to construct a free fatty acid-induced hyperlipidemia model of HepG2 cells to simulate abnormal lipid accumulation in hepatocytes under conditions of lipid metabolism disorder, providing a reliable in vitro pathological model basis for verifying the application of icariin in the preparation of drugs that improve lipid metabolism disorder and have muscle protective function.
[0042] Example 2
[0043] Compound cytotoxicity assay
[0044] Cells in logarithmic growth phase were spaced at 3 × 10⁻⁶ cells per well. 4 ~5×10 4 Cells were seeded at a density of 10 cells / mL, and control, experimental, and drug-treated groups were set up. All cells were incubated in a cell culture incubator for 24 h. The culture medium was discarded. For drug-treated groups, 100 μL of prepared drug at different concentrations (5, 10, 20, 40, 60, 80 μM) was added to each well. For control and experimental groups, 100 μL of complete culture medium was added to each well. Cells were incubated for another 24 h. Subsequently, 10 μL of MTT assay solution was added to each well, and incubation was continued for 4 h. The liquid in the wells was aspirated, and 150 μL of DMSO was added to each well. The cells were shaken thoroughly for 10 min, and the absorbance of each well was measured at 570 nm. Cell viability was calculated using the following formula, and a concentration with cell viability >80% was selected as the safe concentration for subsequent experiments.
[0045] Survival rate % = (Drug-treated group - Blank group) / (Control group - Blank group) × 100%
[0046] See attached Figure 1 When the concentration of icariin was not higher than 10 μM, the cell viability was greater than 80%, indicating that the drug had no significant toxicity to cell growth within this concentration range. When the concentration reached 20 μM and above, the cell viability decreased significantly, showing obvious growth inhibition. Therefore, 10 μM was selected as the maximum dosage concentration for subsequent cell experiments. This embodiment used the MTT assay to evaluate the effect of different concentrations of icariin on the viability of HepG2 cells, and determined its safe dosage concentration for subsequent lipid-lowering and muscle protection experiments, thereby providing a cellular-level safety basis for the application of icariin in the preparation of safe and effective lipid-lowering drugs.
[0047] Example 3
[0048] Determination of total cholesterol and triglyceride content
[0049] After the model was established, the drug-treated group was cultured in a drug-containing medium for another 24 hours, while the model group and the blank control group were simultaneously replaced with their respective drug-free media. After the drug intervention, the media were discarded, the cells were washed twice with pre-cooled PBS, and the cells were collected from each group. The cells were lysed according to the kit instructions, and the triglyceride (TG) and total cholesterol (TC) levels were measured. Simultaneously, the total protein concentration of each group was determined using the BCA method, and all lipid data were standardized to the total protein concentration.
[0050] See attached Figure 2 Compared with the blank control group (TG: 0.39±0.008 mmol / g prot, TC: 0.28±0.013), the intracellular TG (1.25±0.026 mmol / g prot) and TC (0.72±0.008 mmol / g prot) levels in the free fatty acid-induced model group were significantly increased (p<0.0001), indicating the successful establishment of the hyperlipidemic cell model. Icariin intervention can reverse lipid accumulation in a dose-dependent manner. Regarding the reduction of triglycerides, different concentrations of icariin (2.5 μM, 5 μM, 10 μM) significantly reduced intracellular TG levels in hyperlipidemic cells. Among them, the 10 μM icariin intervention showed the most outstanding effect, reducing TG levels to 0.49±0.028 mmol / g prot, an effect even superior to the positive control group treated with atorvastatin. In terms of cholesterol reduction, all treatment groups showed a highly significant reduction in intracellular total cholesterol (TC) levels in hyperlipidemic cells (p<0.0001), with the positive control group showing the best effect (0.33±0.011 mmol / g prot). The high-concentration icariin group (0.35±0.009 mmol / g prot) was comparable to the positive control group. This embodiment quantitatively detected the regulatory effect of icariin on intracellular triglyceride and total cholesterol levels in a free fatty acid-induced hyperlipidemic cell model, directly demonstrating that icariin can improve lipid metabolism disorders and supporting its application in the preparation of lipid-lowering drugs.
