**3. Results**

#### *3.1. Liquid Chromatography-Mass Spectrophotometry Analysis of Bee Bread*

Five phenolic compounds were identified in the bee bread used in the present study. Isorhamnetin showed highest level of mass spectrum (317.07 *m*/*z*), followed by kaempferol (287.06 *m*/*z*), apigenin (271.06 *m*/*z*), ferulic acid (195.09 *m*/*z*), and ca ffeic acid (181.12 *m*/*z*) (Table 2).


**Table 2.** Liquid chromatography-mass spectrophotometry analysis of bee bread.

#### *3.2. Lee Obesity Index, Weight Gain, Food, and Calorie Intake*

After 6 weeks, all rats in HFD, HFD + BB, and HFD + O groups were obese, as their Lee obesity indices were more than 315 compared to the N group. The Lee obesity index was significantly lower in the HFD + BB group compared to HFD group. There were no significant differences in weight gain and food intake among all groups. Calorie intake was significantly higher in all groups which received the HFD compared to the N group, but it was not significantly different among all groups that received the HFD (Table 3).

**Table 3.** The effects of bee bread on Lee's obesity index, weight gain, food, and calorie intake.


Data are presented as means (with standard deviations) (*n* = 8 per group). N, normal; HFD, high fat diet; HFD + BB, high-fat diet and 0.5 g/kg bee bread; HFD + O, high-fat diet and 10 mg/kg orlistat. a *p* < 0.05 compared with N group; b *p* < 0.05 compared with the HFD group (one-way ANOVA followed by Tukey's post-hoc test).

#### *3.3. The E*ff*ects of Bee Bread on Lipid Profile and Atherogenic Index*

TC and LDL levels were found to be significantly higher in HFD group compared to N group and were significantly lower in HFD + BB group compared to HFD group. No significant differences were found for TG and HDL levels among all the groups. Atherogenic index was significantly lower in the groups supplemented with bee bread (HFD + BB) and orlistat (HFD + O) compared to the HFD group. However, the indices were not significantly different between HFD + BB and HFD + O groups (Table 4).

**Table 4.** The effects of bee bread on serum lipid profiles and the atherogenic index.


Data are presented as means (with standard deviations), *n* = 8 per group. N, normal; HFD, high fat diet; HFD + BB, high-fat diet and 0.5 g/kg bee bread; HFD + O, high-fat diet and 10 mg/kg orlistat. TC, total cholesterol; TG, triglyceride; LDL, low-density lipoprotein; HDL, high-density lipoprotein. a *p* < 0.05 compared with N group; b *p* < 0.05 compared with the HFD group (one-way ANOVA followed by Tukey's post-hoc test).

#### *3.4. The E*ff*ects of Bee Bread on Aortic Oxidant-Antioxidant Status and FAS Activity*

Aortic oxLDL and MDA levels were significantly higher in the HFD group compared to the N group and significantly lower HFD + BB compared to the HFD group. OxLDL was significantly lower in the HFD + O group compared to the HFD group. GPx and CAT activities were significantly lower in the HFD group compared to the N group. Meanwhile, significant increases of SOD and GPx activities were seen in HFD + BB group when compared to the HFD group. GPx activity was significantly higher in HFD + O group compared to N, HFD, and HFD + BB groups. The HFD group had significantly more FAS compared to the N group. However, in the HFD+BB group, the FAS level was significantly lower than for the HFD group (Table 5).

**Table 5.** The effects of bee bread on aortic oxidant/antioxidant biomarkers and fatty acid synthase.


Data are presented as means (and standard deviation), *n* = 8 per group. N, normal; HFD, high fat diet; HFD + BB, high-fat diet and 0.5 g/kg bee bread; HFD + O, high-fat diet and 10 mg/kg orlistat. OxLDL, oxidised LDL; MDA, malondialdehyde; SOD, superoxide dismutase; GPx, glutathione peroxidase; CAT, catalase; FAS, fatty acid synthase. a *p* < 0.05 compared with N group; b *p* < 0.05 compared with HFD group; c *p* < 0.05 compared with HFD + BB group (one-way ANOVA followed by Tukey's post-hoc test).

#### *3.5. The E*ff*ects of Bee Bread on the Presence of Atherosclerotic Plaque*

Figure 1 shows representative pictures of aortic arch segments in Oil Red O staining. Aortic arch segments from N, HFD + BB, and HFD + O groups were intact without the presence of atherosclerotic plaque. Whereas an aortic arch segmen<sup>t</sup> from HFD group showed a red-stained atherosclerotic plaque within the curvature of aortic arch.

**Figure 1.** *Cont.*

**Figure 1.** Macroscopic findings of aortic arch segmen<sup>t</sup> in Oil Red O staining under a stereomicroscope. Aortic arch segments from N (**a**), HFD + BB (**c**), and HFD + O (**d**) groups show intact aorta arch segments without atherosclerotic plaque. However, the segmen<sup>t</sup> from HFD group shows the presence of atherosclerotic plaques (yellow arrow) in the aortic arch (**b**). N, normal; HFD, high fat diet; HFD + BB, high-fat diet and 0.5 g/kg bee bread; HFD + O, high-fat diet and 10 mg/kg orlistat.

#### *3.6. The E*ff*ects of Bee Bread on Histology of Adipose Tissue*

Figure 2 shows the difference in adipocyte sizes of adipose tissue from each representative experimental group using H&E staining. Adipocyte size in HFD group was relatively larger compared to the N group. The sizes were observed to be smaller in HFD + BB and HFD + O groups compared to that of the HFD group.

**Figure 2.** Histological findings of adipose tissue in H&E staining under light microscope. HFD group (**b**) has relatively larger adipocyte size compared to N group (**a**). Both HFD + BB (**c**) and HFD + O (**d**) groups had relatively smaller adipocyte size compared to the HFD group. N, normal; HFD, high fat diet; HFD + BB, high-fat diet and 0.5 g/kg bee bread; HFD + O, high-fat diet and 10 mg/kg orlistat. (Magnification ×200, Haematoxylin and Eosin staining).
