Effectiveness and Safety of a Mixture of Nobiletin and Tangeretin in Nocturia Patients: A Randomized, Placebo-Controlled, Double-Blind, Crossover Study.

Nobiletin and tangeretin (NoT) are flavonoids derived from the peel of Citrus depressa, and they have been found to modulate circadian rhythms. Because nocturia can be considered a circadian rhythm disorder, we investigated the efficacy of NoT for treating nocturia. A randomized, placebo-controlled, double-blind, crossover study was conducted. The trial was registered with the Japan Registry of Clinical Trials (jRCTs051180071). Nocturia patients aged ≥50 years who presented nocturia more than 2 times on a frequency-volume chart were recruited. Participants received NoT or a placebo (50 mg once daily for 6 weeks), followed by a washout period of ≥2 weeks. The placebo and NoT conditions were then switched. Changes in nocturnal bladder capacity (NBC) were the primary endpoint, and changes in nighttime frequency and nocturnal polyuria index (NPi) were secondary endpoints. Forty patients (13 women) with an average age of 73.5 years were recruited for the study. Thirty-six completed the study, while four withdrew. No adverse events directly related to NoT were observed. NoT had little effect on NBC compared with the placebo. In contrast, NoT significantly changed nighttime frequency by -0.5 voids compared with the placebo (p = 0.040). The change in NPi from baseline to the end of NoT was significant (-2.8%, p = 0.048). In conclusion, NoT showed little change in NBC but resulted in decreased nighttime frequency with a tendency toward reduced NPi.

Flavonoid Nobiletin Attenuates Cyclophosphamide-Induced Cystitis in Mice through Mechanisms That Involve Inhibition of IL-1ß Induced Connexin 43 Upregulation and Gap Junction Communication in Urothelial Cells.

Bladder inflammatory diseases cause various urinary symptoms, such as urinary frequency and painful urination, that impair quality of life. In this study, we used a mouse model of cyclophosphamide (CYP)-induced bladder inflammation and immortalized human urothelial (TRT-HU1) cells to explore the preventive potential of nobiletin (NOB), a polymethoxylated flavone enriched in citrus fruit peel, and investigate its mechanism of action in the bladder. Prophylaxis with PMF90 (60% NOB) attenuated the development of bladder inflammation and urinary symptoms in CYP-treated mice. PMF90 also reduced the upregulation of connexin 43 (Cx43), a major component of gap junction channels, in the bladder mucosa of CYP-treated mice. Stimulation of TRT-HU1 cells with the pro-inflammatory cytokine IL-1ß increased Cx43 mRNA and protein expression and enhanced gap junction coupling-responses that were prevented by pre-treatment with NOB. In urothelium-specific Cx43 knockout (uCx43KO) mice, macroscopic signs of bladder inflammation and changes in voiding behavior induced by CYP treatment were significantly attenuated when compared to controls. These findings indicate the participation of urothelial Cx43 in the development of bladder inflammation and urinary symptoms in CYP-treated mice and provide pre-clinical evidence for the preventive potential of NOB through its anti-inflammatory effects on IL-1ß signaling and urothelial Cx43 expression.

Effects of Baccharin Isolated from Brazilian Green Propolis on Adipocyte Differentiation and Hyperglycemia in ob/ob Diabetic Mice.

Propolis is a honeybee product with various biological activities, including antidiabetic effects. We previously reported that artepillin C, a prenylated cinnamic acid derivative isolated from Brazilian green propolis, acts as a peroxisome proliferator-activated receptor γ (PPARγ) ligand and promotes adipocyte differentiation. In this study, we examined the effect of baccharin, another major component of Brazilian green propolis, on adipocyte differentiation. The treatment of mouse 3T3-L1 preadipocytes with baccharin resulted in increased lipid accumulation, cellular triglyceride levels, glycerol-3-phosphate dehydrogenase activity, and glucose uptake. The mRNA expression levels of PPARγ and its target genes were also increased by baccharin treatment. Furthermore, baccharin enhanced PPARγ-dependent luciferase activity, suggesting that baccharin promotes adipocyte differentiation via PPARγ activation. In diabetic ob/ob mice, intraperitoneal administration of 50 mg/kg baccharin significantly improved blood glucose levels. Our results suggest that baccharin has a hypoglycemic effect on glucose metabolic disorders, such as type 2 diabetes mellitus.

