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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.

Background Nobiletin exerts beneficial effects on cognitive function in various animal models of Alzheimer's disease. The present study aimed to investigate the benefits and safety of a combination food of nobiletin‐rich extract from C. depressa peel for healthy elderly subjects. Methods The nobiletin‐containing test food (Nobilex®) comprised high‐purity nobiletin powder combined with dried root powder of K. parviflora and dried lead powder of P. japonicum and was administered to elderly Japanese subjects once a day for 16 weeks. The Japanese version of the Wechsler Memory Scaled‐Revised (WMS‐R) was used as a primary evaluation item for the assessment of global memory. Data from a protocol‐matched population (Per Protocol Set: PPS) (n = 108) were analyzed. Results The scores of “general memory” or “visual memory” in the indices of WMS‐R were significantly higher in the nobiletin‐containing test food group than in the placebo group. The difference in the total WMS‐R score was significantly higher in the test‐food group (9.0 ± 7.20) than in the placebo group (5.9 ± 7.70). An age‐stratified analysis of the WMS‐R test showed similar changes in subjects aged ≦74 years to those in the overall subject population. In the stratified analysis involving subjects with an MMSE‐J score of between 24 and 28, the “figural memory” subscale score in WMS‐R was significantly higher in the test food group than in the placebo group. Conclusion The present results indicate that the nobiletin‐containing test food is beneficial for improving memory dysfunction in healthy elderly subjects., Nobiletin exerts beneficial effects on cognitive function in various animal models of Alzheimer's disease. The present results indicate that the nobiletin‐containing test food is beneficial for improving memory dysfunction in healthy elderly subjects.

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.

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-1β 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-1β signaling and urothelial Cx43 expression.

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.

Alcohol consumption causes the accumulation of reactive oxygen species in liver, which leads to alcoholic fatty liver and hepatocyte injury. In this study, we investigated the effects of an ethanolic Citrus depressa extract and those of its main components on alcohol-induced liver damage using a mouse model. Four polymethoxylated flavonoids, namely, nobiletin, tangeretin, 5-O-demethylnobiletin, and sinensetin, were isolated from C. depressa extract. Treatment of ethanol fed mice with C. depressa extract, nobiletin, tangeretin, or 5-O-demethylnobiletin at 300 mg/kg for 8 weeks by oral administration alleviated the accumulation of lipid droplets in liver and significantly decreased the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (markers of liver damage). Also, in mice treated with ethanol plus nobiletin, tangeretin, or 5-O-demethylnobiletin, liver level of glutathione (an antioxidant) increased whereas levels of tumor necrosis factor-alpha (TNF-α), hepatic malondialdehyde, and hepatic cytochrome P450 2E1 (CYP2E1) mRNA decreased as compared with ethanol fed controls. These findings suggest C. depressa extract and polymethoxylated flavonoids had a protective effect on alcohol-induced liver injury, and that the mechanism involved is related to the regulation of hepatic CYP2E1-mediated oxidative stress.

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.

Acinetobacter baumannii is a nosocomial pathogen that can survive unfavorable conditions, such as, desiccation, nutrient starvation, and antimicrobial treatment, and this is primarily due to its ability to form biofilms on biotic and abiotic surfaces like tissues and medical devices. In this study, honokiol and magnolol were investigated for antibiofilm activity against A. baumannii ATCC 17978. Both were found to inhibit biofilm formation dose-dependently and to disperse matured biofilms. Honokiol and magnolol were found to inhibit biofilm formation by five and four of eight additional clinical A. baumannii isolates, respectively. Furthermore, honokiol and magnolol effectively suppressed pellicle formation and the surface motilities of the A. baumannii and prolonged the survival of infected nematode Caenorhabditis elegans. These results demonstrate that honokiol and magnolol may be useful for controlling A. baumannii infections.

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.

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.