[0051] Example 4
[0052] Oil Red O staining
[0053] After the model was established and the drug intervention was completed, the supernatant was discarded, and the cells were washed twice with PBS. The Oil Red O staining stock solution was diluted according to the reagent manufacturer's instructions to prepare the working solution. Cells were first fixed with 4% paraformaldehyde for 15 min, then stained with Oil Red O staining solution for 30 min. After staining, excess dye was washed away with 60% isopropanol, counterstained with hematoxylin solution for 1 min, and then washed with PBS to remove excess dye. The results were then observed under a microscope and photographed.
[0054] See attached Figure 3 A. After FFA induction, numerous large, deeply red lipid droplets were observed in the model group cells, mostly distributed around the cell nucleus, indicating the successful establishment of the cellular fatty degeneration model. In contrast, almost no obvious red lipid droplets were observed in the blank control group cells. After intervention with icariin, the morphology and distribution of intracellular lipid accumulation changed. Compared with the model group, the lipid droplet volume in the drug-treated group cells was significantly reduced, the staining was lighter, and the lipid droplets were mostly distributed and migrated to both sides of the cell. The results suggest that icariin can not only reduce the total amount of intracellular lipids but also may inhibit the abnormal fusion and growth of lipid droplets.
[0055] A semi-quantitative analysis of the Oil Red O staining results was performed, and the results are as follows: Figure 3 B. The percentage of lipid surface area in the model group (34.98% ± 3.41%) was significantly higher than that in the blank control group (p < 0.0001). Icariin significantly reversed lipid accumulation in a dose-dependent manner. When the concentration was 10 μM, the percentage of lipid surface area decreased to 14.30% ± 1.97%, and the positive control group also showed a significant effect (15.73% ± 1.78%). The quantitative results were consistent with the morphological observations, both indicating that icariin has a clear effect on reducing lipid accumulation in hepatocytes. In this example, Oil Red O staining was used to visually demonstrate the inhibitory effect of icariin on lipid droplet formation in hyperlipidemic cells, further confirming its lipid-lowering effect from a morphological perspective and strengthening its technical value in the preparation of drugs with both lipid-lowering and muscle-protective functions.
[0056] Example 5
[0057] Bodipy staining
[0058] After model establishment and drug intervention, the supernatant was discarded, and the cells were washed twice with PBS. A staining solution containing BODIPY493 / 503 and Hoechst 33342 was added, and the cells were stained at room temperature in the dark for 15 minutes. The cells were then washed three times with PBS and observed and photographed under a fluorescence microscope.
[0059] See attached Figure 4 A. After FFA induction, the model group cells showed dense and bright green fluorescence signals, indicating a large accumulation of neutral lipid droplets within the cells, further confirming the successful construction of the hyperlipidemic cell model. In contrast, the blank control group showed only weak background fluorescence. After intervention with icariin, the intensity of green fluorescence in the cells decreased, and the size of the fluorescent lipid droplets decreased, suggesting that icariin can effectively reduce the content of neutral lipids in the cells.
[0060] Further quantitative analysis results of fluorescence intensity ( Figure 4B) showed that the average fluorescence intensity of the model group was significantly higher than that of the blank control group (p < 0.0001). All concentrations of icariin reduced fluorescence intensity, with the 10 μM dose showing the most significant effect. Its effect was comparable to that of the positive control drug. This quantitative result is consistent with fluorescence microscopy observation, jointly confirming, from the perspective of specifically labeling neutral lipid droplets, that icariin has a clear effect in alleviating lipid accumulation in hepatocytes.
[0061] Example 6
[0062] Nematode Culture and Cryopreservation
[0063] Culture: After being removed from the -80℃ freezer, the frozen nematodes were immediately thawed in a 37℃ water bath, then transferred to centrifuge tubes and collected by centrifugation. After discarding the supernatant, the nematodes were resuspended in 200 μL of sterile M9 buffer, transferred to NGM plates coated with OP50, and cultured in a 20℃ incubator, with regular observation of their growth status.
[0064] Cryopreservation: Culture dishes containing nematodes in the Dauer stage were selected, and the nematodes were collected by rinsing with M9 buffer. After centrifugation, the supernatant was discarded, and the washing was repeated three times to remove residual culture medium and bacterial suspension. The nematode precipitate was mixed thoroughly with cryopreservation solution (S-Buffer solution and 30% glycerol mixed in a 1:1 ratio) at a 1:1 ratio, aliquoted, and then gradually cooled before storage at -80°C. This example establishes a standardized culture and cryopreservation system for *C. elegans*, providing stable experimental materials for subsequent high-glucose-induced high-fat models and muscle function evaluation, and verifying the application effect of icariin in the preparation of drugs with both lipid-lowering and muscle-protective functions.
