*3.2. Differences in Royal Jelly Phospholipid and Sphingolipid Composition*

Phospholipids and sphingolipids are important components of cell membranes and have a variety of physiological functions that are beneficial to human health [20]. To further understand the effects of the BP-Pa and BP-Am diets on the composition of phospholipids and sphingolipids in royal jelly, we quantified SMs, Cers, PCs, and PEs in the two types of royal jelly (RJ-Am and RJ-Pa). There were clear trends in the relative content of SMs, Cers, PCs, and PEs in RJ-Am and RJ-Pa (Figure 1). In their natural state, bee colonies gather various kinds of pollen for food. After honeybees were fed with a single pollen source instead of natural pollen for six days, levels of SMs, Cers, PCs, and PEs were significantly lower in the single-pollen royal jelly compared to the control. Lipid levels in the singlepollen royal jelly began to increase after 12 days of feeding; the highest lipid levels occurred on day 18 or 24, but were not as high as lipid levels in the control royal jelly. On day 30, the lipid content of royal jelly began to decrease significantly. Notably, the average PC content was significantly higher in RJ-Am than in RJ-Pa, which might be attributed to the significantly higher PC content in BP-Am than in BP-Pa.

**Figure 1.** Changes in levels of phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), ceramides (Cers), and sphingomyelins (SMs) in royal jelly secreted by honeybees fed bee pollen from a single plant source, *Acer mono* Maxim. (BP-Am) or *Phellodendron amurense* Rupr. (BP-Pa). Samples were collected on days 6, 12, 18, 24, and 30 after honeybees began feeding on single bee pollen. RJ-Am, royal jelly secreted by honeybees fed BP-Am; RJ-Pa, royal jelly secreted by honeybees fed BP-Pa. Significant differences in lipid content of RJ-Am or RJ-Pa secreted by honeybees fed BP-Am or BP-Pa at different timepoints are indicated with lowercase letters (a, b, c, d) and uppercase letters (A, B, C), respectively.
