New Insights into the Biological and Pharmaceutical Properties of Royal Jelly

International Journal of Molecular Sciences Review New Insights into the Biological and Pharmaceutical Properties of Royal Jelly Saboor Ahmad 1, Maria Graça Campos 2,3 , Filippo Fratini 4,5, Solomon Zewdu Altaye 1and Jianke Li 1,* 1Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China; [email protected] (S.A.); [email protected] (S.Z.A.) 2Coimbra Chemistry Centre, (CQC, FCT Unit 313), Faculty of Sciences Technology, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal; [email protected] 3Observatory of Drug-Herb Interactions, Sciences Health Campus, Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal 4Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy; [email protected] 5Interdepartmental Research Center “Nutraceuticals and Food for Health”, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy *Correspondence: [email protected]; Fax: +86-10-8210-6448 Received: 23 November 2019; Accepted: 6 January 2020; Published: 8 January 2020   Abstract: Royal jelly (RJ) is a yellowish-white and acidic secretion of hypopharyngeal and mandibular glands of nurse bees used to feed young worker larvae during the first three days and the entire life of queen bees. RJ is one of the most appreciated and valued natural product which has been mainly used in traditional medicines, health foods, and cosmetics for a long time in different parts of the world. It is also the most studied bee product, aimed at unravelling its bioactivities, such as antimicrobial, antioxidant, anti-aging, immunomodulatory, and general tonic action against laboratory animals, microbial organisms, farm animals, and clinical trials. It is commonly used to supplement various diseases, including cancer, diabetes, cardiovascular, and Alzheimer’s disease. Here, we highlight the recent research advances on the main bioactive compounds of RJ, such as proteins, peptides, fatty acids, and phenolics, for a comprehensive understanding of the biochemistry, biological, and pharmaceutical responses to human health promotion and life benefits. This is potentially important to gain novel insight into the biological and pharmaceutical properties of RJ. Keywords: royal jelly; bioactive compounds; functional properties; proteins; fatty acids; phenolics 1. Introduction Royal jelly (RJ) is known as a “superfood” which is produced by nurse bees to feed young worker larvae and queen bees [ 1 , 2 ]. The major components of RJ are (60–70% w/w) water, (9–18% w/w) proteins, (7–18% w/w) sugars, and (3–8% w/w) lipids [ 3 , 4 ]. RJ also contains minor components, such as minerals (Fe, Na, Ca, K, Zn, Mg, Mn, and Cu), amino acids (eight essential amino acids Val, Leu, Ile, Thr, Met, Phe, Lys, and Trp), vitamins (A, B complex, C, and E), enzymes, hormones, polyphenols, nucleotides, and minor heterocyclic compounds [ 3 , 5 , 6 ]. RJ is an active research domain because it is essential for larval development and queen reproduction in honeybee colonies through the metabolism of sugars, lipids, and proteins [ 7 , 8 ]. Thereby, the larger body size, longer lifespan, and fertility of queens compared to worker bees are potentially correlated to the special diet of RJ [9]. RJ has been produced in large scale for commercial purposes to date, and its market value is significantly higher than other bee products, such as honey or pollen, thus, it is a major income Int. J. Mol. Sci. 2020,21, 382; doi:10.3390/ijms21020382 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020,21, 382 2 of 26 source for beekeepers [ 4 , 10 ]. Beekeepers have made great efforts to develop the technique to improve RJ production and to select for high-producing strains of honeybees. For instance, the increase in the production of RJ in China over the last 40 years has been achieved by the development of genetic selection of high RJ-producing bees (RJBs) from Italian bees [ 11 – 13 ], and the development and implementation of production techniques to increase and optimize RJ production [ 14 – 16 ]. At present RJBs have the potential to produce more than 10 kg RJ/colony/year, which is 10 times more than for non-selected Italian bees [ 15 – 18 ]. Notably, China is the largest producer and exporter of RJ around the world, producing more than 4000 tons annually, with more than $2.5 billion market, which is 90% of the total RJ production globally and mostly exported to Japan, Europe, and the United States [12,19]. The health-promoting benefits and pharmaceutical properties of RJ from animal models to humans have been widely investigated. RJ is a nutritional modification of honey and bee bread (Figure 1), and it is commercially available on a large scale as health food and cosmetics in Asia, especially in China and Japan [ 9 , 10 ]. Moreover, RJ is used to explore further applications as a drug and traditional consumption as “remedies” for humans and animals [ 20 ]. To date, the importance of RJ has attracted attention around the world, which is evidenced by the growth in the