Disuse muscle atrophy is an urgent public healthcare issue in aging societies because it is closely associated with the bedridden state 
. However, medications for the relief of this condition are not available. Our previous study 
revealed that periodic injection of the flavonol-type flavonoid quercetin into the GM was effective for prevention of the loss of muscle weight in mice that underwent tail suspension (which was used as a model of disuse muscle atrophy). Therefore, skeletal muscle is one of the target tissues of flavonoids for exerting their pharmacological functions. We first demonstrated that a prenylflavonoid prevented the loss of function of the GM in mice that underwent sciatic denervation (which is an alternative model of disuse muscle atrophy) (). The prenylflavonoid 8-PN was administered orally to mice by mixing with the diet for 18 days before denervation. Hence, 8-PN was assumed to prevent disuse muscle atrophy after intestinal absorption and transport into the target muscle tissue. It can be deduced that prenylation increases the bioavailability of a continuously ingested flavonoid and promotes its accumulation in muscle because the plasma concentration of total 8-PN and its content in the GM were much higher than those of naringenin (). Enhancement of accumulation in muscle tissue is how flavonoids exert preventive effects upon disuse muscle atrophy. Several reports have shown that orally administered naringenin is mostly metabolized into conjugated metabolites in intestinal epithelial cells and hepatic cells before entering the blood circulation, resulting in quite low (or undetectable) levels of its aglycone in plasma 
. In general, conjugation reactions are known to diminish a wide variety of the biological activities of flavonoids because conjugated metabolites are characterized as hydrophilic, stable products to be excreted into urine 
. One study using quercetin indicated that all tissues (including muscle) contained 30–100% of the deconjugated aglycone of total flavonoids after long-term supplementation of quercetin to pigs 
even though the aglycone could have been derived from deconjugation during the extraction procedure. In contrast, neither the GM nor plasma accumulated naringenin in the present study. Interestingly, herein we demonstrated that a considerable amount of the 8-PN aglycone was present in the plasma and GM. In particular, the content of the 8-PN aglycone was almost identical to that of total naringenin in the GM (). The ratio of the aglycone to total 8-PN in the GM was 0.15 without denervation and 0.17 with denervation, and was nearly tenfold higher than that in the plasma (0.016). Therefore, it is plausible that in situ
deconjugation of 8-PN precedes its accumulation in the GM for it to exert preventive effects. Some authors have argued that deconjugation of dietary flavonoids must occur for these compounds to exert their functions in vivo
. Further studies are required to clarify the role of deconjugation in the preventive effects of prenylated flavonoids.
We selected 8-PN for evaluation of the structural significance of prenylation in dietary flavonoids for exertion of their physiological functions. Binding of the prenyl group to the molecule should accelerate intestinal absorption by passive diffusion because such binding increases lipophilicity and allows the molecule to have a higher affinity to cellular membranes 
. High accumulation of 8-PN in muscle tissue and the appearance of preventive effects may be derived from its high bioavailability based on effective absorption in the intestine. However, the time-courses of the plasma concentrations of 8-PN and naringenin in a single dose () and their values of Cmax
and AUC shown in indicated that the prenylation of flavonoids lowered their effectiveness with regard to intestinal absorption and blood circulation. Pang et al. 
reported that a prenylated chalcone exhibits little efflux into the basolateral side when incorporated into Caco-2 cells (a cellular model of intestinal absorption). It is therefore likely that prenylation suppresses the intestinal absorption of flavonoids by interfering with efflux into the portal vein. In contrast, the plasma concentration of total 8-PN 24 h after administration was significantly higher than that seen with naringenin (), indicating that prenylation reduces the rate of elimination into urine. This may be why 8-PN was present in plasma at a considerable level after its ingestion for 22 days (). Our study on uptake using cultured mouse C2C12 myotubes () strongly suggested that prenylation significantly enhanced the accumulation of flavonoids within cells and/or their association to cellular membranes in mouse C2C12 myotubes because it increased passive transport into cells and interaction with cellular membranes owing to increased lipophilicity. Therefore, prenylation seems to be an effective tool to improve the physiological functions of dietary flavonoids by increasing the concentration to target tissues through a reduction in the rate of elimination from the circulation and higher uptake into cells.
