Application of eugenol in preparation of products for promoting myoblast differentiation

Abstract

The invention belongs to the technical field of medicines, and particularly provides novel application of eugenol in preparation of products for promoting myoblast differentiation. Cell experiment evaluation proves that the eugenol can remarkably promote C2C12 myoblast differentiation and promote myotube formation, and the eugenol is mainly characterized by increasing the myotube diameter, increasing the myotube length, improving the myotube fusion index and increasing the cell total protein content. Therefore, the eugenol provided by the invention can be applied to preparation of products for promoting myoblast differentiation.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines and specifically discloses use of eugenol in preparing a product for promoting myoblast differentiation. Background Art

[0002] Skeletal muscle is one of the main muscle types in the human body and an important organ related to movement and metabolism. It is of great significance for maintaining movement balance, regulating protein metabolism and energy metabolism. Skeletal muscle has a high degree of plasticity and strong regeneration ability. After mild damage, it can usually restore normal structure and function within 2 to 3 weeks. Myoblasts refer to muscle precursor cells containing myofilaments in the cytoplasm. They have the ability to self-renew, proliferate, differentiate, fuse, and form muscle fibers to maintain the normal physiological function of skeletal muscle. Under normal physiological conditions, muscle satellite cells are in a quiescent state; when skeletal muscle is injured, muscle satellite cells are activated and transformed into myoblasts, and express myogenic markers; then myoblasts irreversibly exit the cell cycle, proliferate, differentiate and fuse to form multinucleated myotubes, and the expression level of regulatory factors also changes accordingly; finally, new muscle fibers are formed. Myoblast proliferation and differentiation are the basis of skeletal muscle tissue development and regeneration, and are of great significance for the treatment of skeletal muscle injury.

[0003] Eugenol is a phenolic compound naturally present in aromatic plants such as cloves and cinnamon. As an aromatic ingredient, eugenol is widely used in the production of cosmetics and spices, and is approved as a synthetic flavor for food. In recent years, research on the biological activity of eugenol has mainly focused on antibacterial and antioxidant capabilities. Existing studies have shown that eugenol has significant antibacterial activity in vitro, and exhibits antibacterial ability against bacteria and fungi through mechanisms such as destroying cell membranes, inhibiting enzyme activity, and affecting biofilm formation, indicating that eugenol has the potential to be developed as an antibacterial drug. In addition, in vitro studies have shown that eugenol has DPPH free radical scavenging activity and reducing ability, which can block hydroxyl free radical-induced DNA oxidation and lipid peroxidation. However, the beneficial effects of eugenol on skeletal muscle growth and development have not been reported. The present invention innovatively conducts research on the effect of eugenol on the in vitro differentiation of mouse skeletal muscle myoblasts C2C12 from the perspective of cell biology, in order to provide a theoretical basis for the application of eugenol in products that promote myoblast differentiation. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of eugenol, specifically, to provide the use of eugenol in preparing a product for promoting myoblast differentiation.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] Use of eugenol in preparing a product for promoting myoblast differentiation.

[0007] Preferably, the eugenol has no significant effect on the cell viability of C2C12 myoblasts within a concentration range of 0 to 50 μM.

[0008] Preferably, the eugenol can increase the diameter of myotubes of C2C12 cells.

[0009] Preferably, the eugenol can increase the length of myotubes of C2C12 cells.

[0010] Preferably, the eugenol can increase the myotube fusion index of C2C12 cells.

[0011] Preferably, the eugenol can increase the total protein content of C2C12 cells.

[0012] The present invention also provides a health food composition for promoting myoblast differentiation, comprising any of the eugenols described above, and a food-acceptable auxiliary material.

[0013] Preferably, the health food composition has no significant effect on the cell viability of C2C12 myoblasts, and can significantly increase the myotube diameter, length, fusion index, and total protein content of C2C12 cells.

[0014] The present invention also provides a pharmaceutical composition for promoting myoblast differentiation, comprising any of the eugenols described above, and a pharmaceutically acceptable excipient or carrier.

[0015] Preferably, the medicine can be an oral solution, powder, tablet, capsule or injection.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention has discovered a new use of eugenol. Cell experiment results show that eugenol can significantly increase the diameter of myotubes, increase the length of myotubes, improve the myotube fusion index, and increase the total protein content of cells when treating C2C12 myoblasts, indicating that eugenol can promote myoblast differentiation. The present invention provides a new use of eugenol in the preparation of related health foods and medicines that have the effect of promoting myoblast differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of eugenol on the viability of C2C12 cells.

[0019] Figure 2 The morphology of myotubes after 3 days of differentiation of C2C12 cells.

[0020] Figure 3This is the effect of eugenol on the diameter of C2C12 cell myotubes. ** p<0.01.

[0021] Figure 4 This is the effect of eugenol on the length of myotubes in C2C12 cells. *** p<0.001.

[0022] Figure 5 This is the effect of eugenol on the myotube fusion index of C2C12 cells. * p<0.05.

[0023] Figure 6 This is the effect of eugenol on the total protein content of C2C12 cells. * p<0.05.

[0024] Note: The present invention Figures 2 to 6 middle:

[0025] EU group represents the eugenol-treated group, and C2C12 cells were treated with eugenol during differentiation. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the embodiments of the present invention and the accompanying drawings, but the protection scope of the present invention is not limited thereto.