number of publications and citations in the core collection of the Web of Science (Figure 2). Recently, the origin and function of RJ, such as major royal jelly proteins (MRJPs) for the development of the larvae [ 21 ], antimicrobial properties [ 9 ], medicinal value [ 20 , 22 ], proteins and peptides [ 23 ], the potential applications for cancer treatment [ 24 ], and health aging and longevity [ 25 ] have been reported. To better understand the biochemistry, biological, and pharmaceutical response to health and life benefits of RJ, we update the knowledge from the research advances of the biological activities and pharmaceutical applications of RJ and its bioactive ingredients that are associated with farm animals, micro-organisms, laboratory animals, insects, and clinical trials in humans. Here, our major focus is on the bioeffects of RJ, such as antimicrobial, antioxidant, anti-inflammatory, wound healing, anti-aging, immunomodulatory, anti-cancer, anti-diabetic, anti-hyperlipidemic, anti-hypertension, hepato-renal protective, neuroprotective, estrogenic, and fertility effects. This evidence is a potentially valuable resource for further studies of the health potential properties of RJ for both humans and honeybees. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 27 Figure 1. Forager bees transport pollens in their hind leg corbiculae to which they add nectar to form pollen pellets. Forager bees deposit and pack the pollen pellets into cell surrounding the brood area and forming bee bread. Nurse bees develop enlarged food glands and produce RJ by consuming honey and bee bread (Photos taken by Prof. Dr. Jianke Li). Figure 2. Numbers of publications on RJ that appear from international journals are increasing every year (data from the core collection of the Web of Science). Figure 1. Forager bees transport pollens in their hind leg corbiculae to which they add nectar to form pollen pellets. Forager bees deposit and pack the pollen pellets into cell surrounding the brood area and forming bee bread. Nurse bees develop enlarged food glands and produce RJ by consuming honey and bee bread (Photos taken by Prof. Dr. Jianke Li). Int. J. Mol. Sci. 2020,21, 382 3 of 26 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 27 Figure 1. Forager bees transport pollens in their hind leg corbiculae to which they add nectar to form pollen pellets. Forager bees deposit and pack the pollen pellets into cell surrounding the brood area and forming bee bread. Nurse bees develop enlarged food glands and produce RJ by consuming honey and bee bread (Photos taken by Prof. Dr. Jianke Li). Figure 2. Numbers of publications on RJ that appear from international journals are increasing every year (data from the core collection of the Web of Science). Figure 2. Numbers of publications on RJ that appear from international journals are increasing every year (data from the core collection of the Web of Science). 2. Bioactive Substances RJ is a rich source of nutrients and bioactive compounds with the potential to play a vital part in their biological activities and pharmaceutical applications [ 26 ]. It has been confirmed that proteins, peptides, lipids, phenolics, and flavonoids are the main bioactive compounds responsible for the various pharmaceutical properties of RJ (Figure 3). The natural variation of bioactive compounds depend upon the biodiversity of flora species present in the different ecosystem [26]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 27 2. Bioactive Substances RJ is a rich source of nutrients and bioactive compounds with the potential to play a vital part in their biological activities and pharmaceutical applications [26]. It has been confirmed that proteins, peptides, lipids, phenolics, and flavonoids are the main bioactive compounds responsible for the various pharmaceutical properties of RJ (Figure 3). The natural variation of bioactive compounds depend upon the biodiversity of flora species present in the different ecosystem [26]. Figure 3. A schematic representation of the main biological substances in RJ and their functional activities. For detailed information refer to Table 1. 2.1. Proteins and Peptides The investigation of novel proteins in RJ has been a long-term perusal for biochemical experts and apicultural biologists. Proteins are the most abundant components of RJ, accounting for more than 50% of the dry weight and MRJPs are the most important components constituting 80%–90% of the total protein content [21,27]. Others are glucose oxidase [27], α-glucosidase, and α-amylase [28]. The MRJPs share a common developmental genesis with the yellow protein family [29]. The YELLOW/MRJPs are named according to their molecular weight or simply numbered by the order in which they are discovered. So far, MRJPs (1–9) are well-described with molecular mass 49–87 kDa, which are encoded by nine different genes [23,30]. MRJP-1 is a weak acidic glycoprotein, accounting for 48% of water-soluble RJ proteins and the secondary structure consists of 