**4. Discussion**

In the present study, the phenolic compounds and the anti-atherogenic effect of bee bread were evaluated in HFD-induced obese rats. After 6 weeks of continuous administration of HFD, Lee obesity index was significantly higher (more than 315) in the HFD group compared to N group, suggesting that the HFD used in the present study successfully induced an animal model of obesity. As compared to normal diet, the HFD contained higher caloric and fat contents, but lower carbohydrate and protein contents, as shown in Table 1. These differences were due to the mixture of 68 g normal diet with 32 g ghee, 300 mg calcium, 100 IU vitamin D, and 12% cholesterol powder based on the previous study [22] with slight modifications. Hence, the obesity in the HFD group could be due to high composition of fat component in HFD regime ingested, as significantly higher calorie intake was found in this group. HFD + BB group had a significant decrease in Lee obesity index when compared to HFD group, suggesting the anti-obesity effect of bee bread. Although Lee obesity index and weight gain in HFD + O group were observed to be lower compared to HFD group, the difference was not statistically significant. This may sugges<sup>t</sup> bee bread at 0.5 g/kg/day has a better anti-obesity effect compared to orlistat at 10 mg/kg/day in this animal model.

Hypercholesterolemia is closely associated with pathogenesis of atherosclerosis. In the present study, the levels of TC and LDL were significantly higher in the HFD group compared to the N group, similarly to a previous study [29]. A previous study hypothesized that elevated TC and TG levels have been reported to be a crucial factor in lipoprotein metabolism, and its higher concentration was attributed to increased LDL formation and deposition, which is potently atherogenic [30]. Meanwhile, levels of TC and LDL were significantly decreased in the HFD + BB group compared to the HFD group, suggesting a hypocholesterolaemic property of bee bread. A similar finding was also found in a study in which there were significant decreases of TC and LDL levels in obese and overweight patients when combined with honey [15]. Significant low level of LDL was also observed in the HFD + O group, which is consistent with a previous study, due to its action towards inhibition of gastric and pancreatic lipase enzymes [31]. The atherogenic index values of HFD + BB and HFD + O groups were significantly lower (by 40%) than the HFD group's, indicating that bee bread and orlistat are able to reduce the risk of CVD as high atherogenic index is an indicator of high risk to develop CVD [32]. Hence, it is plausible to sugges<sup>t</sup> that both bee bread and orlistat have LDL-lowering effects which might reduce the progression of atherosclerosis.

Growing evidence supports that increased oxidative stress is attributed by the presence of excessive free radicals which interplay between hypercholesterolemia and atherosclerosis [33]. Oxidative modification of LDL plays an immense role in the initial development of atherosclerosis and promotes further accumulation of free radicals in the arterial wall. A significant increase of aortic oxLDL and MDA levels in the HFD group might sugges<sup>t</sup> an excessive formation of oxidative stress in the aortic tissue following HFD ingestion as a consequence of the lipid oxidation process [34]. Meanwhile, these oxidative stress markers demonstrated significantly lower levels in the HFD + BB group compared to the HFD group. The HFD + O group had significantly lower oxLDL level compared to the HFD group, without any changes in MDA level. This might indicate that bee bread may have a greater ability to reduce lipid oxidation compared to orlistat. Antioxidants have been shown to stabilize free radicals, thereby reducing oxidative damage within biochemical, cellular and molecular levels [35]. HFD group had significantly lower levels of aortic GPx and CAT activities compared to N group which could be due to suppression or deactivation of these enzymes by oxidative stress. Significant increase of SOD and GPx activities in the HFD + BB group could be responsible for the lower oxLDL and MDA levels found in this group, which sugges<sup>t</sup> the involvement of antioxidants in reducing the increase of lipid oxidation. This antioxidant effect could partly due to the presence of phenolic compounds such as isorhamnetin, apigenin, caffeic acid, ferulic acid, and kaempferol found in the bee bread which have antioxidant properties [36]. However, it is suggested to further quantify these identified phenolic compounds in future study. In addition, previous studies have reported that bee bread also contains other compounds that have antioxidant properties, such as amino acids, vitamins, and minerals [9,37,38]. As reported

in our previous study, the bee bread used in the present study consists of carbohydrate (59.55%), protein (18.37%), and fat (4.51%) [38]. Hence, the antioxidant e ffect of bee bread found in the present study could also partly due to the interaction among these compounds, which needs further study. Orlistat has also shown to exert an antioxidant e ffect by evidence of significant increase in GPx activity compared to the HFD group. The previous study has shown involvement of antioxidant activity following orlistat administration in high-fed diet rats, whereby a significant increase of testicular and brain SOD activity was demonstrated [39]. High levels of oxLDL and MDA, suggesting high oxidative stress, with concomitant high TC and LDL levels, might explain the presence of atherosclerotic plaque in HFD group. Meanwhile, both HFD + BB and HFD + O groups showed an absence of atherosclerotic plaque, suggesting that supplementation of bee bread and orlistat for 6 weeks might protect against the formation of atherosclerotic plaque. Similarly, a study has reported the protective e ffect of orlistat in reducing the progression of atherosclerotic changes in rats fed with HFD [40].

The above results indicate that supplementation of bee bread for 6 weeks demonstrated anti-atherogenic e ffect as it significantly improved Lee obesity index, TC, and LDL levels; the atherogenic index; aortic oxidant-antioxidant status; and showed an absence of atherosclerotic plaque formation. To further assess the possible underlying mechanism of anti-atherogenic e ffect of bee bread, we also evaluated the level of aortic FAS activity. Significant low levels of TC and LDL in HFD + BB group might be related to the presence of ferulic and ca ffeic acids which are reported to exert hypocholesterolemic properties by significantly reducing TC, TG, and LDL levels. This, in turn, may reduce the risk of atherosclerosis [41,42]. The abundance of oxLDL in circulatory system is permeable to subendothelial layer, which further promotes inflammatory response that subsequently a ffects the endothelial wall integrity and whole vascular function [39]. A previous study has reported that the increase in the levels of adhesive molecules, such as intracellular and vascular cell adhesion molecule, is mediated by platelet-activating factor, which is crucial for adhesion and migration of leucocytes into subendothelium layer [43]. As a result, more leucocytes engulf the oxLDL, and subsequently form foam cells, which further triggers higher inflammation cascades for development of an atherosclerotic plaque [44]. Kaempferol, a flavonoid derivative, has been shown to exert an anti-inflammatory e ffect by reducing the aortic levels of inflammatory markers, intracellular and vascular cell adhesion molecule, monocyte chemotactic protein 1, and E selectin in rabbits as the model of atherosclerosis [45]. Hence, the anti-atherogenic e ffect of bee bread could also be attributed to the anti-inflammatory e ffect of kaempherol and the hypocholesterolemic e ffects of ferulic and ca ffeic acids that are present in the bee bread. In addition, it is also suggested to determine the role of inflammatory cytokines in a future study.

Higher fatty acids in circulation are associated with higher formation of polyunsaturated fatty acids mediated by FAS [46]. In the present study, significant higher aortic FAS in the HFD group, which represents higher fatty acid formation, could be attributed to high TC and LDL levels found in this group. This is further supported by the finding of larger adipocyte size observed in this group. A significant low activity of FAS in the HFD + BB group might explain the low levels of TC and LDL, and the presence of smaller adipocyte size in this group. Taken together, these findings may indicate the potential mechanism of anti-atherogenic e ffects of bee bread, which needs further study to determine its exact molecular mechanism of action.