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 glutamate‑induced cell death in hippocampal HT22 cells through the upregulation of HO‑1 expression. These effects were also associated with nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the activation of phosphoinositide 3‑kinase/protein kinase B. Taken together of the efficacy researches, this study determines that the Nrf2/HO‑1 pathways denotes a biological mark and that acerogenin C might contribute to prevention of neurodegenerative disorders.

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.

Obesity is a continuous chronic metabolic disorder and has adverse effects on health, such as dyslipidemia, hyperglycemia, and insulin resistance. This study evaluated whether Rumex japonicus Houtt. ethanol extract (RU) and its active component nepodin (NE) affect obesity and its related complications, such as dyslipidemia and hyperglycemia in high-fat diet (HFD)-fed obese mice. The NE and RU supplements did not produce significant differences in body weight and adipose tissue weight compared to HFD group, while plasma lipid profiles were only improved by the RU supplement. However, both the NE and RU supplement had beneficial effects on glucose homeostasis and insulin resistance through reduction of plasma free fatty acid (FFA) (p &lt, 0.05), insulin (p &lt, 0.05), homeostatic model assessment for insulin resistance (p &lt, 0.05), and C-peptide (p &lt, 0.05) levels. In particular, the RU supplement decreased the area under the curve (AUC) of intraperitoneal glucose tolerance test (IPGTT) (p &lt, 0.05), and improved glucose intolerance. Taken together, NE and RU supplements can contribute to improvements in HFD-induced hyperglycemia, while dyslipidemia was only improved by the RU supplement.

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.

Honokiol and magnolol are neolignans contained in the Chinese medicinal herb Magnolia officinalis that exert anti-tumor and anti-inflammatory effects. Both compounds have been reported to enhance glucose uptake. Little is known about effects when used in combination. The effects of honokiol, magnolol, and a combination of both compounds on lipid and glucose metabolism in highfat diet-induced obese mice were investigated, and underlying mechanisms were examined. All 3 treatments significantly (p

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.

cope Bone homeostasis is ensured by the balance between bone formation and resorption. Thus, control of the recruitment, proliferation, and differentiation of bone cells is essential to maintain bone mass. The aim of this study was to elucidate the effects of rosmarinic acid as a potential therapeutic agent on bone metabolism using bone cells and a mouse model. Methods and results Rosmarinic acid increased alkaline phosphatase activity and induced mineralization in osteoblasts. Addition of rosmarinic acid to cultures of calvarial osteoblastic cells prepared from T-cell factor/β-catenin TOP-GAL mutant mice strongly induced the expression of LacZ and promoted stabilization of β-catenin in the cytoplasm of ST2 cells, suggesting that rosmarinic acid affects the canonical Wnt signaling pathway. Moreover, rosmarinic acid inhibited not only osteoclast formation in cocultures of mouse bone marrow cells and osteoblasts, but also receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation in bone marrow-derived macrophages. RANKL-induced p38 mitogen-activated protein kinase and the expression of nuclear factor of activated T cell, c-Jun, and c-Fos were inhibited by rosmarinic acid in bone marrow macrophages. Finally, we confirmed that rosmarinic acid improved bone mass in a soluble RANKL-induced bone loss mouse model. Conclusion Rosmarinic acid has dual regulatory effects on bone metabolism and may control the bone functions by controlling osteoblastic and osteoclastic differentiation.

Platelet-derived growth factor (PDGF)-BB is one of the most potent factors in the development and progression of various vascular disorders, such as atherosclerosis and restenosis. PDGF is a major stimulant for vascular smooth muscle cells (VSMCs) proliferation via the mitogenesis signaling pathway. In the present study, we investigated the effect of acerogenin C, a macrocyclicdiarylhep-tanoid, on PDGF-BB-stimulated human aortic smooth muscle cells (HASMCs) proliferation. Acer-ogenin C significantly inhibited PDGF (20 ng/mL)-BB-induced [3H]-thymidine incorporation into DNA at concentrations of 0.1, 1 and 10 μM without any cytotoxicity. Acerogenin C also blocked PDGF-BB-stimulated phosphorylation of PLCγ1 and Akt but had no effect on extracellular signal-regulated kinase 1/2 (ERK1/2) and PDGF beta-receptor (Rβ) activation. In addition, acerogenin C (0.1 - 10 μM) induced cell-cycle arrest in the G1 phase, which was associated with the down-regulation of cyclins and the up-regulation of p27kip1. These results suggest that acerogenin C blocks PDGF-BB-stimulated HASMCs proliferation via G0/G1 arrest in association with the induction of p27kip1 and the suppression of PLCγ1 and phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathways. Furthermore, acerogenin C may be used for prevention and treatment of atherosclerosis during restenosis after coronary angioplasty.