9.6% α-helices, 38.3% βsheets, and 20% β-turns [6,31]. Particularly, MRJP-1 occurs as a monomer (mono MRJP-1) or as an oligomer known as apisin by polymerization with apisimin [31]. Apisin could be used to determine the quality of RJ [32]. MRJP-2 and MRJP-3 produced by Chinese bees (Apis cerana cerana) are less polymorphic compared to European bees (Apis mellifera ligustica) and Africanized bees (Apis mellifera scutellate) [33,34], and MRJP-4 was first time confirmed by two-dimensional gel electrophoresis (2DE) analysis during the comparison in the RJ of Africanized and European bees [34]. The important feature of MRJP-5 is a wide repeated region located between amino acid residues 367 and 540 [33]. The most significant post-translational modification of the MRJPs is methylation which triggers Figure 3. A schematic representation of the main biological substances in RJ and their functional activities. For detailed information refer to Table 1. Int. J. Mol. Sci. 2020,21, 382 4 of 26 2.1. Proteins and Peptides The investigation of novel proteins in RJ has been a long-term perusal for biochemical experts and apicultural biologists. Proteins are the most abundant components of RJ, accounting for more than 50% of the dry weight and MRJPs are the most important components constituting 80%–90% of the total protein content [ 21 , 27 ]. Others are glucose oxidase [ 27 ], α -glucosidase, and α -amylase [ 28 ]. The MRJPs share a common developmental genesis with the yellow protein family [ 29 ]. The YELLOW/MRJPs are named according to their molecular weight or simply numbered by the order in which they are discovered. So far, MRJPs (1–9) are well-described with molecular mass 49–87 kDa, which are encoded by nine different genes [ 23 , 30 ]. MRJP-1 is a weak acidic glycoprotein, accounting for 48% of water-soluble RJ proteins and the secondary structure consists of 9.6% α -helices, 38.3% β -sheets, and 20% β -turns [ 6 , 31 ]. Particularly, MRJP-1 occurs as a monomer (mono MRJP-1) or as an oligomer known as apisin by polymerization with apisimin [ 31 ]. Apisin could be used to determine the quality of RJ [ 32 ]. MRJP-2 and MRJP-3 produced by Chinese bees (Apis cerana cerana) are less polymorphic compared to European bees (Apis mellifera ligustica) and Africanized bees (Apis mellifera scutellate) [ 33 , 34 ], and MRJP-4 was first time confirmed by two-dimensional gel electrophoresis (2-DE) analysis during the comparison in the RJ of Africanized and European bees [ 34 ]. The important feature of MRJP-5 is a wide repeated region located between amino acid residues 367 and 540 [ 33 ]. The most significant post-translational modification of the MRJPs is methylation which triggers polymorphism of MRJP 1–5 in the RJ [ 35 ]. MRJPs 6–9 are recognized in RJ through proteomic analysis [ 36 , 37 ]. Furthermore, 1-peroxiredoin and 1-glutathione S-transferase are identified in RJ [ 11 ]. RJ also contains a calcium-binding protein, known as regucalcin, and a lipid-binding protein, such as apolipophorin-III [ 38 ]. Phosphorylated icarapin (venom protein-II) and apolipophorin-III-like proteins are identified in RJ may promote the strength of immunity [ 39 ]. There are 53 N-glycosylation sites residing on 25 N-glycosylated proteins in RJ. Most of the glycosylated proteins are associated with metabolic activities and health benefits [12,28]. RJ is rich in amino acids, including lysine, proline, cysteine, aspartic acid, valine, glutamic acid, serine, glycine, cysteine, threonine, alanine, tyrosine, phenylalanine, hydroxyproline, leucine, isoleucine, and glutamine [ 22 , 40 ]. These high amounts of amino acids in the MRJPs family is essential for developing of both queen bees and larvae. Amino acids, such as arginine, leucine, isoleucine, histidine, lysine, threonine, tryptophan, methionine, valine, and phenylalanine, are most commonly present in MRJPs, with MRJP-1 to 9 contains 48%, 47%, 39.3%, 44.5%, 51.4%, 42%, 48.3%, 49.5%, and 47.3% of these amino acids, respectively. The major amino acids in MRJP-1, MRJP-2, and MRJP-4 are valine and leucine. MRJP-3 is rich with arginine and lysine while the prominent amino acids in MRJP-5 is methionine and arginine. Furthermore, leucine is the major amino acid in MRJP-(6–8) and isoleucine is the rich one in MRJP-9 [ 41 , 42 ]. The MRJPs provide nutritive components such as essential amino acids to RJ. Similar to proteins, peptides represent a specific sequence of amino acids in RJ that has biological activity with health effects and potential applications. They can be identified by proteomics, such as jelleines-I, jelleines-II, jelleines-III, jelleines-IV, and jelleines, are identical to the C-terminal of the MRJP-1 [ 43 ]. Moreover, RJ also contains peptides including apidaecin, defensin, hymenoptaecin, jelleine-II, jelleine-II (pT), and jelleine-II (pS) [ 39 , 44 ]. Phosphorylated jelleine-I (pS), jelleine-II (pS), and jelleine-IV (pS) are found in Apis cerana RJ while jelleine-II (pT) and jelleine-IV (pT) in Apis mellifera RJ [39]. 