*2.2. Lipids*

Among the principal bioactive constituents of RJ, lipids constitute 7–18% of RJ content; 90% of these lipids are rare and unique short hydroxy fatty acids with 8–12 carbon atoms in the chain and dicarboxylic acids. The most prominent RJ fatty acids in order are 10-hydroxydecanoic acid (10-HDA), 10-hydroxy-2-decenoic acid (10H2DA), and sebacic acid (SA) [18]. The 10-HDA exerts epigenetic control over caste differentiation of *Apis mellifera* by inhibiting histone deacetylases, which catalyze the hydrolysis of ε-acetyl-lysine residues of histones [25,26]. Due to the low pH of RJ, 10-HDA acts as a strong bactericidal. It therefore, protects bee larvae against virulent bacterial infections that affect bee hives such as those caused by some strains of *Paenibacillus* larvae [27]. In mammals, it protects mice against pulmonary damage induced by lipoteichoic acid, a toxin from *Staphylococcus aureus* [28]. It also exerts an antibacterial effect against various pathogenic bacterial species in human cancer colon cells [29]. The 10-HDA may be used to treat age-related neurodegenerative disorders given its documented neurogenic activity—it stimulates neuronal differentiation from progenitor cells (PC12) cells through mimicking the effect of brain-derived neurotrophic factor [30]. In addition, it possesses neuroprotective effects against glutamate- and hypoxia-induced neurotoxicity [31]. The 10-HDA may also be used for manufacturing cosmetics and anticancer drugs, given that it increases skin-whitening and exerts antiproliferative effects on B16F10 melanoma cells by inhibiting the expression of microphthalmia-associated transcription factor and tyrosinase-related protein 1 (*TRP-1*) and *TRP-2* [32]. Moreover, 10-HDA has been identified as an inhibiting factor of matrix metalloproteinases (*MMPs*)—proenzymes activated by proteolytic cleavage under inflammatory

conditions, which degrade matrix and non-matrix proteins and contribute to tissue aging (e.g., skin) and cause serious disabling diseases such as rheumatoid arthritis [33,34].

SA, 10-HDA, and 10H2DA, demonstrate anti-inflammatory effects through regulation of several proteins involved in the mitogen-activated protein kinase (*MAPK*) and nuclear factor kappa-B signaling [29,35]. Moreover, these acids mediate estrogen signaling by enhancing the activity of estrogen receptors (*ERs*) *ER*α, *ER*β [36], which can benefit bone, muscle, and adipose tissue in a sex-dependent manner [31]. A derivative of 4-hydroperoxy-2-decenoic acid known as 4-hydroperoxy-2-decenoic acid ethyl ester (HPO-DAEE) prevents 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells through triggering slight emission of reactive oxygen species (ROS), which stimulates the production of antioxidants via activation of antioxidant pathways: nuclear factor erythroid 2 (*NRF2*)-antioxidant response element (*ARE*) and eukaryotic initiation factor 2 (*eIF2*α), an upstream effector of the activating transcription factor-4 (*ATF4*) [37]. HPO-DAEE also demonstrates anticancer effects through accumulation of intracellular ROS and activation of proapoptotic CCAAT-enhancer-binding protein homologous protein expression [38]

#### *2.3. Proteins*

Proteins are the dominant ingredient of RJ (50% of its dry matter) and more than 80% of total RJ proteins are composed of nine major RJ proteins (MRJPs, 49–87 kDa)—the first five MRJPs constitute up to 82–90% of MRJPs. Glycosylation and phosphorylation of MRJPs is essential for biological processes that involve glycoproteins, such as cell adhesion, cell differentiation, cell growth, and immunity. MRJPs modulate the development of female larvae, not only through their high nutritional value but mainly through physiological activity of their highly homologous 400–578 amino acids that contribute to RJ's role in cell proliferation, cytokine suppression, and antimicrobial activity [12].

Research documents anti-senescence activity of MRJPs for human cells in vitro [39]. MRJP1 is the most dominant among all MRJPs; essential amino acids constitute 48% of its content. Circular dichroism measurements indicate that the secondary structure of MRJP1 consists of 9.6% α-helices, 38.3% β-sheets, and 20% β-turns [22]. MRJP1 exists in two distinct forms: oligomer and monomer. Oligomer MRJP1 is highly heat-resistant and it is considered a predominant proliferation factor compared with MRJP2 and MRJP3 [40]. High-performance liquid chromatography and SDS-PAGE analyses of MRJP1 revealed the presence of a 57 kDa monomeric glycoprotein, which can be degraded at 40 ◦C, known as royalactin. Royalactin mimics the effect of epidermal growth factor (*EGF*) in rat hepatocytes and modulates the development of bee larvae [41]. Royalactin is reported to bind with the most sensitive regions in mouse embryonic stem cell culture, resulting in activation of a pluripotency gene network that enables self-renewal of stem cells [42]. MRJP1 exerts nematicidal activity against *C. elegans* via constant downregulation of a rate-controlling enzyme of the citric acid cycle known as isocitrate dehydrogenase encoding the idhg-1 gene [43]. On the other side, MRJP2 and its isoform X1 exhibit potent anticancer effects and protect hepatocytes against CCl4 toxicity by inducing caspase-dependent apoptosis, scavenging intracellular free radicals, inhibiting tumor necrosis factor (*TNF*)-α, and mixed lineage kinase domain-like protein [44].

RJ contains proteins other than MRJPs, albeit in small amounts, such as royalisin, jelleines, and aspimin. Royalisin and jelleines are common RJ antimicrobial peptides that enhance efficiency of the immune response of bee larvae to various infections. The structure of royalisin is highly compact due to its high cysteine content, which boosts its stability at low pH and high temperature. On the other hand jelleines are thought to stem from trypsin digestion of MRJP1 by the action of exo-proteinase of the hypopharyngeal glands on C-terminal to N-terminal tryptic fragment. Peptides of royalisin and jelleines possess hydrophobic residues, which contribute to their antimicrobial properties by affecting functions of bacterial membranes. RJ also contains apolipophorin III-like protein, a lipid binding protein that exerts antimicrobial effect by carrying lipids into aqueous environments through the formation of protein–lipid complexes. In addition, the antibacterial effect of RJ is partially attributed to its glucose oxidase enzyme, which catalyzes the oxidation of glucose to hydrogen peroxide [45]. Recently, examination of whole RJ and single protein bands by off-line LC-MALDI-TOF MS glycomic analyses, complemented by permethylation, Western blotting, and arraying data, revealed the presence of glucuronic acid termini, sulfation of mannose residues, core β-mannosylation of the N-glycans, and a fairly scarce zwitterionic modification with phosphoethanolamine, which may contribute to the development of honey bees and their innate immunity [46].