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 ± 7.53 μM and 12.3 ± 4.79 μM, respectively, and the corresponding Vmax values were 156 ± 15.0 μM and 109 ± 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.

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.

Many active components derived from edible natural resources such as plant extracts have recently attracted attention for their potential use as functional foods or drugs for preventing and treating metabolic diseases such as diabetes. To obtain a novel modulator of glucose metabolism, we conducted screening of a small compound library in cultured L6 myotubes. We identified nepodin that stimulated glucose uptake dose-dependently in differentiated L6 myotubes. The stimulatory effect of nepodin on glucose uptake was abrogated by a 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibitor. In addition, nepodin stimulated the phosphorylation of AMPK. Nepodin also stimulated the translocation of GLUT4 to the plasma membrane in L6 myoblasts transfected with a Glut4 cDNA-coding vector and in differentiated L6 myotubes. In in vivo study, nepodin suppressed the increases in fasting blood glucose levels and improved the glucose intolerance of C57BL/KsJ-db/db mice, a type 2 diabetic animal model. Nepodin rescued the impaired phosphorylation of AMPK in the skeletal muscle of db/db mice. These results suggest that nepodin has an antidiabetic effect, which is at least partly mediated by stimulation of GLUT4 translocation via AMPK activation by nepodin.

Obesity and osteoporosis are associated with estrogen deficiency following menopause. Therefore, it is important to prevent and treat both disorders to maintain a healthy life in postmenopausal women. Nobiletin, a polymethoxylated flavone, exhibits various pharmacologic effects, including anti-tumor and anti-inflammatory activities. Therefore, in this study, we examined the effects of nobiletin on obesity, obesity-related metabolic disorders, and bone mass in ovariectomized (OVX) mice. Mice were divided into four groups and underwent sham operation or OVX. OVX mice were treated with 50 or 100 mg/kg nobiletin, or received vehicle alone (0.3% carboxyl methyl cellulose/0.5% dimethyl sulfoxide). Nobiletin decreased body weight gain and white adipose tissue weight in OVX mice. Nobiletin also decreased triglyceride levels, and tended to reduce plasma total cholesterol and glucose levels. Additionally, nobiletin prevented the reduction in bone mineral density of the trabecular region of the femur in OVX mice. Taken together, our results suggest that nobiletin improves adiposity, dyslipidemia, hyperglycemia, and prevents bone loss in OVX mice. Therefore, nobiletin is expected to have beneficial effects for the prevention and improvement of metabolic disorders and osteoporosis in postmenopausal women.

Nobiletin and tangeretin are polymethoxy flavonoids that are abundantly present in the pericarp of Citrus unshiu (family Rutaceae) and the fruit of Citrus depressa (family Rutaceae). They exhibit various biological activities, including anti-inflammatory and anti-asthmatic effects. To evaluate the anti-allergic effects of nobiletin and tangeretin, we measured their inhibitory effects in histamine- or compound 48/80-induced scratching behavioral mice. Nobiletin and tangeretin potently inhibited scratching behavior, as well as histamine-induced vascular permeability. Furthermore, they inhibited the expression of the allergic cytokines, IL-4 and TNF-α as well as the activation of their transcription factors NF-κB, AP-1 and p38 in histamine-stimulated skin tissues. They also inhibited the expression of IL-4 and TNF-α and the activation of NF-κB and c-jun in PMA-stimulated RBL-2H3 cells. Furthermore, nobiletin and tangeretin inhibited protein kinase C (PKC) activity and the IgE-induced degranulation of RBL-2H3 cells. These agents showed potent anti-histamine effect through the Magnus test when guinea pig ileum was used. Based on these results, nobiletin and tangeretin may ameliorate scratching behavioral reactions by inhibiting the action of histamine as well as the activation of the transcription factors NF-κB and AP-1 via PKC.