2.2. Lipids and Fatty Acids A distinctive feature of RJ is associated with its lipids and fatty acids content. The lipids are 80%–85% of free fatty acids with few being esterified. This fraction also includes 4–10% phenolic compounds, 5–6% waxes, 3–4% steroids, and 0.4–0.8% phospholipids. RJ contains a medium-chain fatty acids, normally 8–12 carbon atoms, some hydroxylated in terminal or internal position, as mono-hydroxyl fatty acids or dicarboxylic acids, and saturated or unsaturated at the 2-position [ 45 ]. About 80–90% fatty acids have a different structure such as 10-hydroxy-2-decenoic acid (10-HDA), Int. J. Mol. Sci. 2020,21, 382 5 of 26 10-hydroxydecenoicacid(10-HDDA),andsebacicacid(SEA).Thisfractionconsistsof32%trans-10-HDA, 22% 10-HDDA, 24% gluconic acid, 5% dicarboxylic acids, and some other acids [ 46 ]. In addition, fatty acids, such as 8-hydroxy octanoic acid (8-HOC), 3,10-dihydroxydecanedioic acid (3,10-HDecDA), 9-hydroxy-2-decenoic acid (9-HDA), 1,10-decanedioic acid (DecDA), 3-hydroxydecanoic acid (3-HHDA), and 2-decene-1,10-dioic acid (2-DecDA), can also found in RJ [ 47 ]. Among all lipids and fatty acids, 10-HDA is a stable compound representing 3.5% of freeze-dried RJ which is considered an international standard for quality [ 5 , 46 , 48 , 49 ]. In the lipid fraction sterols should be included, even if they are only in trace amounts. For instance, 24-methylene cholesterol (24-MET) contribute with 49–58% for total sterols in RJ. Other similar compounds include β -sitosterol (19–24%), isofucosterol (9–16%), campesterol (67%), and desmosterol (0.5–4.5%) [3]. 2.3. Other Constituents RJ contains some other bioactive compounds, such as 23.3 ( µ g/mg) of phenolics and 1.28 ( µ g/mg) of total flavonoids [ 20 , 49 , 50 ]. The phenolic compounds comprise phenol and carboxylic groups [ 51 ]. From flavonoid compounds, various structures could be distinguished, such as flavones (apigenin and its glycosides, luteolin, chrysin, and acacetin), flavanones (naringenin, hesperetin, and isosakuranetin), flavonols (kaempferol and isorhamnetin glycosides), and isoflavonoids (genistein and formononetin). Coumestrol is an isoflavonoid phytoalexin that can also be found in RJ [ 52 ]. Furthermore, flavonoids are mostly present in the form of glycosides, and the aglycones are linked by glycosidic bonds to the osidic group [ 53 ]. Another unique compound of RJ is adenosine N1-oxide, which is an oxidized product of adenosine at the N1 position of adenine base moiety [ 54 , 55 ]. Adenosine monophosphate (AMP) and adenosine itself are important biomolecules with physiological effects [ 56 – 58 ]. Acetylcholine can also be found with a mean concentration of 1 mg/g dry weight [ 59 ]. Hormones, gonadotropins, pantothenic acid, testosterone, estradiol, progesterone, and prolactin also were identified in RJ [60–64]. 3. Functional Properties of RJ The biological functions of RJ and its application (Table 1) are investigated in vivo and in vitro experimental models, such as laboratory animals (rabbits, mice, rats, and hamsters), microbial organisms (bacteria, fungi, viruses, and nematodes), farm animals (ewes and buffalos), and clinical trials (humans disease treatment), to provides the basis for further developments of its pharmaceutical effects. The biological activities of the RJ are variable and have been correlated to the content of their active ingredients [65]. Table 1. The biological activities and pharmaceutical applications of RJ and their bioactive ingredients. Bioactive Compounds/Experimental Models Effects Sources RJ, MRJP-2, and MRJP-4 (Micro-organisms) Antibacterial, antifungal, anti-yeast Induce damage and dysfunction in microbial cell wall and membrane [66–68] Royalisin and 10-HDA (Micro-organisms) Antibacterial (Gram+, Gram−), antifungal Inhibit growth [9,69,70] Jelleine I-III, jelleine-II (pS), and jelleine-II (pT) (Micro-organisms) Antibacterial (Gram+, Gram−) Cell degranulation, hemolysis, and increase immune defense [39,43] RJ, 10H∆2DA, 3,10-HDA, 11S, 10-HDA, 10-acetooxy-2-DEA, and Native jelleine-11 (Micro-organisms) Antifungal and anti-yeast Strongly inhibit growth [39,43,71–73] Pre and post administration of RJ (Animals) Antioxidant activity Decrease oxidative stress (MDA) and increase antioxidant properties (CAT, GPx, and SOD) [74] RJ (Humans) Anti-cancer effect Inhibit the tumor-induced angiogenesis, activate immune system, metabolism of 2-AF metabolites, and stop the damage of bisphenol A [75–77] Int. J. Mol. Sci. 2020,21, 382 6 of 26 Table 1. Cont. Bioactive Compounds/Experimental Models Effects Sources Intravenously application of 10-HDA and the HuIFN-aN3 (Animals) Anti-cancer effect Decrease the level of glutathione and enhance the level of lipid peroxidation via MDA [78] RJ (Animals and humans) Anti-diabetic effect Improve the serum level of triglycerides, lipoprotein, and cholesterol Decrease glucose level and increase insulin concentration [79–83] MRJP-3 (Animals) Immunomodulatory effect Decrease antigenicity and inhibit IL-4, IL-2, and