Taiki Fushimi, Kenta Aso, Takafumi Shimizu, Chie Hirahata, Kento Hiroki, Daichi Shinmura, Yasuyuki Fujii, Mitsugu Akagawa, S. Ali Abdelhameed, Vittorio Calabrese, Shu Taira, Yoshitomo Suhara and Naomi Osakabe : C-Ring Structure-Dependent Redox Properties of Flavonoids Regulate the Expression of Bioactivity, Antioxidants, 15, 2, 194, 2026.
(Summary)
(-)-Epicatechin (EC), taxifolin (Tax), and quercetin (Q) are flavonoids with different C-ring structures. We compared their physicochemical properties and biological activities. The comparison of their stability and redox properties was conducted at mildly acidic or neutral conditions mimicking the plant vacuole or gut, and their sympathetic hyperactivation ability was examined using catecholamine (CA) excretion and blood flow. At pH 5, flavonoids were stable, but at pH 7, their retention rates decreased in the order EC > Q > Tax. LC-MS analysis detected brown oxidized oligomers in EC, while Tax and Q primarily yielded degradation products. All flavonoids exhibited O2·- scavenging activity after incubation at pH 5. At pH 7 after 45-min, EC and Tax promoted O2·-, while Q only scavenged O2·- after 24-hr incubation, EC's properties decreased but Tax's properties enhanced. Computational chemistry analysis indicated EC has higher reactivity compared to Tax and Q. EC caused a significant increase in CA excretion and blood flow, which were not observed with Tax or Q or 24-h cultured EC. These results suggest that the C-ring structure of flavonoids plays a crucial role in their stability and redox properties. Furthermore, reactive oxygen species generated by flavonoids in the gut may exert beneficial effects through sympathetic activation.
Ryo Hayakawa, Takeshi Ishii, Taiki Fushimi, Yuki Kamei, Ai Yamaguchi, Kenji Sugimoto, Hitoshi Ashida and Mitsugu Akagawa : Luteolin protects human ARPE-19 retinal pigment epithelium cells from blue light-induced phototoxicity through activation of Nrf2/Keap1 signaling, Free Radical Research, 59, 4, 356-368, 2025.
(Summary)
Age-related macular degeneration (AMD), a serious physical and mental health problem worldwide, is the leading cause of irreversible, severe vision impairment and loss in older people. AMD is associated with multiple risk factors, many of which are closely linked to increased oxidative stress. Some studies have suggested that long-term and excessive exposure to blue light may be a potential risk factor for the development or progression of AMD. Recently, we demonstrated that blue light irradiation caused oxidative stress in all-trans-retinal (atRAL)-exposed human ARPE-19 retinal pigment epithelium cells by generating singlet oxygen (1O2), leading to apoptotic cell death. Luteolin, a flavonoid found in various edible plants, has been reported to possess divergent health-promoting properties including anti-oxidative and chemopreventive effects by up-regulating anti-oxidative and phase II detoxifying enzymes through activation of Keap1/Nrf2 signaling. Herein, we verified the cytoprotective action of luteolin against blue light irradiation using atRAL-exposed ARPE-19 cells. Our results established that luteolin effectively prevented blue light-induced apoptosis of ARPE-19 cells by mitigating oxidative stress. We also confirmed that luteolin suppressed intracellular accumulation of 1O2 and formation of atRAL-derived lipofuscin by increased expression of heme oxygenase-1 and aldehyde dehydrogenase 1A1 through activation of Keap1/Nrf2 signaling. Furthermore, our data implied that the luteolin-provoked activation of Keap1/Nrf2 signaling might be due to covalent binding of luteolin o-quinone to the critical cysteinyl thiol in Keap1. The present results suggest that luteolin could be helpful in the prevention and amelioration of blue light-induced retinal degeneration, including AMD.