cope This study investigated the effect of honokiol (HON) and magnolol (MAG), phenolic compounds in Magnolia plants, on adiposity and adiposity-related metabolic disturbances in mice fed high-fat diet (HFD), and the potential underlying mechanisms focusing on the lipid metabolism and inflammatory response. Method and results C57BL/6J mice were fed HFD (45 kcal% fat) with or without HON (0.02%, w/w) or MAG (0.02%, w/w) for 16 wk. Despite no changes in body weight, food intake, and hepatic fat accumulation, HON and MAG significantly lowered the weight of white adipose tissue (WAT) as well as adipocyte size and protected against insulin resistance induced by HFD. These effects were associated with increases in energy expenditure and adipose fatty acid oxidation and decreases in fatty acid synthase activity and expression of genes related to fatty acid synthesis, desaturation, and uptake, as well as adipocyte differentiation in WAT. Moreover, HON and MAG significantly lowered the expression of proinflammatory genes in WAT and elevated the plasma IL-10 level. Particularly, HON significantly decreased the plasma resistin level and increased the plasma adiponectin level compared to the control group. Conclusion HON and MAG have potential as novel agents for amelioration of adiposity and associated insulin resistance and inflammation.

Liquorice is one of the botanicals used frequently as a traditional medicine in the West and in the East. Platelet-derived growth factor (PDGF)-BB is involved in the development of CVD by inducing abnormal proliferation and migration of vascular smooth muscle cells. In our preliminary study, dehydroglyasperin C (DGC), an active compound of liquorice, showed strong antioxidant activity. Since phytochemicals with antioxidant activities showed beneficial effects on chronic inflammatory diseases, the present study aimed to investigate the effects of DGC on PDGF-induced proliferation and migration of human aortic smooth muscle cells (HASMC). Treatment of HASMC with DGC for 24 h significantly decreased PDGF-induced cell number and DNA synthesis in a dose-dependent manner without any cytotoxicity, as demonstrated by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide test and thymidine incorporation. Upon cell cycle analysis, DGC blocked the PDGF-induced progression through the G0/G1 to S phase of the cell cycle, and down-regulated the expression of cyclin-dependent kinase (CDK); 2, cyclin E, CDK4 and cyclin D1. Furthermore, DGC significantly attenuated PDGF-stimulated phosphorylation of PDGF receptor-β, phospholipase C-γ1, AKT and extracellular-regulated kinase 1/2, and DGC inhibited cell migration and the dissociation of actin filaments by PDGF. In a rat vascular balloon injury model, DGC suppressed an excessive reduction in luminal diameters and neointimal formation compared with the control group. These results demonstrate the mechanistic basis for the prevention of CVD and the potential therapeutic properties of DGC.