IFN-Υproduction [84] 3,10-DDA (Humans) Immunomodulatory effect Increase the production of IL-12, IL-18, and stimulate the production of IFN-Υ [85] RJ (Animals and humans) Hypocholesterolemic effect Reduced the level of triglyceride, insulin, total lipids, and cholesterol level by decreasing very low-density lipoprotein levels [86–88] RJ and MRJP-1 (Humans) Hypocholesterolemic effect Decreased the total cholesterol and LDL-c level by improving the (HDL-c) level [89,90] RJ, ERJ, And MRJP-1 (Animals) Antihypertension effect Reduce systolic blood pressure, diastolic blood pressure, and increase NO level [62,91–93] RJ (Animals) Anti-inflammatory effect RJ inhibit the TNF-α, IL-1β, and, IL-18 levels in the blood due to its antiradicals and antioxidative effect [94,95] RJ and MRJP-2 (Animals) Hepato-renal protective effect Reduce blood urea, MDA level, leukocyte infiltration, creatinine, adhesion molecule-1 expression, glomerular diameter, and TNF-a Increased SOD and GPx [50,96–99] RJ and 10-HDA Animals Neurotrophic effects Inhibited production of oligodendrocytes, astrocytes, and stimulate neuron differentiation [57,100] RJ and RJPs (Animals and humans) Neuroprotective Decrease cholesterol and amyloid-beta deposition by down-regulation of β-secretase Increase cholinergic response, estrogen level, and antioxidant capacities Improved blood-brain barrier, and autonomic nervous systems [101–103] RJ (Humans) Genoprotective effect Increase of BCL2/BAX ratio for cell survival Enhance in hTERT/BAX for increasing age Increase in NRF2/BAX for antioxidative response [104] RJ and 10-HDA (Humans) Protective effect Protect it from photo-aging by improving collagen production via up-regulation of TGF-β1 expression [105] RJ (Animals and humans) Effect on fertility Increase sperm motility, luteinizing hormones, and testosterone levels [81,106–108] ERJ (Humans) Anti-allergic Significantly reducing IgE-binding capacity of blood [109] Note: RJ (royal jelly); RJPs (purified royal jelly peptides); RJPH (royal jelly protein hydrolysate); MRJP-4 (ajor royal jelly protein 4); 10-HDA (10-hydroxydecanoic acid); 10H ∆ 2DA (10-hydroxy-Delta-2-decenoic acid); 3,10-DDA (3,10-dihydroxy-decanoic acid); MDA (malondialdehyde); GPx (Glutathione peroxidase); SOD (superoxide dismutase); IFNΥ (interferon-gamma); IL-4 (interleukin-4); TNFα (tumor necrosis factor); BCL2: (B-cell lymphoma 2); BAX (BCL2 associated X protein); NRF2 (nuclear factor erythroid 2 related factor 2); 2-AF (2-aminofluorene); BACE1 (β-site amyloid precursor protein cleaving enzymes), and IgE (Immunoglobulin E). 3.1. Biological Activity of RJ RJ has health benefits effects for both humans and honeybees. It is a natural antibiotic and plays an efficient role in developing the larval stages in blood cells and maintains its ovulatory characteristics during the whole life span. Moreover, RJ has antioxidants with the potential of reducing the risk of cancer, high blood pressure, diabetes, and cardiovascular diseases [ 94 , 110 , 111 ]. RJ also affects the Int. J. Mol. Sci. 2020,21, 382 7 of 26 morphological characters, growth, learning, size, and shape variations in various creatures, such as honeybees, mice, and humans [112]. 3.1.1. Antimicrobial Activity RJ demonstrates strong antimicrobial properties against different pathogens [ 39 , 66 , 67 , 70 ], due to the existence of special proteins and peptides [ 9 , 43 ], and the presence of the 10-HDA [ 9 , 113 ]. Moreover, RJcould fightagainstperiodontopathic bacteria, suchasAggregatibacter actinomycetemcomitans,Prevotella intermedia,Fusobacterium mucleatum, and Porphyromonas gingivalis [ 67 ]. MRJPs (2–5 and 7) reveal antibacterial activity against Gram-negative E. coli [ 114 ]. Jellenie I, II, III, and IV are important antibacterial peptides in RJ. Although the difference between jellenie (I–IV) is minor, with only one residue difference in the sequence, this slight difference has a significant impact on their antibacterial activities. Jelleine I–III could inhibit both Gram-positive and Gram-negative bacteria whereas Jelleine-IV doesn’t [ 43 ]. Antibacterial peptides are positively charged due to the existence of lysine, arginine, and histidine residues that allow them to interact with anionic phospholipids of the cell membrane and collapse it [ 115 ]. Royalisin has three intramolecular disulfide bonds between cysteine residues and shows strong antibacterial activity against different types of Gram-positive and Gram-negative bacteria [ 70 ]. In addition, native jelleines could inhibit Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus,Paenibacillus larvae) and Gram-negative bacteria (Escherichia coli,Pseudomonas aeruginosa). Furthermore, the phosphorylated jelleines (Jelleine-II (pT) and Jelleine-II (pS) could fight against E. coli and B. subtilis,P. larvae, and E. coli [ 39 ]. MRJPs 2 and 4 act as antimicrobial agents and have a wide range of activity against bacteria (Gram-positive and Gram-negative), fungi, and yeasts. Recombinant MRJP-2 and MRJP-4 could kill microorganisms