(Keyword)
age-related macular degeneration / blue light / Luteolin / retinal pigment epithelium cells / singlet oxygen
Naomi Osakabe, Toshihiko Shoji, Kurumi Onishi, Chie Hirahata, Kento Hiroki, Taiki Fushimi, Yasuyuki Fujii, M. Ursula Jacob, S. Ali Abdelhameed, Tilman Fritsch, Rosanna Paola Di, Salvatore Cuzzocrea and Vittorio Calabrese : Sensory Characteristics and Impact of Flavanol-Rich Grape and Blueberry Extract on Blood Flow Velocity and Oxidative Stress, Journal of Dietary Supplements, 22, 2, 219-235, 2025.
(Summary)
Several epidemiological studies and intervention trials have demonstrated that grapes and blueberries, which are rich in flavanols, can lower the risk of cardiovascular disease. However, the mechanisms of action of these compounds remain unclear due to their low bioavailability. This study aimed to characterize the sensory properties, blood flow velocity, and oxidative stress of a polyphenol rich grape and blueberry extract (PEGB) containing approximately 16% flavanols (11% monomers and 4% dimers). A sensory property of PEGB was compared with quercetin at uniform concentration using healthy young subjects. In addition, the reactivity of PEGB with O2·- was also compared with quercetin utilizing a luminescence method. Furthermore, the effect of a single administration of PEGB on the blood flow velocity of skeletal muscle arterioles was investigated using a laser Doppler method in rats. At a concentration where quercetin was barely tasteful, flavanol in PEGB exhibited a robust astringent taste. Furthermore, under pH conditions mimicking the oral cavity and intestinal tract, PEGB promoted O2·- production at low concentrations and scavenging O2·- at high concentrations. In contrast, quercetin demonstrated antioxidant activity. A single oral administration of PEGB significantly increased the blood flow velocity of skeletal muscle arterioles. The results demonstrate that PEGB exhibited a pronounced astringent taste, O2·- production at low concentrations in neutral pH environments, and significantly enhanced blood flow to skeletal muscle following a single administration to rats. These findings highlight the necessity for further investigation into the causal relationships between oral perception, redox properties, and bioactivity of polyphenols.
Ryosuke Kamikubo, Hiroki Yoshida, Taiki Fushimi, Yuki Kamei and Mitsugu Akagawa : β-Caryophyllene, a dietary phytocannabinoid, alleviates high-fat diet-induced hepatic steatosis in mice via AMPK activation, Bioscience, Biotechnology, and Biochemistry, 88, 12, 1465-1471, 2024.
(Summary)
β-Caryophyllene (BCP), a dietary phytocannabinoid, significantly suppresses palmitate-induced lipid accumulation in human HepG2 hepatocytes via activation of AMP-activated protein kinase (AMPK) signaling. The objective of the preset research was to assess whether oral administration of BCP alleviates obesity-induced hepatic steatosis in mice through AMPK activation. We examined the protective action of supplementation of 0.3% BCP (w/w) in a high-fat diet (HFD) on C57BL/6J mice for 12 weeks. BCP supplementation evidently ameliorated histological hepatic steatosis features, and significantly reduced triglycerides and cholesterol levels in the liver, and serum levels of aspartate aminotransferase and alanine aminotransferase as compared with non-supplemented HFD-fed mice. Immunoblotting revealed that BCP supplementation in HFD-fed mice also caused hepatic AMPK activation. Furthermore, treatment with BCP in HFD-fed mice significantly suppressed body weight gain and attenuated obesity-related phenotypes relative to the HFD mice. Our results suggest the usefulness of BCP in the prevention of obesity-related liver steatosis and liver injury.
Naomi Osakabe, Taiki Fushimi, Yasuyuki Fujii and Vittorio Calabrese : Procyanidins and sensory nutrition; do procyanidins modulate homeostasis via astringent taste receptors?, Bioscience, Biotechnology, and Biochemistry, 88, 4, 361-367, 2024.