Leaves develop as flat lateral organs from the indeterminate shoot apical meristem. The establishment of polarity along three-dimensional axes, proximal–distal, medial–lateral, and adaxial–abaxial axes, is crucial for the growth of normal leaves. The mutations of ASYMMETRIC LEAVES1 (AS1) and AS2 of Arabidopsis thaliana cause defects in repression of the indeterminate state and the establishment of axis formation in leaves. Although many mutations have been identified that enhance the adaxial–abaxial polarity defects of as1 and as2 mutants, the roles of the causative genes in leaf development are still unknown. In this study, we found that wild-type plants treated with berberine produced pointed leaves, which are often observed in the single mutants that enhance phenotypes of as1 and as2 mutants. The berberine-treated as1 and as2 mutants formed abaxialized filamentous leaves. Berberine, an isoquinoline alkaloid compound naturally produced in various plant sources, has a growth inhibitory effect on plants that do not produce berberine. We further showed that transcript levels of meristem-specific class 1 KNOX homeobox genes and abaxial determinant genes were increased in berberine-treated as1 and as2. Berberine treated plants carrying double mutations of AS2 and the large subunit ribosomal protein gene RPL5B showed more severe defects in polarity than did the as2 single mutant plants. We suggest that berberine inhibits (a) factor(s) that might be required for leaf adaxial cell differentiation through a pathway independent of AS1 and AS2. Multiple pathways might play important roles in the formation of flat symmetric leaves. Electronic supplementary material The online version of this article (doi:10.1007/s11103-012-9929-7) contains supplementary material, which is available to authorized users.

Genistein, an isoflavone, is known to possess diverse biological functions such as antioxidative and anti-inflammatory actions. It also acts like estrogen and inhibits several tyrosine kinases. Genistein was reported to suppress insulin-mediated glucose uptake in adipocytes. In this study, we investigated the effects of genistein on glucose uptake in vitro and in vivo as well as the mechanisms associated with the glucose uptake. We found that genistein decreased nonfasting blood glucose levels in KK-Ay/Ta Jcl mice, a type 2 diabetic animal model. It also dose-dependently induced insulin secretion by Rin-5F cells. In L6 myotubes, it directly stimulated glucose uptake independently of insulin under normal and high glucose conditions in dose-dependent manners. It promoted the translocation of glucose transporter 4 to the cell membrane under both glucose conditions. Based on studies using inhibitors of signaling molecules related to glucose uptake, the stimulatory effect of genistein on glucose uptake appeared to be dependent on the phosphatidylinositol 3-kinase, mammalian target of rapamycin, protein kinase C and 5'-adenosine-monophosphate-activated protein kinase pathway under both glucose conditions. In addition, O-GlcNAcylation by O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate, an inhibitor of N-acetylglucosaminidase, reduced the stimulatory effect of genistein on glucose uptake under both glucose conditions. Taken together, genistein may regulate glucose uptake by increasing the phosphorylation and decreasing the O-GlcNAcylation of proteins related to glucose homeostasis.

The dried flower buds of Magnolia sp. are widely used as herbal medicines because of their anti-inflammatory, anti-malarial and anti-platelet activities. Here, we found that veraguensin and galgravin, lignan compounds derived from Magnolia sp., dose-dependently inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblastic cells. These compounds also inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells and bone marrow macrophages. In the RANKL-induced signaling pathway, veraguensin and galgravin reduced p38 phosphorylation and suppressed the expression of c-Fos, a key transcription factor for osteoclastogenesis. Veraguensin and galgravin also inhibited osteoclastic pit formation, which was accompanied by decreased mature osteoclast viability. In conclusion, these results indicate that veraguensin and galgravin can inhibit bone resorption and may offer novel compounds for the development of drugs to treat bone-destructive diseases such as osteoporosis.

We used a ProteoChip coated with a calixcrown derivative protein linker to measure adiponectin and monocyte chemoattractant protein-1 (MCP-1) levels and compared the results with commercial enzyme-linked immunosorbent assay (ELISA) kits. Adiponectin and MCP-1 levels in normal human serum and RAW264 cell supernatants, respectively, were measured. The ProteoChip quantification results correlated with those from the ELISA kits; however, the ProteoChip required less sample volume, exhibited higher sensitivity, and had a wider detection range. The ProteoChip was capable of detecting and quantifying small amounts of protein, possibly replacing ELISA kits in evaluating the levels of adiponectin and MCP-1.