[0065] Example 7
[0066] Nematode Synchronization Treatment
[0067] Synchronization is used to obtain nematodes of the same life cycle from the same batch to ensure experimental consistency. Synchronization is performed when most adults are observed to have densely packed eggs and a large number of unhatched eggs are present on the culture plate. First, oviposition-stage adults are collected into 1.5 mL centrifuge tubes using M9 buffer. After centrifugation, the supernatant is discarded, and the tubes are washed 2-3 times to remove residual culture medium and bacterial suspension. Then, 1 mL of freshly prepared lysis buffer (M9 buffer: 10% NaClO: 5M NaOH = 7:2:1) is added, and the tubes are shaken until completely dissolved. After centrifugation, the eggs are collected, washed 3 times with M9 buffer, and resuspended in 50 μL of M9 buffer. The tubes are incubated overnight at 20°C to allow for synchronized hatching. The next day, the obtained L1-stage nematodes are transferred to NGM plates containing fresh OP50 bacterial culture and incubated at 20°C for 36 h to obtain the L4-stage nematode population. In this embodiment, L4-stage nematodes were obtained through alkaline lysis combined with egg hatching to ensure consistent developmental stages of experimental individuals in the high-sugar-induced high-lipid model and subsequent efficacy evaluation, thereby improving the reproducibility of verifying the lipid-lowering and muscle-protective effects of icariin.
[0068] Example 8
[0069] Lifespan experiment of Caenorhabditis elegans
[0070] The experiment consisted of a blank control group, a model group, a positive control group (atorvastatin), a low-dose icariin group (50 μM), a medium-dose icariin group (100 μM), and a high-dose icariin group (150 μM). Synchronized L4-stage nematodes were transferred to high-glucose NGM agar plates containing different drugs, with 50 nematodes per plate and at least three replicates per group. Counting and transfer were performed under a microscope to ensure the nematodes remained from the same batch. Starting from the fifth day of culture, the number of surviving nematodes and the number of dead individuals were recorded under a microscope every other day until all individuals died. No reaction upon light touch of a platinum wire was used as the mortality criterion. Nematodes that died due to handling or crawled onto the plate walls were lost from the records.
[0071] See attached Figure 5The average lifespan of nematodes in the high-sugar model group was significantly shortened by 10.36% compared to the blank control group (p<0.0001), indicating that a high-sugar environment can have a significant negative impact on nematode lifespan. Icariin intervention showed a significant lifespan-prolonging effect. Compared with the model group, the 50 μM, 100 μM, and 150 μM icariin treatment groups prolonged nematode lifespan by 4.67% (p<0.01), 11.85% (p<0.0001), and 8.00% (p<0.01), respectively. The 100 μM concentration group showed the best lifespan-prolonging effect, significantly superior to the positive control drug atorvastatin group (5.16%, p<0.05). This indicates that icariin can effectively resist the lifespan shortening of nematodes caused by a high-sugar environment. This embodiment uses a high-sugar-induced high-lipid model of *C. elegans* to investigate the effect of icariin on prolonging the life cycle, demonstrating its successful application in improving lipid metabolism disorders and supporting the drug application value of icariin.
[0072] Example 9
[0073] Measurement of nematode locomotion behavior
[0074] L4-stage nematodes, after being synchronized and induced to develop a high-lipidemia model by high glucose for 48 h, were transferred to high-glucose NGM medium containing different drugs and cultured for 5 days. The nematode motility was assessed by measuring body bending frequency and head wagging frequency. Body bending frequency was assessed by recording the number of sine curves generated by the nematodes during movement on sterile NGM medium over 30 seconds. Head wagging frequency was recorded by recording the number of times the head waggled from one side to the other over 30 seconds in M9 buffer. Thirty nematodes were randomly selected from each group for the measurements.