by attaching to the cell wall of fungi, yeast, and bacteria that damage the structure of the cell wall [ 66 , 68 ]. RJ aqueous fraction has reported a strong inhibition of the growth of Fusarium species [ 73 ]. RJ has also exhibited antifungal properties against Syncephalastrum racemosum,Aspergillus fumigants, and A. niger [ 72 ]. Royalisin also indicates an anti-fungal response against necrotrophic fungus, such as Botrytis cinerea [ 69 ]. The native jelleine-ll protein presents an inhibitory effect on Candida albicans [ 39 , 43 ]. Moreover, 10H ∆ DA has antifungal potential in inhibiting the growth rate of Neurospora sitophila [ 116 ]. RJ is effective against C. albicans and as an alternative agent to fight this yeast [ 117 ]. Fatty acids such as 3,10-HDA, 11S, 10-HDA and 10-acetooxy-2-DEA could strongly inhibit the growth of yeasts, such as C. tropicalis,C. albicans, and C. glabrata [ 71 ]. Moreover, RJ could fight against herpes 2 virus, influenza virus, heart virus coxsackie B3, herpes simplex virus type 1 (HSV-1), and certain rhabdoviruses [118,119]. 3.1.2. Antioxidant Activity The antioxidant activity of RJ could be explored as the prevention and treatment of various chronic and degenerative diseases. In the diet of Sprague–Dawley rats fed with contaminated fumonisin (FB) (200 mg/kg) and RJ (150 mg/kg) for three weeks, RJ attenuates the harmful effect of FB via improving glutathione peroxidase formation and reducing the effects of lipid peroxidation and free radical generation [ 120 ]. RJ could also recover from cadmium-induced genotoxicity and oxidative stress in mice, which improves the antioxidant status via glutathione (GSH) and reduces malondialdehyde (MDA) production [ 121 ]. After rats exposed to cisplatin and carbon tetrachloride, RJ administration could resist against oxidative stress in liver and renal tissues, which is achieved by decreasing MDA production and increasing the concentration of cellular antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) [ 122 ]. In radiation-induced lung and liver damage of Sprague–Dawley rats, preand post-administration of RJ are effective in reducing oxidative stress and increasing antioxidant properties [ 74 ]. The antioxidant response of enzyme-treated RJ (ERJ) is confirmed by the reduction of nitric oxide (NO) and intracellular reactive oxidative species, and increased the effect of the antioxidant glutathione and antioxidant SOD levels. Moreover, ERJ has the potential as an oxidative agent to be used for human, as well as animal, diets [ 123 ]. Similarly, MRJP-2 has potential action as an antioxidant to protect mammalian and Int. J. Mol. Sci. 2020,21, 382 8 of 26 insect cells via decreasing the levels of caspase-3 activity and oxidative stress-induced cell apoptosis followed by increase cell viability [ 68 ]. Hydroxyl radicals and hydrogen-peroxide scavenging activity were verified with 29 antioxidant peptides isolated from RJ hydrolysate, in which 12 small peptides having 2–4 residues (Ala-Lys, Phe-Arg, Ile-Arg, Lys-Phe, Lys-Leu, Lys-Tyr, Arg-Tyr, Tyr-Asp, Tyr-Tyr, Leu-Asn-Arg, and Lys-Asn-Tyr-Pro) having the strongest activity. Moreover, three dipeptides (Lys-Tyr, Arg-Tyr, and Tyr-Tyr) in RJ indicate strong scavenging activity due to a donation of the hydrogen atom from their phenolic hydroxyl group [124]. 3.1.3. Wound Healing Activity Woundhealing isanimportant healthissueand awiderange of in vivo and in vitro studiesindicate that RJ seems play a significant role [125,126]. The development of atopic dermatitis-like skin lesions in picryl chloride treated NC/Nga mouse is suppressed after treatment with RJ. This is achieved by the down-regulating protein of antigen-specific interferon-gamma (IFNΥ ) production and up-regulation of NO synthase [ 127 ]. The application of dose-dependent RJ improves the healing effect of severe oral mucositis in hamsters induced by chemotherapy drug 5-fluorouracil [ 128 ]. Oral treatment with RJ could increase the wound healing process in diabetic mice [ 125 ]. Moreover, RJ in 5 µ g/mL concentration promotes the fibroblasts migration in human beings by altering the level of different lipids and enhance the level of sphingolipids that promote wound healing [ 125 ]. Moreover, an RJ dressing is a good way of treating diabetic foot ulcer patients along with other standard methods. Furthermore, this method creates vasodilation effects around the wound which could dilate blood vessels to increase blood flow and prevent the wound from infection by other microbial organisms [ 129 ]. In addition, RJ promotes the wound healing response to control dermal infection induced by methicillin-resistant S. aureus (MRSA) [ 126 ]. Water-soluble proteins of RJ and its fractions induce proliferative and migratory effects on a human epidermal keratinocyte in a scratch wound model. A protein fraction, mainly containing MRJP-2,3,7 have the potential to influence wound healing bioactivity by stimulating keratinocyte growth and migration suggests that these proteins promote the development of new wound healing medication [ 130 ]. The defensin-1 peptide in RJ contributes to skin regeneration and cutaneous wound closure by increasing matrix metalloproteinase-9 secretion and keratinocyte migration [131]. 