[0027] Example 1

[0028] Effects of eugenol on the viability of C2C12 cells

[0029] C2C12 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution at 37°C in the presence of 5% CO2. When the cell confluence reached more than 60%, the cells were passaged, washed with PBS, and then treated with 0.25% trypsin-ethylenediaminetetraacetic acid solution to detach the adherent cells. The cell suspension was inoculated in a 96-well plate and treated with different concentrations of eugenol, namely 3.125, 6.25, 12.5, 25, and 50 μM, and cultured for 24 hours. In addition, a control group containing only culture medium but no cells was set up. After incubation in CCK8 reagent for 1 hour, the optical density (OD) of each well in the 96-well plate was measured at 450 nm using an enzyme reader. The cell viability (X, %) was calculated as follows:

[0030]

[0031] Wherein: A2 is the OD value of each test well, A1 is the average OD value of the group without eugenol treatment, and A0 is the average OD value of the control group.

[0032] according to Figure 1As shown, eugenol had no significant effect on C2C12 cell viability within the concentration range of 0 to 50 μM, so a 50 μM eugenol concentration was used in subsequent experiments.

[0033] Example 2

[0034] Effects of eugenol on the diameter of myotubes in C2C12 cells

[0035] C2C12 cells were divided into a control group and a eugenol-treated group, with 3 replicates in each group. The cells were cultured in DMEM medium containing 10% FBS and 1% PS until the cells reached 90% confluence, and then replaced with differentiation medium and continued to be cultured for 3 days to induce differentiation into myotubes. During the differentiation process, the eugenol-treated group was treated with 50 μM eugenol. The cells in each group were collected, washed with PBS 3 times, fixed with methanol at room temperature for 10 minutes, dried for 15 minutes, and stained with an appropriate amount of Swiss-Giemsa stain for 30 minutes; washed with PBS 3 to 5 times, and an appropriate amount of PBS was added. At least 9 myotube cells in each field of view were randomly selected, and images were taken under a 200X microscope. The diameter of the myotubes in the images was measured using ImageJ software.

[0036] according to Figure 2 , 3 As shown, the diameter of myotubes in the eugenol-treated cells was significantly increased compared with that in the control group.

[0037] Example 3

[0038] Effects of eugenol on the length of myotubes in C2C12 cells

[0039] For each group of C2C12 cells stained in Example 2, at least 9 fields of view were randomly selected from each group, and images were taken under a 200X microscope. The length of myotubes in the images was measured using ImageJ software.

[0040] according to Figure 2 , 4 As shown, the myotube length of cells in the eugenol-treated group was significantly increased compared with that in the control group.

[0041] Example 4

[0042] Effects of eugenol on the myotube fusion index of C2C12 cells

[0043] At least 9 fields of view were randomly selected from each group of C2C12 cells differentiated and cultured in Example 2, and images were taken under a 200X microscope. The nuclei of all cell nuclei and myotubes in the captured images were counted using ImageJ software. Muscle cells containing three or more nuclei were considered myotubes. The fusion index was calculated as the number of nuclei in the myotube divided by the total number of nuclei counted.

[0044] according to Figure 5As shown, the fusion index of cells in the eugenol-treated group was significantly increased compared with that in the control group.

[0045] Example 5

[0046] Effects of eugenol on total protein content in C2C12 cells

[0047] The culture medium of each group of C2C12 cells cultured in Example 2 was discarded, the cells were rinsed once with PBS, 100 μL of lysis buffer was added, and the cells were lysed on ice for 5 min. The cells were scraped and transferred to a 1.5 mL EP tube and rotated at 12 000 r·min. -1 Centrifuge at 1000rpm for 10 minutes. Aspirate the supernatant and measure the OD value of each well at a wavelength of 562nm using an ELISA reader according to the BCA kit operating procedures. Calculate the sample concentration according to the standard concentration calculation formula, which is the protein content of the stock solution.

[0048] according to Figure 6 As shown, the total protein content of cells in the eugenol-treated group was significantly increased compared with the control group.

[0049] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. Use of eugenol in preparing products for promoting myoblast differentiation.

2. The use according to claim 1, characterized in that The eugenol can promote the differentiation of C2C12 myoblasts and the formation of myotubes.

3. The use according to claim 2, characterized in that: The eugenol has no significant effect on the cell viability of C2C12 myoblasts within a concentration range of 0 to 50 μM.

4. The use according to claim 2, characterized in that: The eugenol can increase the diameter of the myotubes of C2C12 cells.

5. The use according to claim 2, characterized in that: The eugenol can increase the length of myotubes of C2C12 cells.

6. The use according to claim 2, characterized in that: The eugenol can increase the myotube fusion index of C2C12 cells.

7. The use according to claim 2, characterized in that: The eugenol can increase the total protein content of C2C12 cells.

8. The use according to claim 1, characterized in that The product is a health food composition or a pharmaceutical composition.

9. A health food composition for promoting myoblast differentiation, characterized in that: The invention comprises the eugenol according to any one of claims 1 to 7, and an auxiliary material acceptable to food.

10. The health food composition according to claim 9, characterized in that The health food composition has no significant effect on the cell viability of C2C12 myoblasts, and can significantly increase the myotube diameter, length, fusion index, and total protein content of C2C12 cells.

11. A pharmaceutical composition for promoting myoblast differentiation, characterized in that: The invention comprises the eugenol according to any one of claims 1 to 7, and a pharmaceutically acceptable auxiliary material or carrier.

12. The drug for promoting myoblast differentiation according to claim 11, characterized in that: The medicine can be oral solution, powder, tablet, capsule or injection.

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