[0075] See attached Figure 6 In the model group, the frequency of body bending and the number of head swings in nematodes were significantly inhibited (p<0.0001), suggesting that fat accumulation in the model group may be related to the inhibition of motor behavior. By administering different concentrations of icariin, it was found that compared with the model group, the frequency of body bending and the number of head swings increased to varying degrees with increasing concentration. When the concentration reached 150 μM, the frequency of body bending increased by 19.12% (p<0.001) and the number of head swings increased by 6.35% (p<0.01) compared with the model group, both showing better improvement than the positive control group (13.35%, p<0.05 and 5.94, p<0.01, respectively). However, compared with the blank control group, there was a significant difference, and the levels did not return to normal. This example evaluates the effect of icariin on improving muscle motor function by measuring the frequency of body bending and the number of head swings in high-lipid nematodes, demonstrating its functional efficacy in the preparation of drugs with both lipid-lowering and muscle-protecting functions.
[0076] Example 10
[0077] Determination of triglyceride content in nematodes
[0078] After establishing a high-lipidemia model by inducing high glucose for 48 h using synchronized L4-stage nematodes, they were transferred to high-glucose NGM medium containing different drugs and cultured for 5 days. The nematodes on the NGM culture plates were collected into centrifuge tubes after washing with M9 buffer. The supernatant was discarded after centrifugation, and the nematodes were repeatedly washed with M9 buffer to remove residual OP50. After washing, M9 buffer was added, and the nematodes were sonicated to disrupt their structure. The triglyceride (TG) content in the nematodes was determined according to the instructions of the BCA protein quantification kit and TG kit.
[0079] See attached Figure 7 The triglyceride content in the model group of nematodes was significantly increased by 61.91% compared with the blank control group (p<0.0001), indicating that high glucose culture conditions successfully induced lipid deposition in nematodes. Icariin treatment showed a significant lipid-lowering effect, exhibiting a clear concentration-dependent effect. At a concentration of 150 μM, the triglyceride content in nematodes decreased by 30.27% compared with the model group (p<0.0001). The positive control group showed a more significant reduction, decreasing by 38.97% compared with the model group (p<0.0001). This indicates that icariin can effectively improve lipid metabolism abnormalities induced by high glucose culture conditions, significantly reduce triglyceride content in nematodes, and reduce lipid deposition.
[0080] Example 11
[0081] Nematode Oil Red O Staining Experiment
[0082] Nematodes from different drug-treated groups were washed with M9 buffer and collected. They were fixed in 4% paraformaldehyde solution for 15 min, frozen at -80℃ for 2 min, thawed in water at room temperature, and repeated 3 times, discarding the supernatant. They were washed 3 times with M9 buffer and the supernatant was discarded. They were dehydrated in 60% isopropanol for 10 min and the supernatant was discarded. Oil Red O working solution (stock solution: distilled water = 3:2) was added and stained in the dark for 20 min, then washed with M9 buffer until the liquid in the centrifuge tube was colorless. The nematodes were placed on a glass slide, and the accumulation of lipid droplets within the nematodes was observed under a microscope. The images were taken and the stained area was calculated using ImageJ software.
[0083] See attached Figure 8 A. Compared with the blank control group, the Oil Red O staining area of nematodes in the model group was significantly increased, and the overall color was significantly redder, indicating an increased lipid droplet content. After intervention with different concentrations of icariin, the lipid staining area of nematodes decreased in a dose-dependent manner. Quantitative analysis of the staining area was performed using ImageJ software, and the results are as follows: Figure 8As shown in Figure B, lipid deposition in the model group was significantly increased by 92.52% compared to the control group (p<0.0001), proving the successful establishment of the high-lipidemia model. Compared to the model group, the nematode lipid droplet staining area was significantly reduced in the epimedium-treated group. When the drug concentration was 50, 100, and 150 μM, the staining area decreased by 31.42%, 42.62%, and 58.25%, respectively (p<0.0001). Among them, the lipid-lowering effect of the high-concentration drug group was better than that of the positive drug group (57.40%). The photographic results were consistent with the Image J quantitative analysis results, indicating that epimedium glycoside can effectively reduce the lipid droplet accumulation in high-sugar-induced high-lipidemia nematodes. This example visually demonstrates the effect of epimedium glycoside in reducing lipid droplet accumulation in nematodes through Oil Red O staining, further confirming its lipid-lowering effect in vivo from a morphological perspective, and its application in the preparation of drugs with both lipid-lowering and muscle-protective functions is reliable.
