*2.4. Phenols, Flavonoids, and Free Amino Acids*

The antioxidant potency of RJ, at least in part, is attributed to its polyphenolic compounds and flavonoids, which are measured based on gallic acid and rutin equivalent (GAE and RE), respectively [47]. RJ contains 23.3 ± 0.92 GAE μg/mg and 1.28 ± 0.09 RE μg/mg of total phenolics and flavonoids, respectively. The vast phenolic content of RJ consists of pinobanksin, organic acids (e.g., octanoic acids, dodecanoic acid, 1,2-benzenedicarboxylic acid), and their esters. Meanwhile, flavonoids of RJ can be differentiated into four groups: (1) flavanones e.g., hesperetin, isosakuranetin, and naringenin; (2) flavones e.g., acacetin, apigenin and its glucoside, chrysin, and luteolin glucoside; (3) flavonols e.g., isorhamnetin and kaempferol glucosides; and (4) isoflavonoids e.g., coumestrol, formononetin, and genistein. The antioxidant activity of these components contributes to the antiapoptotic and anti-inflammatory properties of RJ [18]. Age of larvae that produce RJ (1–14 days) and harvesting time affect RJ content of phenols and amino acids; RJ harvested from the youngest larvae (one-day-old) within 24 h contains higher proteins and polyphenolic compounds and exhibits stronger free radical scavenging effect compared with RJ harvested from older larvae or later than 24 h [47]. Small peptides such as di-peptides (Lys-Tyr, Arg-Tyr, and Tyr-Tyr) obtained from RJ proteins hydrolyzed by protease N possess high antioxidative activity owing to their phenolic hydroxyl groups, which scavenge free radicals by releasing a hydrogen atom [48].

RJ is rich in amino acids, including essential ones [22]. Concentrations of free amino acids in RJ increase with harvesting time from 4.30 mg/g at 24 h to 9.48 mg/g at 72 h, whereas levels of total amino acids decrease by time from 197.96 mg/g at 24 h to 121.32 mg/g at 72 h [49]. LC/MS method analysis and hydrophilic interaction liquid chromatography-tandem mass spectrometry indicate that lysine is the most prominent free amino acid in RJ (62.43 mg/100 g), followed by proline (58.76 mg/100 g), cystine (21.76 mg/100 g), and aspartic acid (17.33 mg/100 g). RJ contains less than 5 mg/100 g of other amino acids such as valine, glutamic acid, serine, glycine, cysteine, threonine, alanine, tyrosine, phenylalanine, hydroxyproline, leucine-isoleucine, and glutamine [18,50].

The amino acid content in protease-treated RJ (pRJ)—obtained by removal of two allergen proteins to convert RJ hydrolysates into shorter chain monomers that are easy to absorb—is greater than in crude RJ [51]. Moreover, eluted water pRJ with 30% MeOH contains higher levels of dipeptides and tripeptides than pRJ. These components are likely to possess a lifespan-prolonging activity since pRJ increases lifespan of *C. elegans* compared with RJ [52]. Evidence signifies that amino acids of RJ can prolong lifespan in mammals. Long-term dietary supplementation of branched-chain amino acid-enriched mixture (BCAAem), which contains 3 branched-chain amino acids that can be found in RJ (leucine, isoleucine, and valine), enhances mitochondrial biogenesis and sirtuin 1 expression, and reduces ROS production in cardiac and skeletal muscle, which is associated with alleviation of age-related muscle dysfunction, resulting in an increase of the average lifespan of male mice. The effect of these amino acids on mitochondrial biogenesis involved activation of signaling pathways of endothelial nitric oxide synthase (*eNOs*) and *mTOR* and its substrates: *S6K* and eukaryotic translation initiation factor (*eIF4E*)-binding protein (*4E-BP1*) [53].