(Summary)
Long-term intake of procyanidins has been suggested to reduce the risk of cardiovascular disease, dementia, and sensory function decline associated with aging. However, most of the ingested procyanidins are not absorbed and are excreted in the feces, so the mechanism of their beneficial impact is unknown. Procyanidins are the components of astringency in plant foods and their stimulation appears to be directly transmitted to the central nervous system via sensory nerves. Recent attention has been focused on the taste receptors expressed in the extra-oral gastrointestinal tract may regulate homeostasis via the neuroendocrine system. In this paper, we have reviewed recent findings on the relationship between the astringency of procyanidins and their bioregulatory effects.
Naomi Osakabe, Takafumi Shimizu, Yasuyuki Fujii, Taiki Fushimi and Vittorio Calabrese : Sensory Nutrition and Bitterness and Astringency of Polyphenols., Biomolecules, 14, 2, 2024.
(Summary)
Recent studies have demonstrated that the interaction of dietary constituents with taste and olfactory receptors and nociceptors expressed in the oral cavity, nasal cavity and gastrointestinal tract regulate homeostasis through activation of the neuroendocrine system. Polyphenols, of which 8000 have been identified to date, represent the greatest diversity of secondary metabolites in plants, most of which are bitter and some of them astringent. Epidemiological studies have shown that polyphenol intake contributes to maintaining and improving cardiovascular, cognitive and sensory health. However, because polyphenols have very low bioavailability, the mechanisms of their beneficial effects are unknown. In this review, we focused on the taste of polyphenols from the perspective of sensory nutrition, summarized the results of previous studies on their relationship with bioregulation and discussed their future potential.
(Keyword)
Polyphenols / Taste / Astringents / Diet / Nutritional Status
Takafumi Shimizu, Taiki Fushimi, Rio Ohno, Fujii Yasuyuki, Kenta Aso, M. Ursula Jacobs, Osamu Nureki, Yoshitomo Suhara, Vittorio Calabrese and Naomi Osakabe : Verification of the interaction between human bitter taste receptor T2R46 and polyphenols; Computational chemistry approach, Current Research in Food Science, 9, 2024.
Orie Muta, Shiori Oyama, Minayu Odaka, Kenta Shimizu, Sae Katsuragawa, Kenta Suzuki, Taiki Fushimi, Yasuyuki Fujii, Ryota Akagi and Naomi Osakabe : Cinnamtannin A2, (-)-epicatechin tetramer, attenuates skeletal muscle wasting in disuse atrophy model mice induced by hindlimb suspension., Journal of Clinical Biochemistry and Nutrition, 73, 2, 124-130, 2023.
(Summary)
The impact of repeated administration of cinntamtannin A2 (A2, 25 μg/kg) on skeletal muscle disuse atrophy model mice induced by hindlimb suspension for 14 days was examined. In soleus, weight loss and a reduction in the average myofibre size with shifting to the smaller side of the peak were observed in the suspension-vehicle group, but A2 reduced these changes. Average myofibre size significantly increased in ground-A2 compared to ground-vehicle. A marked increase in the dephosphorylation of forkhead box O (FoxO) 3a by the suspension was reduced by A2. The phosphorylation of protein kinase B (Akt) and eukaryotic translation initiation factor 4E-binding protein (4EBP)-1 were significantly increased by the treatment of A2. In addition, a single dose of A2 increased dramatically in the 24-h excretion of catecholamines in urine. These results suggest that A2 administration results in sympathetic nerve activation and promotes hypertrophy while inhibiting the progress of disuse muscle atrophy.
Orie Muta, Minayu Odaka, Yasuyuki Fujii, Taiki Fushimi, Hiroki Sato and Naomi Osakabe : Difference in endocrine and behavior between short-term single- and paired-housing mice in metabolic cage., Neuroscience Letters, 806, 2023.
