**3. Results**

#### *3.1. Anti-Glycation E*ff*ects of Brazilian Propolis In Vitro*

The inhibitory activity of Brazilian propolis against formation of AGEs was evaluated by measurement of fluorescent AGEs formed by glyceraldehyde and bovine serum albumin (Figure 1). Propolis inhibited the formation of fluorescent AGEs (0 mg/mL, 0 ± 4.53%; 0.1 mg/mL, 31.1 ± 2.87%; 1.0 mg/mL, 84.5 ± 2.00%; 10 mg/mL, 118 ± 0.62%; 100 mg/mL, 122 ± 1.79%, means ± SE, *n* = 4/group).

#### *3.2. The E*ff*ect of Brazilian Propolis on Body Weight, Food and Fluid Intake, and Muscle Weight*

Body and muscle weights and food and fluid intake are presented in Table 2 and Figures S1–S3. Repeated measures ANOVA did not reveal significant di fferences in the body weights among the groups (*p* = 0.070). Food intake was significantly di fferent among the groups (*p* = 0.0004); specifically, MGO + PRO group had lower food intake than all other groups (*p* = 0.007 vs. N; *p* = 0.003 vs. PRO; *p* = 0.001 vs. MGO). Fluid intake was significantly di fferent among the groups (*p* = 0.0004); in that, the PRO (*p* = 0.010), MGO (*p* = 0.043), and MGO + PRO (*p* = 0.0003) groups had lower fluid intakes than N group. Two-way ANOVA revealed that MGO, but not propolis, had a significant main effect on EDL muscle weight normalized to tibia length (propolis, *p* = 0.69; MGO, *p* = 0.039) (Table 2) and muscle cross sectional area (CSA) (propolis, *p* = 0.95; MGO, *p* = 0.042) (Table S1). No significant main effects were observed for soleus muscle weight normalized to tibia length (propolis, *p* = 0.054; MGO, *p* = 0.086) (Table 2) and muscle CSA (propolis, *p* = 0.18; MGO, *p* = 0.13) (Table S1). However, propolis and MGO showed a large (η<sup>2</sup> = 0.16) and moderate (η<sup>2</sup> = 0.12) effect size in soleus muscle mass as calculated using η2, respectively.

**Figure 1.** The inhibitory effect of Brazilian propolis used at different concentrations (0, 0.1, 1.0, 10, and 100 mg/mL) on formation of advanced glycation end products (AGEs). Values are means ± SE; *n* = <sup>4</sup>/group. Values are expressed as percent change relative to the value of aminoguanidine.


**Table 2.** Body weight, food intake, fluid intake, and muscle weight.

EDL, extensor digitorum longus; MGO, methylglyoxal; *n* = 4–6/group; \* and † indicates *p* < 0.05 vs. Normal and MGO + propolis group, respectively.

#### *3.3. Brazilian Propolis Suppressed the Accumulation of MGO-Derived AGEs in the Skeletal Muscle In Vivo*

The content of MGO-derived AGEs in EDL and soleus muscles was measured to evaluate the effect of Brazilian propolis on accumulation of AGEs in the skeletal muscle in vivo. In the EDL muscle, two-way ANOVA revealed a significant interaction (*p* = 0.020); specifically, the content of MGO-derived AGEs following MGO treatment tended to increase (*p* = 0.08), but it had a large effect size as calculated using Cohen's d (d = 1.54). Brazilian propolis ingestion suppressed this accumulation (*p* = 0.003) (Figure 2). In the soleus muscle, no significant alterations in the content of MGO-derived AGEs was observed according to ANOVA (MGO, *p* = 0.59; propolis, *p* = 0.97) (Figure 2).

**Figure 2.** The content of methylglyoxal (MGO)-derived advanced glycation end products (AGEs) in skeletal muscles. The extensor digitorum longus (EDL) and soleus muscles were dissected from mice treated with or without Brazilian propolis (0.1%)-containing diet or MGO (0.1%)-containing drinking water for 20 weeks. Values are means ± SE; *n* = 5–6/group. \* *p* < 0.05 between the groups.

#### *3.4. Brazilian Propolis Enhanced Glyoxalase 1 Activity in The Skeletal Muscle*

To evaluate the ability of Brazilian propolis to detoxify MGO in the skeletal muscle, the activity of glyoxalase 1, a dicarbonyl compound eliminating enzyme, was measured in the EDL and soleus muscles. In the EDL, two-way ANOVA revealed a significant main effect of propolis, but not MGO (propolis, *p* = 0.038; MGO, *p* = 0.49) (Figure 3). In the soleus muscle, no significant alterations in glyoxalase 1 activity was observed via ANOVA (MGO, *p* = 0.25; propolis, *p* = 0.47) (Figure 3).

**Figure 3.** The activity of glyoxalase 1 in skeletal muscles. EDL and soleus muscles were dissected from mice treated with or without Brazilian propolis (0.1%)-containing diet or MGO (0.1%)-containing drinking water for 20 weeks. Values are means ± SE; *n* = 5–6/group. †, significant main effect between diets (normal and propolis).

#### *3.5. Brazilian Propolis Suppressed MGO-Induced mRNA Expression of Inflammatory-Related Molecules in The Skeletal Muscle*

To evaluate the effect of propolis on inflammatory responses, themRNA expression of proinflammatory cytokines, IL-1β and IL-6, and AGEs-related receptors, TLR4 and RAGE, were measured in the EDL and soleus muscles (Figure 4). In the EDL muscle, two-way ANOVA revealed significant e ffects on IL-1β, Il-6, and TLR4 expression. MGO treatment significantly increased the mRNA expression of IL-1β (*p* = 0.037); however, the ingestion of Brazilian propolis suppressed this increase (*p* = 0.006). MGO treatment tended to increase the mRNA expression of IL-6, and propolis ingestion suppressed this effect in MGO-treated mice (*p* = 0.036). MGO treatment significantly increased the mRNA expression of TLR4 (*p* = 0.028), but no change was observed under propolis ingestion (*p* = 0.20). The mRNA level of RAGE in the EDL muscle was not altered by either MGO (*p* = 0.28) or propolis (*p* = 0.41). In the soleus muscle, no significant alterations in the mRNA expression of IL-1β (MGO, *p* = 0.078; propolis, *p* = 0.44), IL-6 (MGO, *p* = 0.80; propolis, *p* = 0.34), TLR4 (MGO, *p* = 0.21; propolis, *p* = 0.38), and RAGE (MGO, *p* = 0.33; propolis, *p* = 0.16) were observed via ANOVA (Figure 4).

**Figure 4.** mRNA expression of interleukin (IL)-1β, IL-6, toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in skeletal muscles. The EDL and soleus muscles were dissected from mice treated with or without propolis (0.1%)-containing diet or MGO (0.1%)-containing drinking water for 20 weeks. Data of IL-1β in the EDL muscle were log-transformed for normal distribution before analysis of variance (ANOVA). Values are means ± SE; *n* = 3–6/group. \* *p* < 0.05 between the groups.