3.1.4. Immunomodulatory Activity Immunomodulatory response plays a significant role in allergy, cancer, and inflammation by activation of antibody formation or inhibition of white blood cell activities [ 132 ]. The first human study in systemic lupus erythematosus (SLE) in children reveals that the effect of RJ treatment in SLE indicates a significant improvement after three months of administration [ 133 ]. The anti-allergic factors of RJ inhibit interleukin-4 (IL-4) production which is induced by anti-CD3 activated spleen cells derived from ovalbumin (OVA)/alum-immunized mice. MRJP-3 (70 kDa) glycoprotein inhibits IL-4, IL-2, and IFNΥ production by T cells associated with the suppression of cell proliferation. Intra-peritoneal MRJP-3 administration indicates inhibition in immune serum level of anti-OVA IgG1 and IgE in OVA/alum-induced allergic mice while heat-treated soluble MRJP-3 administration decreases antigenicity and maintains its inhibitory effect on antibody response to ovalbumin. Both in vivo and in vitro studies demonstrate that MJRP-3 has strong immunomodulatory activities [ 84 ]. Moreover, the lower concentration of water extract of RJ and 3,10-HDA activate the T-cell proliferation by triggering concanavalin A (Con-A) and enhances the production of IL-2 while a higher concentration of water extract of RJ, dry powder of RJ, and trans-10-HDA inhibit T-cell proliferation by decreasing IL-2 and NO production. Water extract from RJ possesses the complexity of biological and strongest immunomodulatory activities [ 134 ]. Fatty acids, such as 10-HDA and 3,10-DDA, have strong immunomodulatory activities exhibited commonly by the dendritic cell-associated reduction of allogeneicT-cellproliferationandIL-2production in vitro , aswellas theinhibition ofthe antigen-specific immune response in vivo [ 135 ]. A fatty acid 3,10-dihydroxy-decanoic acid (3,10-DDA) of RJ stimulates the maturation of monocyte-derived dendritic cells (MoDCs) by up-regulating the expression of Int. J. Mol. Sci. 2020,21, 382 9 of 26 allogeneic CD1a, CD40, CD54, and CD 86, and also boosting the allostimulatory potential in co-culture with allogeneic CD4+T cells. The 3,10-DDA administrations to monocyte-derived dendritic cells (MoDCs) increase the production of IL-12, IL-18, and stimulate the production of IFNΥ in allogeneic CD4+T cells in co-culture. Therefore, 3,10-DDA encourages maturation and Th1 polarizing potential of human MoDCs in vitro that could have an anti-viral and anti-tumor response [ 85 ]. 10-HDA has various immunomodulatory effects depending on applied concentrations. The high 10-HDA concentration could stop the function and maturation of human MoDCs and lower doses support the Th1 immune response [136]. 3.1.5. Anti-Aging Activity RJ is associated with an increase in the lifespan of queen honeybees as well as several other species [ 9 ], and improves the quality of life in old age rats [ 137 ]. RJ and ERJ administration have the potential to delay aging, age-related disorder, and promote longevity and stress resistance in Caenorhabditis elegans [ 138 ]. Furthermore, ERJ and enzyme-untreated RJ (NRJ) influence in an age focusing motor disorder in genetically heterogeneous male mice. Age-related variations affect muscle fiber size at an advanced age, muscle satellite cell markers, and catabolic genes in RJ-treated mice, thus, RJ may be useful to improve the quality of life during aging through regulating the motor functions [ 139 ]. Royalactin, a glycoprotein from RJ, extends the life span of C. elegans by promoting epidermal growth factor (EGF) and its receptors’ signaling [ 140 ]. MRJPs are longevity-promoting substances that increase the longevity period of Drosophila through promoting the anti-epidermal growth factor receptor (EGFR)-mediated signaling pathway [ 141 ]. Protein and lipid components in RJ have the potential to extend the life span in various living beings, including honeybees, crickets, silkworms, nematodes, mice, and inhibit senescence of human tissues in cell cultures via down-regulation of insulin-like growth factors and up-regulation of epidermal growth factor signaling [ 25 ]. Moreover, 10-HDA is used to increase the longevity of C. elegans via reduced insulin-like signaling (ILS) and increase the lifespan by dietary restriction signaling and the target of rapamycin (TOR) components in C. elegans [ 142 ]. The biological activities of RJ and their underlying possible mechanisms are shown in Figure 4. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 27 response [85]. 10-HDA has various immunomodulatory effects depending on applied concentrations. The high 10-HDA concentration could stop the function and maturation of human MoDCs and lower doses support the Th1 immune response [136]. 