(Summary)
Metabolic cage housing which is exposed to a number of environmental stressors is often used in pharmacokinetic studies. In this study, we compared the difference in stress response between single- and paired-housing in metabolic cages by evaluating the alteration of urinary stress hormones and behavior. Mice were randomly divided into single- or paired-housing groups and placed in a metabolic cage with wire mesh. Their urine was collected every 24 h for consecutive 4 days to determine excreted catecholamine and corticosterone. The change in body weight was significantly decreased at 3 and 4 days in the single-housing group compared with that before the experiment, but not paired-housing group. The level of urinary catecholamines, such as noradrenaline, adrenaline, and their metabolite vanillylmandelic acid, was significantly increased in the single-housing compared with paired housing group and urinary corticosterone increased as well. Next, for the two similarly housed groups, we observed spontaneous behavior on the fourth day and conducted an elevated plus-maze test on the fifth day. Spontaneous behavior was not different between experimental groups. In the elevated plus-maze test, the proportion of time spent in the open arms was significantly prolonged in the paired-housing group compared to that of the single-housing group. Short-term social isolation stress loading in metabolic cages was suggested to exhibit endocrinological and behavioral changes in mice. To reduce such interference due to stress exposure, it was suggested to keep two mice in a metabolic cage.
(Keyword)
Mice / Animals / Corticosterone / Endocrine System / Social Isolation / Catecholamines / Housing, Animal / Behavior, Animal
Taiki Fushimi, Shiori Oyama, Ryo Koizumi, Yasuyuki Fujii and Naomi Osakabe : Impact of cyanidin 3-O-glucoside on rat micro-and systemic circulation, possibly thorough angiogenesis., Journal of Clinical Biochemistry and Nutrition, 72, 2, 132-138, 2022.
(Summary)
Cyanidin 3-glucoside (C3G), an antioxidant, is one of the most abundant anthocyanin in plant foods. Intervention trials and subsequent meta-analyses have suggested that anthocyanins could reduce the risks of cardiovascular diseases. This study investigated hemodynamic alterations following a single intragastric dose of C3G by measuring blood flow in rat cremaster muscle arteriole for 60min. Next, in excised aortas, we performed western blotting to measure the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). A single oral dose of C3G significantly increased blood flow soon after ingestion, and it was maintained throughout the experimental period. In addition, aortic Akt phosphorylation increased. Then, we examined the impact of repeated oral administrations of C3G for 14 days. The mean blood pressure was significantly reduced at 7 and 14 days after treatment, with a slight increase in aortic eNOS expression. Immunohistochemical analyses of the soleus showed that the level of CD31, an angiogenesis-marker protein, was significantly increased with C3G. These results suggested that an oral dose of C3G increased blood flow, which promoted angiogenesis within skeletal muscle, and consequently, blood pressure was reduced.
Naomi Osakabe, Taiki Fushimi and Yasuyuki Fujii : Hormetic response to B-type procyanidin ingestion involves stress-related neuromodulation via the gut-brain axis: Preclinical and clinical observations., Frontiers in Nutrition, 9, 2022.
(Summary)
, but animal and clinical studies are limited. Hormesis of hemodynamic and metabolic responses to B-type procyanidins was recently confirmed in animal studies, however, and our work has linked these effects to the CNS. Here, we evaluate the hormetic response elicited by B-type procyanidins, recontextualizing the results of intervention trials. In addition, we discuss the possibility that this hormetic response to B-type procyanidins arises via CNS neurotransmitter receptors. We have verified the direction of future research for B-type procyanidins in this review.
Yuko Ishii, Orie Muta, Tomohiro Teshima, Nayuta Hirasima, Minayu Odaka, Taiki Fushimi, Yasuyuki Fujii and Naomi Osakabe : Repeated Oral Administration of Flavan-3-ols Induces Browning in Mice Adipose Tissues through Sympathetic Nerve Activation., Nutrients, 13, 12, 2021.
(Summary)
were increased in epididymal adipose (eWAT) by FLs. A multilocular morphology with cell size reduction was shown in the inguinal adipose (iWAT), together with increasing the level of Ucp-1 by FLs. These results exert that FLs induce browning in adipose, and this change is possibly produced by the activation of the SNS.