Prenylflavonoids from fruit of Macaranga tanarius promote glucose uptake via AMPK activation in L6 myotubes.

Skeletal muscle is a major tissue of glucose consumption and plays an important role in glucose homeostasis. Prenylflavonoids, a component of Macaranga tanarius fruits, have been reported to have antioxidant, antibacterial, and anticancer effects. However, the effects of these compounds on skeletal muscle glucose metabolism are unclear. Here, we isolated five prenylflavonoids from M. tanarius fruits, and investigated the mechanism of action of these compounds on skeletal muscle cells using L6 myotubes. We found that isonymphaeol B and 3'-geranyl naringenin increased glucose uptake in a dose-dependent manner. Furthermore, both isonymphaeol B and 3'-geranyl naringenin increased AMPK phosphorylation but did not affect PI3K-Akt phosphorylation. Isonymphaeol B and 3'-geranyl naringenin also increased Glut1 mRNA expression and plasma membrane GLUT1 protein levels. These results suggest that isonymphaeol B and 3'-geranyl naringenin have beneficial effects on glucose metabolism through AMPK and GLUT1 pathway. Isonymphaeol B and 3'-geranyl naringenin may be potential lead candidates for antidiabetic drug development.

Effect of pinocembrin isolated from Alpinia zerumbet on osteoblast differentiation.

Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Osteoporosis is a bone metabolism disorder in which bone mass decreases due to increased bone resorption rather than bone formation. We focused on the traditional plant Alpinia zerumbet in Okinawa, Japan, and searched for promising compounds for the prevention and treatment of osteoporosis. Pinocembrin isolated from the leaves of A. zerumbet showed enhanced alkaline phosphatase (ALP) activity and mineralization and increased mRNA expression of osteoblast-related genes Alp and Osteocalcin (Ocn) in MC3T3-E1 cells. Pinocembrin increased the mRNA expression of Runx2 and Osterix, which are important transcription factors in osteoblast differentiation, and the mRNA expression of Dlx5 and Msx2, which are enhancers of these transcription factors. The bone morphogenetic protein (BMP) antagonist noggin, its receptor kinase inhibitor LDN-193189 and p38 MAPK inhibitor SB203580 attenuated pinocembrin-promoted ALP activity. Pinocembrin increased the mRNA of Bmp-2 and its target gene Id1. In addition, the estrogen receptor (ER) inhibitor ICI182780 suppressed pinocembrin-stimulated ALP activity. Pinocembrin may increase BMP-2 expression via ER. Then, the BMP-2 promotes osteoblast specific genes expression and mineralization through both Smad-dependent and independent pathway following Runx2 and Osterix induction. Our findings suggest that pinocembrin has bone anabolic effects and may be useful for the prevention and treatment of bone metabolic diseases such as osteoporosis.

Effect of methoxyflavones contained in Kaempferia parviflora on CRE-mediated transcription in PC12D cells.

The cAMP-response element (CRE) is critical in the formation of long-term memory. To prove the pharmacological effects of the methoxyflavones-rich residue (MRR) and its constituent methoxyflavones (1-9) extracted from the rhizomes of Kaempferia parviflora on the nervous system, we examined the effects of the MRR and methoxyflavones (1-9) on CRE-mediated transcription in PC12D cells. The MRR increased CRE-mediated transcription in PC12D cells. In addition, among methoxyflavones (1-9) isolated from MRR, compounds 1-4 increased CRE-mediated transcription. These results suggest that K. parviflora and methoxyflavone might be very useful materials for preventing and recovering from cognitive decline.

Molecular mechanism for nobiletin to enhance ABCA1/G1 expression in mouse macrophages.

