*5.5. Oxidative Stress*

According to the free radical theory of aging, aging is the result of accumulation of molecular damages that are induced by reactions of free radicals and reactive species that inevitably form during the course of metabolism, which cause errors in cellular processes that are conducive to various age-related disorders [117]. This notion is supported by findings from bee queen studies. Despite the fact that bee queens live longer compared with worker bees, queens enjoy youthful and vigorous cellular functioning. This is attributed to the peroxidation-resistant cell membrane and the lower expression of oxidative stress genes [118,119]. The latter has been attributed, at least in part, to the effect of queen food consumption on microbiota composition and microbiota-derived metabolites, whereas gut microbiota of the short-lived workers—that feast on honey or pollens—is aging i.e., deficient in bacteria that produce metabolites that prevent the expression of oxidative stress genes such as lactobacillus and bifidobacterium [119]. Hence, another possible contributor to the longevity effect of RJ is its enhancement of antioxidation capacity and resistance to oxidative stress, which foster scavenging of free radicals as well as phosphorylation and retention of *DAF-16*/*FOXO* in the cytoplasm, through the involvement of 3-phosphoinositide-dependent kinase-1. As noted above, RJ supplementation to *C. elegans* increased their resistance to various types of stress [66,67]. Similarly, RJ supplementation to *Drosophila* at low concentrations regulated the gene expression related to oxidative stress and catabolism [16]. In particular, MRJPs fostered the gene expression of *CuZn-SOD*, which was reflected by increased activity of the antioxidant SOD and decreased levels of MDA [12]. Similarly, RJ supplementation to mice decreased kidney DNA and serum levels of 8-hydroxy-2-deoxyguanosine, an age-related marker of oxidative stress [2].

#### **6. Discussion**

Aging is characterized by progressive functional decline and increased vulnerability to various pathologies, which result from long-term alterations of numerous physiological processes [4]. Therefore, apart from prolonging life, it is necessary to find intelligent anti-aging agents that target age-related genetic pathways and biochemical processes in order to prevent or delay the detrimental effect of age-related physiological changes. Despite the slight discrepancy across studies examined in this review—possibly out of differences in model organisms and experimental designs—the findings indicate that consumption of an appropriate dose of RJ and its ingredients exerts proliferative effects that promote health, increase stress resistance, and prolong lifespan in numerous diverse species. RJ seems to have a potent effect on the functioning of various healthspan and longevity pathways: *IIS*, *mTOR*, *EGFR*, oxidative stress, and dietary restriction eat-2. It might be necessary to further examine the effect of RJ on mechanisms related to microbiota since microbiota composition can interact with pathways of oxidative stress to exert longevity effects [119]. It is also necessary to examine the possibility that such mechanisms may be applicable to humans.

Though several animal models demonstrate extended lifespan as a result of RJ treatment, it is not clear which constituents of RJ are responsible for the longevity effect. MRJPs and royalactin appear to be probable candidates [41,68]. Yet, longevity effects were obtained in many studies that did not include MRJPs, which signifies that other ingredients also enhance health and prolong lifespan. It seems that pRJ [51,67], as well as certain RJ constituents such as RJP60 [15]; lipids e.g., 10-HDA [66]; and vitamins e.g., pantothenic acid [54] exhibit healthspan and lifespan-extending activities greater than crude RJ.

For RJ to exert an anti-aging effect, it should be regularly used for long periods of time. Therefore, safety of prolonged use of RJ represents another issue of concern. Bee products such as RJ, honey, pollen, and propolis have long been used as multifunctional substances with various biological activities. Despite the limited possibility of occurrence of allergic reactions, most bee products are relatively nontoxic e.g., high doses of propolis (1400 mg/kg/day) in mice had no effect level [120], whereas oral consumption of RJ in high doses (20 g) in humans produced no adverse effects [86]. Nonetheless, most of the observed positive anti-aging effects of RJ in model organisms were dose-dependent—extremely high doses were associated with unfavorable outcomes in some studies. Yet, with the exception of one study that supplemented bee larvae with RJ 100% [60], the definition of "a high dose" varied between studies and outcomes also varied: no effect [52], increased fertility [60] and survival [64], increased developmental time, and decreased survival [16]. Apart from variation of the studied organisms, as well as nutrient contents of culture media used for in vitro breeding [16,25,41] (which may, in part, explain the discrepancy noticed between these studies), very few attempts were made