Ryo Koizumi, Taiki Fushimi, Yuki Sato, Yasuyuki Fujii, Hiroki Sato and Naomi Osakabe : Relationship between hemodynamic alteration and sympathetic nerve activation following a single oral dose of cinnamtannin A2., Free Radical Research, 55, 5, 491-498, 2021.
(Summary)
We previously found that a single dose of B-type procyanidin mixture increase in skeletal muscle blood flow (BF). We compared BF changes following administration of (-)-epicatechin (EC, monomer) and the B-type procyanidins procyanidin B2 (B2, dimer), procyanidin C1 (C1, trimer), and cinnamtannin A2 (A2, tetramer). Each chemical was administered orally to rats, followed by BF measurement in cremaster arteriole for 60 min. About 10 and 100 µg/kg of B2 and C1 elicited BF increase, the effect was potent at 100 µg/kg. BF also increased significantly after administration of 10 µg/kg A2, but not with the administration at 100 µg/kg. EC yielded no BF changes. Co-treatment with the nonselective adrenaline blocker carvedilol attenuated the BF increase seen with 10 µg/kg A2 treatment. This outcome suggested the involvement of sympathetic nerve activation in the BF increase by this dose of A2. Co-treatment of 100 µg/kg A2 with the α2 blocker yohimbine exhibited an increase of BF significantly. The α2 adrenaline receptor in the vasomotor centre is an inhibitory receptor and it regulates hemodynamics. This result suggested that high doses of A2 did not alter BF because of activating the α2 adrenergic receptor. Phosphorylation of aortic endothelial nitric oxide synthase (eNOS) increased with 10 µg/kg A2 alone or co-treatment with 100 µg/kg A2 and yohimbine, but not with co-treatment of 10 µg/kg A2 and carvedilol or 100 µg/kg A2 alone. These results imply that A2 does not directly activate eNOS, but that shear stress from the increased BF might be associated with eNOS phosphorylation.
Taiki Fushimi, Yasuyuki Fujii, Hideaki Koshino, Kodai Inagawa, Akiko Saito, Ryo Koizumi, Masahiro Shibata and Naomi Osakabe : Method for detecting hemodynamic alterations following a single gavage in rats., Experimental Animals, 70, 3, 372-377, 2021.
(Summary)
It is known that administering a gavage to rodents evokes a cardiac reflex, due to gastrointestinal stimulation. Consequently, it is difficult to evaluate changes in hemodynamics after a single oral dose of a pungent or astringent, which alters the circulation by increasing sympathetic activity. In the present study, we developed a method for administering a gavage without significantly affecting hemodynamics measurements. We marked a gastric tube at 10 cm from the tip, to mark the distance from the oral cavity to the stomach body of Wistar male rats. Rats were intubated under urethane anesthesia.After 10-15 min of stabilization, we measured the mean blood pressure (MBP), heart rate (HR), and blood flow (BF) in the cremaster arteriole under two different conditions; condition 1: a pointed gastric tube, room temperature distilled water, and injected at normal speed (approximately 3 ml/min); condition 2: a rounded gastric tube, 37°C distilled water, and injection at 1.0 ml/min. Under condition 1, we observed striking hemodynamic alterations, due to the somatic afferent reflex. In contrast, under condition 2, these hemodynamic changes were nearly eliminated. In addition, we could clearly detect hemodynamic changes in rats after a single gavage treatment of pungent (capsaicin) or astringent (cinnamtannin A2). We observed transient increases in the HR and MBP soon after treatment with capsaicin. Moreover, cremasteric BF was elevated with cinnamtannin A2. These results confirmed the utility of the gavage method developed in this study.
Kenta Suzuki, Nayuta Hirashima, Yasuyuki Fujii, Taiki Fushimi, Ayaka Yamamoto, Tomoya Ueno, Ryota Akagi and Naomi Osakabe : Theaflavins decrease skeletal muscle wasting in disuse atrophy induced by hindlimb suspension in mice., Journal of Clinical Biochemistry and Nutrition, 68, 3, 228-234, 2020.
