[0198] 5.3 Experimental Example 3 The effect of ginsenosides on glucose consumption of C2C12 cells
[0199] 5.3.1 Materials
[0200] 5.3.1.1 Main reagents
[0201] Penicillin and streptomycin were purchased from Sigma; DMEM culture medium, Fetal bovine serum (FBS) and Horse serum (HS) were purchased from Hyclone; Ginseng monomer saponins were obtained from laboratory separation .
[0202] 5.3.1.2 cell line
[0203] The C2C12 myoblast cell line was provided by the Department of Pharmacology, Bethune Medical College, Jilin University.
[0204] 5.3.1.3 Main instruments
[0205] Microplate reader; vortex mixer; electronic balance; 24-well cell culture plate, etc.
[0206] 5.3.2 Experimental method
[0207] 5.3.2.1 C2C12 myoblast culture and myotubule differentiation
[0208] Normal mouse skeletal muscle C2C12 myoblasts were inoculated in a culture flask, in DMEM medium containing 10% fetal bovine serum (containing penicillin 100IU/mL, streptomycin 100IU/mL) at 37℃, 5% CO 2 Cultured under conditions, the cells were cultured under 80% fusion and passaged once, and passaged to a 24-well plate according to 1:4. When the cells were fused to 80-90%, use DMEM medium containing 2% horse serum (containing penicillin 100IU/mL, streptomycin 100IU/mL) induce differentiation (day 0), and change the medium every 48 hours; after 5-6 days, more than 90% induce differentiation into mature skeletal muscle cells, which can be used for experiments.
[0209] 5.3.2.2 Glucose uptake test
[0210] 1. After the cells have differentiated and matured, wash them twice with sterile PBS, and then incubate them in a serum-free medium for 2 hours, taking care not to shed the cells. Discard the serum-free DMEM and replace it with DMEM low-sugar medium (sugar 5.55mmol/L) configured with 200μM ginsenoside in a 24-well plate, 0.5mL per well, 4 replicate wells for each concentration, and 4 replicate wells without adding Insulin, measure the glucose uptake rate of cells in the basal state, 100nM insulin as a positive control, at 37℃, 5% CO 2 Cultivate for 24h under conditions.
[0211] 2. Determination of glucose concentration by glucose oxidase method
[0212] After 24 hours of action, draw the supernatant to be measured glucose content from each well; C2C12 cell sugar consumption (mmol/L) = initial concentration of medium (5.55 mmol/L)-remaining glucose concentration in culture.
[0213] 3. Determination of protein concentration
[0214] Cells at the bottom of a 24-well plate were washed 3 times with PBS, lysed with cell lysate in an ice bath for 15 minutes, centrifuged, and cell debris was removed to collect protein. After staining with Coomassie brilliant blue, the protein content was measured at 492nm with a microplate reader (for calibration Number of cells)
[0215] 5.3.2.3 Statistical methods
[0216] Use software SPSS11.5 for data analysis. P<0.05 means the difference is statistically significant.
[0217] 5.3.2.4 Results and discussion
[0218] Table 3-1 Effects of ginsenosides on sugar uptake in C2C12 cells (n=4)
[0219]
[0220] Note: The sugar consumption of the blank control group is set to 1, and the other groups are all its multiples. Compared with the blank control group " * "P<0.05
[0221] Insulin resistance (IR) is manifested by the low sensitivity and/or responsiveness of target cells to insulin, which is the common pathophysiological basis of many component diseases of metabolic syndrome (MS). Since the 1990s, the research on the pathogenesis and prevention methods of IR has been a hot spot in the medical field. As an important target of insulin action, skeletal muscle is not only an important place for glucose and lipid metabolism, but also an important link in the occurrence of IR. It is currently believed that lipid accumulation in skeletal muscle is closely related to the occurrence and development of skeletal muscle IR. Muscle cells transport glucose mainly by glucose transporter-4 (GLUT-4), which is regulated by insulin. Through comparative studies, it is found that ginseng secondary saponins can significantly promote cellular sugar uptake (P<0.05), and can improve diabetes insulin resistance .
[0222] 5.4 Example 4
[0223] Take 1g of the above-mentioned ginseng secondary saponin or its fatty acid ester, add 10g of excipient medicinal cyclodextrin, mix uniformly, and granulate to obtain tablets, each containing 10mg of active ingredient.
[0224] 5.5 Example 5
[0225] Take 1g of the above-mentioned ginseng secondary saponin or its fatty acid ester, add 10g of excipient medicinal cyclodextrin, mix uniformly, granulate, and fill into capsules, each containing 10mg of active ingredient.