BACKGROUND AND AIMS: Nobiletin (NOB), a functional ingredient found in citrus peel, is said to act against diabetes, obesity, and atherosclerosis. It has been reported to activate AMPK pathway, as well as increase SREBP1c, PPARα and PPARγ expression. However, no molecular mechanism has been elucidated to be able to integrate these sporadic findings with some controversies to lead to concrete outcomes. In this study, regulation of HDL biogenesis by NOB was investigated modulating ABCA1 and ABCG1 expression. METHODS AND RESULTS: Regulation of ABCA1/G1 by NOB was investigated in mouse macrophages J774.1. NOB increased mRNA and protein levels of ABCA1/G1, and cell cholesterol release by these factors. It also increased mRNA of PPARγ and LXRα but not PPARα. The increase in ABCA1/G1 mRNA levels by NOB was suppressed by antagonists of PPARγ and LXRα. The increase in PPARγ mRNA levels by NOB was suppressed by an LXRα antagonist, and the increase in LXRα mRNA levels was suppressed by a PPARγ antagonist. NOB increased CD36 mRNA and this was suppressed by an LXRα antagonist. The increase in ABCA1 mRNA by a PPARγ agonist was also suppressed by an LXRα antagonist. NOB did not influence LPL1 mRNA expression levels. NOB stimulated AMPK phosphorylation, and the increase in ABCA1/G1, LXRα and PPARγ mRNA levels and ABCA1/G1 protein levels by NOB was reversed by an AMPK inhibitor. AMPK siRNA suppressed ABCA1 expression. CONCLUSIONS: NOB activates AMPK and subsequently LXRα to promote the expression of ABCA1 and ABCG1, and an LXRα - PPARγ loop pathway amplifies these signals.

Supplementation of the Flavonoid Myricitrin Attenuates the Adverse Metabolic Effects of Long-Term Consumption of a High-Fat Diet in Mice.

The flavonoid myricitrin exhibits various pharmacological and physiological effects. However, studies on the effects of myricitrin on obesity are limited. We hypothesized that dietary myricitrin would attenuate the adiposity and metabolic dysfunction that occur in obesity. To test this hypothesis, mice were randomly fed a high-fat diet (HFD) or HFD supplemented with myricitrin for 16 weeks. Myricitrin significantly reduced white adipose tissue (WAT) mass, adipocyte size, and plasma leptin levels, and also attenuated dyslipidemia. These changes appeared to result from increased energy expenditure and activation of the carnitine acyltransferase (CPT) and ß-oxidation in WAT. Expressions of the proinflammatory genes NF-κB, TLR2, MCP1, and TNF-α were also lower in the WAT of myricitrin-supplemented mice. Moreover, myricitrin markedly reduced hepatic triglyceride accumulation and plasma aspartate transaminase levels by increasing CPT activity and reducing fatty acid synthase activity in the liver. Myricitrin-supplemented mice also showed improved glucose tolerance, insulin sensitivity, and decreased hyperinsulinemia, along with decreased levels of circulating resistin. In conclusion, long-term consumption of a myricitrin-supplemented diet may effectively protect against HFD-induced obesity and related metabolic disorders.

Antimicrobial and antibiofilm activities of prenylated flavanones from Macaranga tanarius.

BACKGROUND: The emergence of antibiotic resistant microorganisms presents a worldwide problem that requires novel antibiotic and non-antibiotic strategies, and biofilm formation is a mechanism of drug resistance utilized by diverse microorganisms. The majority of microorganisms live in biofilms that help their survival against starvation, antimicrobial agents, and immunological defense systems. Therefore, it is important novel compounds be identified that inhibit biofilm formation and cell survival without drug resistance. STUDY DESIGN: In this study, the antimicrobial and antibiofilm activities of five prenylated flavanones (Okinawan propolins) isolated from fruits of Macaranga tanarius (L.) were investigated against 14 microorganisms including 10 pathogens. RESULTS: Of these five propolins, propolin D at 5-10 µg/ml significantly inhibited biofilm formation by three Staphylococcus aureus strains, a Staphylococcus epidermidis strain, and a Candida albicans with MICs from 10 to 50 µg/ml, and in C. albicans, propolin D was found to inhibit biofilm formation by reducing cell aggregation and downregulated the expressions of hypha/biofilm-related genes including ECE1 and HWP1. Interestingly, at sub-MIC concentrations (10-50 µg/ml), propolin D significantly inhibited biofilm formation by enterohemorrhagic E. coli O157:H7, uropathogenic E. coli O6:H1, and Acinetobacter baumannii without affecting planktonic cell growth, but did not inhibit biofilm formation by a commensal E. coli K-12 strain, three probiotic Lactobacillus plantarum strains, or two Pseudomonas aeruginosa strains. And, propolin D reduced fimbriae production by E. coli O157:H7 and repressed gene expression of curli fimbriae genes (csgA and csgB). Also, propolin D was minimally toxic in a Caenorhabditis elegans nematode model. CONCLUSION: These findings show that prenylated flavanones, especially propolin D from Macaranga tanarius (Okinawan propolis), should be considered potential candidates for the development of non-toxic antibacterial and antifungal agents against persistent microorganisms.