[0084] Example 12
[0085] Analysis of nematode body wall muscle fibers
[0086] Five days after high glucose induction treatment, RW1596 nematodes were collected and washed with M9 buffer, and then anesthetized with 1 mL of levamisole hydrochloride solution containing 5 mM. Subsequently, 20 μL of the anesthetized nematode suspension was dropped onto a glass slide, and the morphology of the nematode body wall muscle fibers was observed under an inverted fluorescence microscope.
[0087] See attached Figure 9 In RW1596 (pmyo-3MYO-3:GFP) nematodes, changes in the morphology of body wall muscle fibers were directly observed under a fluorescence microscope using GFP fluorescent labeling. The results showed that the body wall muscle fiber structure of the blank control group was relatively intact, with continuous and regularly arranged muscle fibers in the head and tail. In contrast, the model group exhibited significant muscle damage, with varying degrees of breakage and loss. The body wall muscle fibers in the icariin-treated group remained relatively intact, with good continuity in all parts, significantly reduced breakage compared to the model group, and regular arrangement comparable to the blank control group. Notably, the body wall muscle fibers in the positive control group (atorvastatin) did not improve, and the degree of breakage and loss was comparable to the model group, with some even showing more severe damage. These results indicate that icariin effectively protects muscle fibers from damage under high-glucose culture conditions, while atorvastatin has an adverse effect on nematode muscle tissue. This embodiment utilizes GFP-labeled body wall muscle fibers to systematically compare the effects of icariin and atorvastatin on the structural integrity of muscle tissue under high-fat conditions, demonstrating that icariin can effectively protect muscle tissue while exerting a lipid-lowering effect.
[0088] The above studies, through the construction of a free fatty acid-induced hyperlipidemic cell model and a high glucose-induced Caenorhabditis elegans model, respectively, demonstrated at the cellular and whole-animal levels that icariin can significantly reduce triglyceride and total cholesterol levels and decrease lipid droplet accumulation, thus exerting a clear lipid-lowering effect. Simultaneously, through nematode motility detection and GFP-labeled body wall muscle fiber structure observation, it was proven that icariin can improve muscle function and maintain muscle tissue integrity under hyperlipidemic conditions, and compared with atorvastatin, it avoids the risk of muscle damage. These studies comprehensively validate the application of icariin in the preparation of drugs with both lipid-lowering and muscle-protective functions.
[0089] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.
Claims
1. Application of icariin in the preparation of products with both lipid-lowering and muscle-protecting functions.
2. The application according to claim 1, characterized in that, The product includes any one of the following: medicine, health product, or food; the product is used to prevent and treat symptoms related to lipid metabolism disorders accompanied by the risk of muscle damage.
3. The application according to claim 2, characterized in that, The symptoms associated with lipid metabolism disorders that carry a risk of muscle damage include any one of the following: hyperlipidemia, obesity, non-alcoholic fatty liver disease, or metabolic syndrome.
4. The application according to claim 1, characterized in that, The muscle protection function includes maintaining the structural integrity of muscle cells and improving muscle function.
5. The application according to claim 1, characterized in that, The application is implemented through at least one of the following methods: (a) In a free fatty acid-induced hyperlipidemic cell model, icariin reduced intracellular triglyceride and total cholesterol levels; (b) In a high-sugar-induced high-lipid Caenorhabditis elegans model, icariin reduced triglyceride levels in nematodes and maintained the integrity of their body wall muscle fiber structure.
6. The application according to claim 1, characterized in that, The dosage form of the drug is one of the following: tablets, capsules, granules, powders, oral liquids, injections, and liposomes.
7. The application according to any one of claims 1 to 4, characterized in that, The drug is used to reduce intracellular triglyceride and total cholesterol levels.
8. The application according to claim 5, characterized in that, In (a), the concentration of icariin is 2.5 to 10 μM; in (b), the concentration of icariin is 50 to 150 μM.
9. A pharmaceutical composition, characterized in that, A pharmaceutical composition comprising a therapeutically effective amount of icariin or a pharmaceutically acceptable salt, derivative or prodrug thereof, and one or more carriers or excipients, wherein the pharmaceutical composition is intended to improve lipid metabolism disorders while protecting muscle tissue.