**4. RJ Might Enhance Longevity in Humans by Promoting General Health**

It is an important question whether observed effects of RJ on healthspan and longevity in model organisms (e.g., bees, fruit flies, *C. elegans*, silkworms, crickets, and mice) can be generalized to humans. A small number of in vitro studies examined the antiaging activity of RJ, 10-HDA, and MRJPs on human cell lines, and results support findings reported from studies of model organisms. In two experiments, normal human skin fibroblasts were ultraviolet-irradiated (as a model of skin photoaging) and treated with RJ and 10-HDA. Results revealed that RJ and 10-HDA protected cells against ultraviolet A- and B-induced ROS-related oxidative damage, decreased cellular senescence, stimulated the production of procollagen type I and transforming growth factor-β1 [33,72], and suppressed the expression of *MMP-1* and *MMP-3* at transcriptional and protein levels [33]. In another study, human embryonic lung fibroblast cells were treated with different concentrations of MRJPs (0.1–0.3 mg/mL) versus bovine serum albumin. MRJP-treated cells showed the highest proliferation activity, the lowest senescence, and the longest telomeres. Such effects were associated with upregulation of SOD1 and downregulation of *mTOR*, catenin beta like-1, and tumor protein p53 [39]. Apart from these limited in vitro studies, we could not locate any study that investigated the effect of RJ on lifespan in humans in vivo. However, several studies assessed the effect of RJ on promotion of wellbeing and prevention of severe diseases associated with increased early death—these studies may mirror the healthspan effects of RJ. Herein, we explore how RJ may support healthy aging in the general population.

It is becoming clear that certain metabolic pathways reduce longevity in humans by increasing the risk of serious illnesses that contribute to mortality e.g., diabetes mellitus, metabolic syndrome, cardiovascular diseases, and cancer [73]. The life-expanding effect of RJ possibly originates from its antioxidant and anti-inflammatory properties, which can promote healthy aging by improving glycemic status, lipid profiles, and oxidative stress—and hence can prevent the occurrence of various debilitating metabolic diseases [13,14]. In accordance, administration of RJ in healthy volunteers was associated with improved indicators of physical wellbeing (erythropoiesis and glucose tolerance) [74].

Rheumatoid arthritis (RA) is one of the most common disabling disorders that seriously endanger healthspan. It is a chronic systemic inflammatory arthritis that occurs at an age of onset of 55 years and increases with age i.e., it predominantly affects the older population. Meanwhile, treatment is complicated by age-related decline in organ function (e.g., renal and metabolic), comorbidities, and changes in body composition (e.g., decreased lean mass), which make outcomes of RA treatment (steroids and anti-*TNF* agents) in the elderly highly disappointing [75]. On the other hand, a relatively large number of in vitro studies indicate that 10-HDA can be a safe treatment of RA. The 10-HDA is likely to prevent joint destruction by inhibiting *MMP* production from rheumatoid arthritis synovial fibroblasts through blockage of p38 kinase and c-Jun N-terminal kinase-AP-1 signaling pathways [34,76]. The 10-HDA also prevents cell proliferation of fibroblast-like synoviocytes by inhibiting target genes of *PI3K–AKT* pathway and genes of cytokine-cytokine receptor interaction [77].