3.1.5. Anti-Aging Activity RJ is associated with an increase in the lifespan of queen honeybees as well as several other species [9], and improves the quality of life in old age rats [137]. RJ and ERJ administration have the potential to delay aging, age-related disorder, and promote longevity and stress resistance in Caenorhabditis elegans [138]. Furthermore, ERJ and enzyme-untreated RJ (NRJ) influence in an age focusing motor disorder in genetically heterogeneous male mice. Age-related variations affect muscle fiber size at an advanced age, muscle satellite cell markers, and catabolic genes in RJ-treated mice, thus, RJ may be useful to improve the quality of life during aging through regulating the motor functions [139]. Royalactin, a glycoprotein from RJ, extends the life span of C. elegans by promoting epidermal growth factor (EGF) and its receptors’ signaling [140]. MRJPs are longevity-promoting substances that increase the longevity period of Drosophila through promoting the anti-epidermal growth factor receptor (EGFR)-mediated signaling pathway [141]. Protein and lipid components in RJ have the potential to extend the life span in various living beings, including honeybees, crickets, silkworms, nematodes, mice, and inhibit senescence of human tissues in cell cultures via downregulation of insulin-like growth factors and up-regulation of epidermal growth factor signaling [25]. Moreover, 10-HDA is used to increase the longevity of C. elegans via reduced insulin-like signaling (ILS) and increase the lifespan by dietary restriction signaling and the target of rapamycin (TOR) components in C. elegans [142]. The biological activities of RJ and their underlying possible mechanisms are shown in Figure 4. Figure 4. The biological activities of RJ and their mechanism. SOD (superoxide dismutase); GSH (glutathione); CAT (catalase); GR (glutathione reductase); GPx (glutathione peroxidase); ROS (reactive oxygen species); MMP (matrix metallopeptidases); MDA (malondialdehyde); NO (nitric oxide); IFN-ϒ (interferon-gamma); IL-4 (interleukin-4); TNF-α (tumor necrosis factor); IFN-α (Interferon-α); EGF (epidermal growth factor); AMPK (5′ AMP-activated protein kinase); MAPK (mitogen-activated protein kinase); IGF-1 (insulin-like growth factor-1), and TOR (target of rapamycin). Figure 4. The biological activities of RJ and their mechanism. SOD (superoxide dismutase); GSH (glutathione); CAT (catalase); GR (glutathione reductase); GPx (glutathione peroxidase); ROS (reactive oxygen species); MMP (matrix metallopeptidases); MDA (malondialdehyde); NO (nitric oxide); IFNΥ (interferon-gamma); IL-4 (interleukin-4); TNFα (tumor necrosis factor); IFNα (Interferonα ); EGF (epidermal growth factor); AMPK (5 0 AMP-activated protein kinase); MAPK (mitogen-activated protein kinase); IGF-1 (insulin-like growth factor-1), and TOR (target of rapamycin). Int. J. Mol. Sci. 2020,21, 382 16 of 26 studies and clinical trials) and validation are demanded to reveal the cellular and molecular mechanisms of RJ for health benefits using cutting-edge genomics, transcriptomics, proteomics, and metabolomics. Author Contributions: S.A. prepared the original draft. M.G.C., F.F., and S.Z.A. participated in writing, review, and editing. J.L. conceived the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: The financial support provided by the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2015-IAR), Modern Agro-Industry Technology Research System (CARS-44) in China, and the National Project for Upgrading Overall Bee-Product Quality of the Beekeeping Industry of China. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations ACE Angiotensin 1-converting enzymes AD Alzheimer’s disease AF Aminofluorene AMP Adenosine monophosphate ANO Adenosine N1-oxide ApoA-1 Apolipoprotein A1 BACE1 β-site amyloid precursor protein cleaving enzymes CAT Catalase CCl4 Carbon tetrachloride cGMP Cyclic guanosine monophosphate Con-A Concanavalin A DecDA 1,10-decanedioic acid DOX Doxorubicin EGF Epidermal growth factor EGFR Anti-epidermal growth factor receptor ERJ Enzyme-treated RJ FB Fumonisin FBG Fasting blood glucose FY4 Food yellow 4 GPx Glutathione peroxidase GR Glutathione reductase GSH Glutathione 9-HDA 9-hydroxy-2-decenoic acid 10-HDA 10-hydroxy-2-decenoic acid 3-HHDA 3-hydroxydecanoic acid 10-HDDA 10-hydroxydecenoic acid 3,10-HDecDA 3,10-dihydroxydecanedioic acid 8-HOC 8-hydroxy octanoic acid HSV-1 Herpes simplex virus type 1 HuIFN-aN3 Human interferon-alpha IFN-ΥInterferon-gamma IgE Immunoglobulin E IkBa Inhibitor of kappa B IkB-z IkappaBzeta IL Interleukin ILS Insulin-like signaling JNK-AP-1 C-Jun N-terminal kinases-activating protein-1 MDA Malondialdehyde 24-MET 24-methylene cholesterol MLKL Mixed lineage kinase domain-like protein MoDCs Monocyte-derived dendritic cells MRJPs Major royal jelly proteins NAT N-acetyltransferase NO Nitric oxide NPCs/NSs Neural progenitors or neural stem cells OXM Oxymetholone PC3 Prostate cancer cell line RJ Royal jelly RJBs Royal jelly bees Int. 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