## 3 Results
### Effect of BuOH Fraction on LPS-Induced C2C12 Myotube Atrophy  
As the BuOH fraction showed the highest TPC and total antioxidant capacity among the five fractions from ISE, we further confirmed the effect of the BuOH fraction on muscle atrophy in LPS-induced C2C12 myotubes. We measured the cell viability of the BuOH fraction in C2C12 myotubes and found that more than 95% of cells were viable when treated with 0-100 \(\upmu\)g/mL of BuOH fraction (Figure 3a). Reduced myotube diameter by LPS was significantly increased by the BuOH fraction at 75 and 100 \(\upmu\)g/mL (Figure 3b,c). The diameter of LPS-treated myotubes was reduced by 63.6% compared to the control (LPS-untreated myotubes), while 75 and 100 \(\upmu\)g/mL of the BuOH fraction increased by 43.5% and 56.5%, respectively, compared to the LPS-treated myotubes. The gene expression levels of FoxO3\(\alpha\), MAFbx, and MuRF1 were significantly increased by LPS, and were significantly reduced by the BuOH fraction (Figure 3d). Furthermore, LPS-stimulated  
Figure 1: Inhibitory effects of ISE on muscle atrophy in LPS-induced C2C12 myotubes. C2C12 myotubes were treated with LPS (1 \(\upmu\)g/mL) and ISE (50, 75, and 100 \(\upmu\)g/mL) for 48 h. (**a**) Cell viability of ISE in C2C12 myotubes. C2C12 myotubes were treated with 0–100 \(\upmu\)g/mL of ISE to measure the cell viability. (**b**) Giemsa staining was performed to visualize C2C12 myotube morphology (magnification 20\(\times\), scale bar = 100 \(\upmu\)m). (**c**) Myotube diameters (\(\upmu\)m) were observed from randomly selected fields and were quantified using the ImageJ program. The mRNA expression of (**d**) FoxO3a, MAFbx, and MuRF1 was analyzed by RT-PCR. GAPDH was used as internal controls. Bars with a different letter are significantly different from the control (\(p\) < 0.05). Figure 2: Effects of ISE and BuOH on the protein expression of muscle-specific E3 ubiquitin ligases in LPS-induced C2C12 myotubes. C2C12 myotubes were treated with LPS (1 \(\upmu\)g/mL) and ISE or BuOH fraction of ISE (50, 75, and 100 \(\upmu\)g/mL) for 48 h. The protein expression of MAFbx and MuRF1 was analyzed using Western blot analysis. \(\upbeta\)-Actin was employed as a loading control. protein expression levels of MAFbx and MuRF1 were noticeably attenuated by the BuOH fraction (Figure 2). Anti-Inflammatory Effects of ISE and BuOH Fraction through Inhibition of p38/ERK Signaling Pathway in LPS-Induced C2C12 Myotube Atrophy  
As inflammation is known to be a major factor in skeletal muscle atrophy [14], we measured the expression of pro-inflammatory cytokines and inflammation-inducible enzymes in LPS-induced C2C12 myotube atrophy. LPS significantly increased the expression of pro-inflammatory cytokines (interleukin-6 (IL-6) and IL-1\(\beta\)) and inflammation-inducible enzymes (iNOS and COX2) (Figure 4a,b) in C2C12 myotubes. ISE significantly reduced IL-1\(\beta\) and COX2 mRNA expression, but not IL-6 and iNOS mRNA expression. On the other hand, the BuOH fraction significantly reduced both the mRNA expression of pro-inflammatory cytokines and inflammation-inducible enzymes. In particular, ISE markedly abolished the LPS-stimulated protein expression of iNOS and COX2 in C2C12 myotubes (Figure 5a). However, the BuOH fraction markedly reduced the LPS-stimulated iNOS protein expression in C2C12 myotubes, but not COX2 protein expression. Furthermore, since LPS-stimulated gene expression of pro-inflammatory cytokines and inflammation-inducible enzymes are mainly activated by the mitogen-activated protein kinase (MAPK) signaling pathway [15], we further investigated the effect of both the ISE and the BuOH fraction on the inhibition of the p38/ERK pathway. LPS markedly increased the phosphorylation of p38 and ERK, which was markedly attenuated by the ISE and the BuOH fraction, but had no dose-dependent effects (Figure 5b,c). Figure 3: Inhibitory effects of the BuOH fraction from ISE on muscle atrophy in LPS-induced C2C12 myotubes.