(Summary)
We previously found that a single dose of theaflavins induced skeletal muscle metabolic changes. In this study, we examined the effect of theaflavins on disuse muscle atrophy model mice by hindlimb suspension. Mice were assigned to 4 groups; ground-vehicle, ground-theaflavins, suspension-vehicle, and suspension-theaflavins, dosed with theaflavins (250 mg/kg/day) for 2 weeks. The peak of myotube size of cross sectional area was significantly moved to the smaller side in the suspension-vehicle group compared with the ground-vehicle group, and these shifts were significantly reduced by the treatment with theaflavins in both soleus and extensor digitorum longus. The level of phosphorylated eukaryotic translation initiation factor 4E-binding protein (4EBP)-1, located downstream of the Akt/mTOR pathway, was significantly different between suspension-vehicle and suspension-theaflavins in soleus. The ratio of forkhead box O (FoxO) 3a to phosphorylated FoxO3a significantly increased in soleus or tended to rise in extensor digitorum longus of suspension-vehicle group compared with ground-vehicle. In contrast, these changes were not observed in suspension-theaflavins group. These results suggested that theaflavins inhibited the progress of disuse muscle atrophy through modulation of protein metabolism.
Ayaka Tsutsumi, Yukari Horikoshi, Taiki Fushimi, Akiko Saito, Ryo Koizumi, Yasuyuki Fujii, Qiang Qing Hu, Yoshihisa Hirota, Koichi Aizawa and Naomi Osakabe : Acylated anthocyanins derived from purple carrot (Daucus carota L.) induce elevation of blood flow in rat cremaster arteriole., Food & Function, 10, 3, 1726-1735, 2019.
(Summary)
Acylated anthocyanins are more stable than monomeric anthocyanins, but little is known about their physiological effects. We evaluated the hemodynamic effects of single intragastric doses of purple carrot (Daucus carota L.) anthocyanin (PCA) and two monomeric anthocyanins, cyanidin 3-O-glycoside (C3G) and delphinidin 3-O-ruthenoside (D3R). PCA, C3G, or D3R was administered orally to rat and blood flow in the cremaster artery was measured for 60 min using a laser Doppler blood flow meter. After measurements, the aorta of the animal was removed and the extent of phosphorylation of aortic epithelial nitric oxide synthase (eNOS) and Akt were determined by western blotting. PCA (10 mg kg-1) or C3G (1 mg kg-1) significantly increased rat cremaster arteriole blood flow and phosphorylation of eNOS and Akt; D3G (1 mg kg-1) only slightly altered cremaster arteriole blood flow and did not affect the phosphorylation of eNOS and Akt in the aorta. These results suggest that hemodynamic alterations depend more on the chemical structure of anthocyanins, particularly the aglycon, than on the glycoside. In addition, increase of blood flow by a single oral dose of PCA was practically reduced with treatment of carvedilol (CR), a non-specific adrenaline blocker. Blood concentrations of cyanidin or its glycoside 15, 30, or 60 min after the administration of 10 mg kg-1 PCA were below the limit of detection. These hemodynamic changes may have been associated with an indirect adrenergic action induced following a single dose of PCA.
Shimizu Takafumi, Taiki Fushimi, Ohno Rio, Fujii Yasuyuki, Suhara Yoshitomo, Carablese Vittorio and Osakabe Naomi : Verification of the interaction between human bitter taste receptor T2R46 and polyphenols; Computational chemistry approach, 11th International Conference on Polyphenols and Health, Oct. 2024.
Proceeding of Domestic Conference:
1.
MATSUMOTO Hina, Taiki Fushimi, Yuki Kamei, 越阪部 奈緒美 and Mitsugu Akagawa : 緑茶カテキンによる神経および免疫細胞への作用機構の解析, 日本農芸化学会2026年度大会, Mar. 2026.