Results from studies on denervated mice demonstrated that 8-PN suppressed atrogin-1 content in denervated muscle compared with control-diet mice (). 8-PN is known to be a powerful phytoestrogen 
. Some studies suggest that estrogenic status affects recovery from disuse muscle atrophy by accelerating the Akt signaling pathway 
. That is, estrogen deficiency inhibits the reproduction of muscle, and estradiol supplementation accelerates the regeneration of skeletal muscles. Our results clearly indicated that 8-PN intake accelerated Akt phosphorylation (). Taken together, the mechanism for 8-PN-dependent prevention of muscle atrophy may be related to its estrogenic activity, which can promote post-natal growth and delay the degradation of skeletal muscles. Insulin-like growth factor-1 (IGF1)/phosphatidyl inositol 3-kinase (PI3K)/Akt pathway-mediated phosphorylation of FoxOs by phosphorylated Akt causes their exclusion from the nucleus, thereby preventing their transcription activity 
. It is therefore likely that 8-PN suppresses the expression of atrogin-1 by inhibiting the transcription activity of FoxOs by elevating the phosphorylated Akt level. 8-PN may also induce protein synthesis because hypertrophy in skeletal muscle was also shown to be activated by the IGF1/PI3K/Akt pathway 
. Further study is required to clarify exact mechanism for the preventive effects of 8-PN on disused muscle atrophy.
8-PN is a demethylated metabolite of isoxanthohumol through the CYP450 reaction in the liver 
. This prenylated flavonoid can also be produced from isoxanthohumol by the action of microflora in the digestive tract 
Isoxanthohumol is produced from xanthohumol via
a ring-closing reaction during brewing 
. Therefore, xanthohumol and isoxanthohumol present in H. lupulus
and beer can act as pro-8-PN. Stevens et al. showed levels of isoxanthohumol and 8-PN in beer to be 40–3,440 µg/L and 1–240 µg/L, respectively 
. It has been shown that, in healthy post-menopausal Caucasian women, nearly 25% were moderate producers and 15% were strong producers of 8-PN based on urinary excretion through microbial conversion capacities 
. A clinical study demonstrated that orally administered 8-PN was absorbed rapidly at a very limited level and was associated with decreased concentrations of luteinizing hormone in post-menopausal women 
. Therefore, diets or supplements containing 8-PN (or an 8-PN precursor) could be beneficial for human health. H. lupulus
is known to contain isoxanthohumol and 8-PN at 0.1–1.0% 
. The results regarding intake of H. lupulus
() could suggest the beneficial effects of H. lupulus
on the prevention of disuse muscle atrophy. Bolca et al. found that 8-PN could accumulate in human breast tissue if 8-PN producers were given H. lupulus
for 5 days 
. Our results in mice suggest that daily consumption of 1150–1250 nmol 8-PN/kg bw/day (0.4 mg/kg bw/day) is effective for the prevention of disuse muscle atrophy. For an individual weighing 50 kg, this corresponds to daily consumption of 1 kg dry weight of H. lupulus
and 83–20,000 L of beer. However, continuous consumption of 8-PN or its precursors in food and beverages every day may lead to a gradual increase in 8-PN content in target tissues. It is likely that daily consumption of foods or beverages containing 8-PN or its related compounds could contribute to the prevention of disuse muscle atrophy, although the study was limited to in model animal experiment using denervated mouse.
In summary, prenylation enabled naringenin to prevent disuse muscle atrophy in denervated mice. Prenylation also significantly enhanced accumulation in target tissues. These findings strongly suggest that prenylation of flavonoids induces their preventive effects by increasing their contents in target tissues. Prenylation seems to be a promising tool for developing and enhancing the pharmacological and physiological functions of dietary flavonoids.