It is believed that hormones play a major role in healthspan and longevity by regulating cellular responses, metabolism, and growth [78]. Insulin is one of such hormones that affect every single cell in the body; its signaling pathway interacts with a variety of other pathways and affects their functioning [79]. For instance, overexpression of insulin-like growth factors (*IGFs*) and receptors for insulin is associated with increased cell proliferation and risk of cancer [80]. Meanwhile, downregulation of insulin signaling contributes to proper mitochondrial function, suppression of inflammatory mediators, regulation of cellular metabolism, cellular resistance to stress, activation of DNA repair genes, and reduction of oxidative damage of macromolecules and cellular senescence, which all further enhance health and longevity, both in humans and other species [79,81,82]. In this respect, most anti-aging dietary interventions target growth-promoting pathways i.e., they function by downregulating *IGF-1* and *mTOR-S6K* pathway and activating nutrient sensors (*MAPK* and sirtuins), which signal nutrient scarcity and stimulate catabolism [83]. A majority of the above-reviewed studies indicate that RJ enhanced longevity by targeting insulin signaling; this mechanism is likely to work in humans. In fact, RJ has an insulin-like activity—an insulin-like peptide had been purified from RJ and it is similar to insulin of vertebrates in solubility, chromatographic, immunological, and biological characteristics [84]. Administration of RJ to healthy athletes significantly decreased their insulin and increased thyroxine (T4) hormone levels in plasma [85]. In line with this, healthy adults who consumed a single oral dose of RJ (20 g) immediately before oral glucose tolerance test showed significantly reduced glucose level [86].

Aging is usually associated with reduction of sex hormones [87]. The genetic switch model of aging postulates that end of reproduction entails a genetically programmed inactivation of survival and maintenance pathways, which causes a progressive age-related decline of function [88]. In fact, sex hormones are considered markers of longevity since they have a neuroprotective effect, which originates from their ability to improve insulin resistance and enhance the DNA repair capacity of neurons [74,89]. It is becoming clear that RJ modulates sex hormones. The endocrine stimulation exerted by RJ is necessary for ovary development in bee queens and it increased egg-laying in *Drosophila M.* and silkworms [41,63,65]. RJ and royalactin increased juvenile hormone titre (a fertility hormone) downstream of *EGFR* signaling in *Drosophila M.* [12,17,41]. Several studies indicated that RJ and its lipids exert estrogenic activity both by binding with estrogen receptors and activating the expression of endogenous genes [87,90]. Therefore, sex hormones represent another facet through which RJ may enhance longevity. In humans, RJ supplementation to healthy volunteers increased serum testosterone and the ratio of testosterone/dehydroepiandrosterone sulfate, indicating that RJ accelerates conversion of dehydroepiandrosterone sulfate into testosterone. The authors suggested that RJ-induced improvement of erythropoiesis in their sample could be ascribed to the anabolic effect of testosterone [74].

Menopause is a common inevitable age-related phenomenon that results from reduction of estrogen production in women at an age range of 45 to 55 years [91]. It is often associated with various uncomfortable physical and psychological symptoms. Therefore, menopausal women may receive hormone therapy to alleviate discomfort and lower their risk for various serious diseases such as osteoporosis and cardiovascular disorders. However, hormone therapy is associated with increased risk of cancer [92,93]. Several natural alternatives (including RJ) have been under investigation as safer alternatives. Animal studies document that consumption of RJ by ovariectomized rats improved bone strength [94] and prevented bone loss in a fashion similar to the effect of 17β-estradiol [95]. Cell culture models revealed that the role of RJ in bone formation is due to upregulation of procollagen I α1 gene expression [96,97], enhancement of intestinal calcium absorption, and increased bone

calcium content [95]. Human studies lend further support to this evidence; pRJ at a dose of 800 mg/day significantly decreased lower back pain in healthy menopausal women [93]. Likewise, daily consumption of 150 mg of RJ for three months exerted cardioprotective effects by improving lipid profile of postmenopausal women [92].

The healthspan-inducing effects of RJ in humans are not only limited to enhancement of physical health but also include improvement of general mental health [74], reduction of anxiety symptoms [93], improvement of mood, and mild cognitive impairment in the elderly (>60 years old) [98,99]. The psychological and neurological effects of RJ reflect improvement of biomarkers of physical health such as cholesterol [98]. Hypercholesterolemia, in particular, is documented to foster aggregation of β-amyloid around neurons, which causes neuronal loss—a characteristic feature of Alzheimer's disease. Meanwhile, the reduction of plasma lipids induced by RJ has been associated with enhancement of antioxidative capacities, reduction of β-amyloid deposition, and prevention of neuronal damage [100].
