## 3 Results  
### Anti-Inflammatory Effect of GP in RAW264.7 Cells  
There were 1434 peptides identified in the GP with the molecule of 987.39 Da to 2317.11 Da and nonapeptide accounted for 20%. Most of the peptides were composed by 7-15 amino acids as shown in Figure 1. The peptides' sequences, mass, \(m/z\), and accession proteins information are shown in Table S1. In the treatment of RAW264.7 cells, concentration of 0.1, 0.3, 0.5, 0.7, 1.0, 1.5 and 2.0 mg/mL GP showed no significant effect on cell viability (Figure S1). In the study of [PERSON] et al. [9], cell viability was significantly increased after being treated with 0.125-1.0 mg/mL sturgeon muscle peptides. However, the 2.0 mg/mL treatment decreased cell viability from 100% (NC) to 91%. The decreasing effect of peptides on cell viability may be related to the increased osmotic pressure, which even causes the death of cells. Currently, the concentration of 0.1-2.0 mg/mL GP was measured to be no toxicity on macrophages cells. In the LPS induced RAW264.7 cells, the GP treatment suppressed the inflammatory biomarkers as shown in Figure 2. Compared with PC, the pretreatment of GP inhibited the secretion of NO in 0.1, 0.5 and 1.0 mg/mL (\(p<0.05\)) and there were no differences among GP groups (Figure 2A, \(p>0.05\)). Similarly, the GP pre-treatment also suppressed the TNF-\(\alpha\) from the 2361 pg/mL in PC to be 1498 pg/mL in 1.0 mg/mL of GP groups (Figure 2B, \(p<0.05\)). Here, the 0.5 and 1.0 GP groups showed no differences from each other. To reflect the anti-inflammatory effect of GP, the cytokine mRNA expression was also measured by RT-PCR with the groups of 0.5 and 1.0 mg/mL, respectively. Compared with PC, the GP reduced TNF-\(\alpha\) mRNA expression by 1.79- and 2.29-times in 0.5 and 1.0 mg/mL groups (Figure 2C, \(p<0.05\)). In addition, GP also reduced IL-6 mRNA expression compared with the PC group (Figure 2D, \(p<0.05\)). The 0.5 mg/mL of GP group showed no differences in IL-1\(\beta\) expression compared with PC (\(p>0.05\)), whereas the 1.0 mg/mL of GP group was investigated to exert the suppressing effect on IL-1\(\beta\) (Figure 2E, \(p<0.05\)). Regarded as the potential resource of anti-inflammatory components, the dry-cured ham peptides were also checked to repress the proinflammatory mediators of TNF-\(\alpha\), IL-6, IL-8, and IL-1\(\beta\) in the LPS-induced RAW264.7 macrophage cells, where the isolated sequences of GPPGL, GPAGPL, and GPPGAP were all originated from collagen protein [20]. Derived from dynastid beetle, the bioactive peptides inhibited the cyclooxygenase-2 (COX-2), iNOS, IL-6, and IL-1\(\beta\) expression [21]. Isolated from microalgae protein, the peptides with the MW of \(<\)3 kDa were also investigated to suppress the TNF-\(\alpha\), COX-2, and IL-6 expression in RAW264.7 macrophage cells [22]. The LPS-induced RAW264.7 macrophage cells were widely used to assess the anti-inflammatory effect of bioactive peptides and the suppressing effect on proinflammatory cytokines was the biomarker to reveal the immune level in cells. From the current results, the GP sequences were mainly composed by 7-15 amino acids and the GPP fragment appeared in 397 peptides such as FLPAGYPE, GRGPP, GPPFGGPK, GPPGFPGY, GPPGPGPGPQ, GPPGFPGE, etc. Similarly, the anti-inflammatory peptides in dry-cured hams (GPPGL, GPPGAP) was also composed by GPP fragment, which means the certain sequences play an important role in inflammation function [20].