A chalcone derivative suppresses the induction of TSLP in mice and human keratinocytes and attenuates OVA-induced antibody production in mice.

Thymic stromal lymphopoietin (TSLP) is a key epithelial-derived factor that aggravates allergic diseases. Therefore, TSLP inhibitors are candidate compounds for the treatment of allergic diseases. Previously, we reported that KCMH-1, a mouse keratinocyte cell line, constitutively produces TSLP. In this study, we tried to identify inhibitors of TSLP by screening 2169 compounds in KCMH-1 cells and found one such chalcone derivative (code no. 16D10). 16D10 inhibited TSLP expression and TSLP promoter activation in HaCaT cells, a human keratinocyte cell line. Although nuclear factor kappa-B (NF-κB) is a key transcription factor for the induction of TSLP, 16D10 did not inhibit the activation pathway of NF-κB, such as degradation of inhibitor of κB (IκB) and p65 nuclear translocation. 16D10 activated the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system, although this system was not involved in the inhibitory effect of 16D10. 16D10 also inhibited TSLP production in a lipopolysaccharide (LPS)- or ovalbumin (OVA)-induced air-pouch-type inflammation model. Further, repeated 16D10 administration diminished serum immunoglobulin G1 (IgG1) and IgE concentration in an OVA-induced air-pouch-type sensitization model. Taken together, these results indicate that 16D10 is an inhibitor of TSLP production and has an anti-allergic effect. This inhibitory effect is independent of the activation of NF-κB and the Keap1-Nrf2 system. Therefore, 16D10 could be a new type of candidate drug for allergic diseases.

A steroid alkaloid derivative 02F04 upregulates thymic stromal lymphopoietin expression slowly and continuously through a novel Gq/11-ROCK-ERK1/2 signaling pathway in mouse keratinocytes.

Thymic stromal lymphopoietin (TSLP), a master switch of allergic inflammation, plays an important role in the pathogenesis of allergic diseases. Although many compounds upregulate TSLP expression in vivo or in vitro, most of them are pollutants or toxicants. In the previous study, for the first time, we found that a steroid alkaloid derivative 02F04, which has a unique skeletal structure compared with other TSLP-inducing chemicals, significantly induced TSLP production in mouse keratinocytes. However, it is not investigated thoroughly that how 02F04 produces TSLP and why. In this study, we did a detailed investigation on the inducible effect and underlying molecular mechanism of 02F04 on TSLP production. We found that the peak time of TSLP mRNA level induced by 02F04 at 48 h led to a slow and continuous TSLP production in PAM212 cells. Besides, 02F04-induced TSLP production was significantly suppressed by inhibitors of Rho-associated protein kinase (ROCK), guanine nucleotide-binding protein subunit alpha q/11 (Gq/11) and extracellular signal-regulated kinase 1/2 (ERK1/2) at not only protein but also mRNA levels, and by siRNA-mediated knockdown of Gq or G11. This suggested that ROCK, Gq/11 and ERK1/2 signaling pathways were involved in 02F04-induced TSLP production. Increase in the level of p-ERK1/2 induced by 02F04 was suppressed by both inhibitors of ROCK and Gq/11, indicating that ROCK and Gq/11 molecules were located at the upstream of ERK1/2 to regulate 02F04-induced TSLP production. Gq/11 was located at the upstream of ROCK because the specific Gq/11 inhibitor of YM-254890 significantly reduced 02F04-induced actin stress fiber formation. Taken together, 02F04 upregulates a slow and continuous TSLP production through a novel Gq/11-ROCK-ERK1/2 signaling pathway. The thorough understanding the effect and mechanism of 02F04 on TSLP production is expected to supply it as a novel TSLP-regulating compound and a potential new tool for investigating the role of TSLP in allergic disorders.

Epimagnolin A, a tetrahydrofurofuranoid lignan from Magnolia fargesii, reverses ABCB1-mediated drug resistance.