to explore mechanisms underlying the occurrence of adverse effects when high doses of RJ are used. In this respect, unphysiological exposure of *Drosophila* to high levels of RJ (up to 70%) prolonged development time and shortened lifespan because excess nutrients in high concentrations of RJ altered the expression of genes responsible for the metabolism of amino acids and encoding of glutathione S transferases, which detoxify xenobiotic compounds resulting in distortion of cellular processes [16]. Furthermore, evidence documents that uncontrollably high intake of antioxidants (e.g., vitamin C, vitamin E, N-acetyl cysteine) disturbs the redox balance between processes of oxidation and reduction and induces reductive stress—a shift of body redox levels into an extra reduced state, which may cause severe alterations of cellular functions and lead to pathologies in the same way as oxidative stress [121]. Therefore, adverse effects associated with high doses of RJ may be the result of reductive stress induced by antioxidants in RJ (e.g., phenols, amino acids, peptides, fatty acids, and vitamins). Accordingly, from a cost-effectiveness-oriented point of view, determining an optimal RJ dose would be an important issue in future studies. On the other hand, accumulation of neonicotinoid insecticides and their metabolites in the body and products of honey bees as a result of environmental pollution is associated with occurrence of adverse effects in bees such as oral toxicity, especially during winter—when bee consumption of honey and pollen increases [122]. Nonetheless, several reports indicate that compared with pollen and honey [123,124], RJ contamination with neonicotinoids is very limited (1 to 9.5 μg/kg) [125,126], equivalent to 0.016% of the original concentrations of pesticide fed to bee workers during in vitro breeding [125]. Therefore, RJ maybe considered a safe an anti-aging agent compared with other bee products such as bee pollen.

Preserving the biological activity of RJ is a prerequisite for its use to efficiently promote health. RJ seems to be sensitive to temperature and other methods of handling, which may affect its ingredients and potency e.g., RJ lost most of its bioactive components after 30 days of storage at 40 ◦C [41] and pRJ had a better effect than crude RJ [52]. Further, the effect of commercial RJ was suboptimal compared with RJ from reliable sources [16]. Another concern is the effect of digestive enzymes on bioactive ingredients of RJ when it is orally ingested. For instance, MRJPs prevent senescence of human cells in vitro [39]; however, MRJPs are rapidly digested in the stomach and small intestine except for MRJP2, which can remain in the intestine as a full-length protein for 40 min and it should be resorbed quickly were it to produce any biological effect [127].

Genetic variation is another challenge if we are to identify candidates for pathology prevention in humans. RJ as a dietary supplement may prevent some of the main age-related diseases. However, various types of genetic variation may affect response to RJ treatment. Taking gender as an example, RJ increased the body size of females only in some species such as silkworms [65]—an effect of increased levels of fertility hormones [17,41]. In humans, women live longer than men, yet they have higher genetic risk for some age-related diseases (e.g., Alzheimer's disease) than men, which indicates the possibility for gender-specific treatment targets [128]. Other types of genetic variation are also important; for instance, RJ affects the *EGFR* pathway, which has been shown to be associated with prolonged lifespan in Japanese—but not in whites or Koreans—which highlights a role of ethnic difference in genotype and epigenotype [101]. Other factors, such as other genetic variation factors, diet, and activity level (which affects healthspan and longevity) should be considered in future clinical trials.

#### **7. Conclusions**

Accumulating evidence from studies of honey bees, fruit flies, crickets, silkworms, mice, and humans indicates that RJ has an obvious role in modulating the mechanisms of aging, which can promote healthspan and longevity. Although most studies relate the anti-aging properties of RJ to MRJPs and 10-HDA, it is not exactly clear which fractions or doses are most beneficial. The discovery of predictive biomarkers that take into account individual variation in genotype and epigenotype is necessary in order to conduct sound clinical trials that can test the efficacy of RJ on the rate of biological aging as well as the risk of age-related diseases.

**Funding:** This study was supported by the Strategic Research Program for Brain Sciences from Japan Agency for Medical Research and Development, AMED, Japan (Grant No. 18dm0107100h0003).

**Conflicts of Interest:** The authors declare no conflict of interest.