BACKGROUND: Epimagnolin A is an ingredient of the Chinese crude drug Shin-i, derived from the dried flower buds of Magnolia fargesii and Magnolia flos, which has been traditionally used for the treatment of allergic rhinitis and nasal congestion, empyema, and sinusitis. The pharmacokinetic activity of epimagnolin A remains to be evaluated. PURPOSE: In this study, we examined the possible interactions of epimagnolin A with human ATP-binding cassette (ABC) transporter ABCB1, a membrane protein vital in regulating the pharmacokinetics of drugs and xenobiotics. STUDY DESIGN/METHODS: The interaction of epimagnolin A with ABCB1 was evaluated in calcein, ATPase, and MTT assays by using Flp-In-293/ABCB1 cells and purified ABCB1 and simulated in molecular docking studies. RESULTS: Epimagnolin A inhibited calcein export by Flp-In-293/ABCB1 cells in a concentration-dependent manner in a calcein assay. ATPase assay revealed a concentration-dependent stimulation of the ATPase activity of ABCB1 by epimagnolin A. Epimagnolin A also showed saturation kinetics in the relationship between the compound-stimulated ATPase activity and the compound concentration, suggesting Michaelis-Menten kinetics similar to those of the control drug, verapamil. Km and Vmax values were calculated from Hanes-Woolf plots of (compound concentration) × (compound-stimulated ATPase activity)-1 vs. (compound concentration); the Km of epimagnolin and verapamil was 42.9 ±â€¯7.53  µM and 12.3 ±â€¯4.79  µM, respectively, and the corresponding Vmax values were 156 ±â€¯15.0  µM and 109 ±â€¯3.18  µM. Molecular docking studies on human ABCB1 showed that epimagnolin A docked to the same binding pocket as verapamil, and 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays showed that the sensitivities of Flp-In-293/ABCB1 cells against anti-cancer drugs were enhanced upon exposure to 10  µM epimagnolin A. CONCLUSION: These results strongly suggest that epimagnolin A affects the transport activity of ABCB1 as a substrate.

Harmine promotes molar root development via SMAD1/5/8 phosphorylation.

Tooth and bone are major tissues involved in physiological calcification in the body, and they use similar molecular pathways for development, homeostasis, and regeneration. Harmine (HMN) is a natural small compound that stimulates osteoblast differentiation in vitro and in vivo. Here we examined the biological effect of HMN on the postnatal development of molar tooth roots and periodontal tissues. HMN supported the formation of tooth roots and periodontal tissues in developing tooth germs. In tooth germ organ culture, HMN promoted the elongation of Hertwig's epithelial root sheath (HERS) and stimulated cell proliferation in HERS and dental follicle-derived tissues, including dental papillae and dental follicles. HMN stimulated cell proliferation and cell movement of HERS-derived cells without mesenchymal cells in vitro and directly induced the phosphorylation of SMAD1/5/8 protein in HERS-derived cells. Our results indicated that HMN was the first natural small compound to stimulate postnatal development of tooth germs.

Induction of thymic stromal lymphopoietin by a steroid alkaloid derivative in mouse keratinocytes.

Thymic stromal lymphopoietin (TSLP) plays critical roles in inducing and exacerbating allergic diseases. Chemical compounds that induce TSLP production can enhance sensitization to antigens and exacerbate allergic inflammation. Hence, identifying such chemicals will be important to prevent an increase in allergic diseases. In the present study, we found, for the first time, that a steroid alkaloid derivative, code no. 02F04, concentration and time dependently induced mRNA expression and production of TSLP in a mouse keratinocyte cell line, PAM212. In particular, the activity of 02F04 was selective to TSLP. As an analogue of the liver X receptor (LXR) endogenous ligand, 02F04 rapidly increased ATP-binding cassette transporter A1 (ABCA1) expression by regulating the nuclear receptor of LXR. However, instead of being inhibited by the LXR antagonist, 02F04-induced TSLP production was delayed and markedly suppressed by inhibitors of phospholipase C (PLC), pan-protein kinase C (PKC), PKCδ, Rho-associated protein kinase (ROCK), extracellular signal-regulated kinase (ERK) 1/2, and IκΒ kinase 2 (IKK2). Treatment with 02F04 caused the formation of F-actin filaments surrounding the nucleus of PAM212 cells, which then disappeared following addition of ROCK inhibitor. 02F04 also induced phosphorylation of ERK1/2 from 2h after treatment, with a maximum at 24h, and increased nuclear factor-κB (NF-κB) promoter activity by 1.3-fold. Taken together, these results indicate that 02F04-induced TSLP production is regulated via distinct signal transduction pathways, including PLC, PKC, ROCK, ERK1/2, and NF-κB but not nuclear receptors. 02F04, with a unique skeletal structure in inducing TSLP production, can represent a potential new tool for investigating the role of TSLP in allergic diseases.

Evaluation of raw nepodin extraction from Rumex japonicus and R. obtusifolius and their DNA polymorphisms.

Nepodin, found in the roots of Rumex japonicus Houtt. (Polygonaceae), inhibits osteoclast differentiation and has an antidiabetic effect. We propose nepodin as an ingredient of new functional foods or as a drug candidate for reducing the risk of reduced locomotion resulting from diseases such as osteoporosis. Although there are no previous reports of R. obtusifolius L., which is found throughout Japan, having roots containing nepodin, we found nepodin in the roots of this species. Therefore, R. obtusifolius as well as R. japonicus was considered a candidate raw material for nepodin extraction. We also discuss the suitability of R. japonicus and R. obtusifolius as sources of raw nepodin for cultivation on the Ryukyu Islands. In this study, all specimens on the Ryukyu Islands were identified as R. japonicus. Conversely, all specimens on mainland Japan were R. obtusifolius. The DNA sequence of the chloroplast trnL-trnF intergenic spacer region and partial nuclear internal transcribed spacer was consistent with the identification of R. japonicus and R. obtusifolius by morphological characteristics of the perianth segments. Therefore, to avoid erroneous identification and misuse of the plant species used for extraction of raw materials, it is preferable to develop DNA markers for these two regions. The content of nepodin varied from undetectable to 0.34% of the fresh weight (%FW) in R. japonicus and from undetectable to 0.21%FW in R. obtusifolius. From a pharmacological perspective, as plants that might be suitable as raw materials for nepodin extraction, it became clear that both R. japonicus and R. obtusifolius can be used with the same expected extraction efficiency. Based on our findings, R. obtusifolius could not be confirmed as inhabiting the Ryukyu Islands. For this reason, to conserve the endemic genetic characteristics of the Ryukyu Islands and to prevent genetic pollution by R. obtusifolius, only R. japonicus should be cultivated on the Ryukyu Islands.

Acerogenin C from Acer nikoense exhibits a neuroprotective effect in mouse hippocampal HT22 cell lines through the upregulation of Nrf-2/HO-1 signaling pathways.

Oxidative stress contributes to neuronal death in the brain, and neuronal death can cause aging or neurodegenerative disease. Heme oxygenase 1 (HO-1) serves a vital role in the regulation of biological reactions, including oxidative stress associated with reactive oxygen species. In the present study, acerogenin C isolated from the Aceraceae plant Acer nikoense, which is used as a Japanese folk medicine for hepatic disorders and eye diseases. However, there have been no studies on the mechanisms underlying the antineurodegenerative biological activities of acerogenin C. In the present study, acerogenin C demonstrated neuroprotective action against glutamate­induced cell death in hippocampal HT22 cells through the upregulation of HO­1 expression. These effects were also associated with nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the activation of phosphoinositide 3­kinase/protein kinase B. Taken together of the efficacy researches, this study determines that the Nrf2/HO­1 pathways denotes a biological mark and that acerogenin C might contribute to prevention of neurodegenerative disorders.

Long-term dietary supplementation with low-dose nobiletin ameliorates hepatic steatosis, insulin resistance, and inflammation without altering fat mass in diet-induced obesity.

SCOPE: We evaluated the long-term effect of low-dose nobiletin (NOB), a polymethoxylated flavone, on diet-induced obesity and related metabolic disturbances. METHODS AND RESULTS: C57BL/6J mice were fed a high-fat diet (HFD, 45 kcal% fat) with or without NOB (0.02%, w/w) for 16 weeks. NOB did not alter food intake or body weight. Despite increases in fatty acid oxidation-related genes expression and enzymes activity in adipose tissue, NOB did not affect adipose tissue weight due to simultaneous increases in lipogenic genes expression and fatty acid synthase activity. However, NOB significantly decreased not only pro-inflammatory genes expression in adipose tissue but also proinflammatory cytokine levels in plasma. NOB-supplemented mice also showed improved glucose tolerance and insulin resistance, along with decreased levels of plasma insulin, free fatty acids, total cholesterol, non-HDL-cholesterol, and apolipoprotein B. In addition, NOB caused significant decreases in hepatic lipid droplet accumulation and triglyceride content by activating hepatic fatty acid oxidation-related enzymes. Hepatic proinflammatory TNF-α mRNA expression, collagen accumulation, and plasma levels of aminotransferases, liver damage indicators, were also significantly lower in NOB-supplemented mice. CONCLUSION: These findings suggest that long-term supplementation with low-dose NOB can protect against HFD-induced inflammation, insulin resistance, dyslipidemia, and nonalcoholic fatty liver disease, without ameliorating adiposity.

Acerogenin A from Acer nikoense Maxim Prevents Oxidative Stress-Induced Neuronal Cell Death through Nrf2-Mediated Heme Oxygenase-1 Expression in Mouse Hippocampal HT22 Cell Line.

Oxidative cell damage contributes to neuronal degeneration in many central nervous system (CNS) diseases such as Parkinson's disease, Alzheimer's disease, and ischemia. Inducible heme oxygenase (HO)-1 acts against oxidants that are thought to play a key role in the pathogenesis of neuronal diseases. The stem bark of Acer nikoense Maxim (Aceraceae) is indigenous to Japan; it has been used in folk medicine as a treatment of hepatic disorders and eye diseases. Acerogenin A, a natural compound isolated from Japanese folk medicine A. nikoense, showed neuroprotective effects and reactive oxygen species (ROS) reduction on glutamate-induced neurotoxicity by inducing the expression of HO-1 in mouse hippocampal HT22 cells. Furthermore, acerogenin A caused the nuclear accumulation of nuclear factor-E2-related factor 2 (Nrf2) and the activation of the PI3K/AKT signaling pathways. In this study, we demonstrated that acerogenin A effectively prevents glutamate-induced oxidative damage, and HO-1 induction via PI3K/Akt and Nrf2 pathways appears to play a key role in the protection of HT22 cells. Therefore, this study implies that the Nrf2/HO-1 pathway represents a biological target and that acerogenin A might be a candidate for the prevention of neurodegeneration.

Hyperpigmentation mechanism of methyl 3,5-di-caffeoylquinate through activation of p38 and MITF induction of tyrosinase.

Methyl 3,5-di-caffeoylquinate (3,5-diCQM) has been used for the treatment of various diseases in oriental medicine, but its effect on melanogenesis has not been reported yet. In this study, the molecular mechanism of 3,5-diCQM-induced melanogenesis was investigated. It was found that 3,5-diCQM induced synthesis of melanin pigments in murine B16F10 melanoma cells in a concentration-dependent manner. Treatment of cells with 3,5-diCQM for 48 h increased extracellular and intracellular melanin production and tyrosinase activity. The expressions of tyrosinase, tyrosinase-related protein 1 (TRP1), and TRP2 were up-regulated in a dose-dependent manner 48 h after 3,5-diCQM treatment. Western blot analysis showed that 3,5-diCQM increased the phosphorylation of p38 mitogen-activated protein kinase and cAMP responsive element binding as well as the expression of microphthalmia-associated transcription factor. In addition, 3,5-diCQM-stimulated cAMP production, and 3,5-diCQM-induced tyrosinase activity and melanin synthesis were attenuated by H89, a protein kinase A inhibitor. These results suggested that 3,5-diCQM-mediated activation of the p38 pathway may represent a novel approach for an effective therapy for vitiligo and hair graying.

Melanogenesis-inducing effect of cirsimaritin through increases in microphthalmia-associated transcription factor and tyrosinase expression.

The melanin-inducing properties of cirsimaritin were investigated in murine B16F10 cells. Cirsimaritin is an active flavone with methoxy groups, which is isolated from the branches of Lithocarpus dealbatus. Tyrosinase activity and melanin content in murine B16F10 melanoma cells were increased by cirsimaritin in a dose-dependent manner. Western blot analysis revealed that tyrosinase, tyrosinase-related protein (TRP) 1, TRP2 protein levels were enhanced after treatment with cirsimaritin for 48 h. Cirsimaritin also upregulated the expression of microphthalmia-associated transcription factor (MITF) after 24 h of treatment. Furthermore, cirsimaritin induced phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) in a dose-dependent manner after treatment for 15 min. The cirsimaritin-mediated increase of tyrosinase activity was significantly attenuated by H89, a cAMP-dependent protein kinase A inhibitor. These findings indicate that cirsimaritin stimulates melanogenesis in B16F10 cells by activation of CREB as well as upregulation of MITF and tyrosinase expression, which was activated by cAMP signaling. Finally, the melanogenic effect of cirsimaritin was confirmed in human epidermal melanocytes. These results support the putative application of cirsimaritin in ultraviolet photoprotection and hair coloration treatments.