The mass and function of bones depend on the maintenance of a complicated balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. An inhibitor of osteoclast differentiation and/or function is expected to be useful for treatment of bone lytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis into bone. Biselyngbyaside is a recently isolated macrolide compound from marine cyanobacteria Lyngbya sp. that shows wide-spectrum cytotoxicity toward human tumor cell lines. In this study, we investigated the effects of biselyngbyaside on osteoclast differentiation and function. Biselyngbyaside inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in mouse monocytic RAW264 cells and primary bone marrow-derived macrophages at a low concentration. Similarly, biselyngbyaside suppressed osteoblastic cell-mediated osteoclast differentiation in cocultures. In the RANKL-induced signaling pathway, biselyngbyaside inhibited the expression of c-Fos and NFATc1, which are important transcription factors in osteoclast differentiation. In mature osteoclasts, biselyngbyaside decreased resorption-pit formation. Biselyngbyaside also induced apoptosis accompanied by the induction of caspase-3 activation and nuclear condensation, and these effects were negated by the pancaspase inhibitor z-VAD-FMK. Taken together, the present findings indicate that biselyngbyaside suppresses bone resorption via inhibition of osteoclastogenesis and induction of apoptosis. Thus, biselyngbyaside may be useful for the prevention of bone lytic diseases.

Oral administration of Terminalia catappa extract (TCE; 1,000 mg/kg) for 5 wk suppressed bone weight loss and trabecular bone loss in ovariectomized mice. An in vitro experiment showed that TCE (1.3-20 µg/mL) did not increase alkaline phosphatase activity, which would indicate osteoclast formation, in osteoblast-like 3T3-L1 cells. On the other hand, TCE (12.5 µg/mL) markedly decreased the number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells, which would indicate osteoclast formation, in a co-culture system (bone marrow cells/osteoblastic UAMS-32 cells). A detailed analysis of the stages of osteoclast differentiation revealed that TCE mainly suppressed the differentiation of bone marrow mononuclear cells into osteoclast progenitor cells in the presence of M-CSF and TGF-β. An additional experiment using fractionated TCE revealed that the water-soluble fraction suppressed the bone weight loss in OVX-mice and osteoclast differentiation in vitro. Therefore, the suppressive effects of TCE on bone weight loss in mice might be due to the suppressive effects of highly polar components on the early stage of osteoclast differentiation.

1. Magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl) is a major phenolic compound purified from Magnolia officinalis. The aim of the present study was to elucidate the effects of magnolol on vascular contractions. 2. Rat aortic rings were mounted in organ baths. Magnolol was added cumulatively (0.3-30 μmol/L) to elicit relaxation in endothelium-intact and -denuded rat aortic rings precontracted with U46619 (30 nmol/L, 20 min), NaF (8.0 mmol/L, 40 min), phenylephrine (1.0 or 0.1 μmol/L, 15 min) or phorbol-12,13-dibutyrate (PDBu, 0.3 or 0.1 μmol/L, 40 min). In separate experiments, cumulative concentrationresponse curves were obtained for NaF (2.0-12 mmol/L), U46619 (1.0 nmol/L1.0 μmol/L) or PDBu (1.0 nmol/L1.0 μmol/L) after pretreatment with either magnolol or vehicle for 30 min in endothelium-denuded aortic rings. After completion of the functional study, we measured the amount of guanosine triphosphate (GTP) RhoA by using a G-LISA RhoA Activation Assay, as well as the phosphorylation of 20 kDa myosin light chain (MLC₂₀), myosin phosphatase-targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI-17) by immunobloting. 3. Magnolol (0.3-30 μmol/L) reduced vascular tension induced by the thromboxane A₂ agonist U46619 (30 nmol/L), sodium fluoride (NaF) (8.0 mmol/L) and the α₁ -adrenoceptor agonist phenylephrine (1.0 or 0.1 μmol/L) in both endothelium-intact and -denuded rings. The magnitude of the relaxation effects of magnolol on the contraction induced by each of the drugs were similar. The magnitude of the effect of magnolol in endothelium-intact and -denuded rings were similar. 4. Pretreatment of rat aortic rings with 1.0, 3.0 or 10 μmol/L magnolol for 30 min dose-dependently inhibited the maximum response on the concentration-response curves to NaF and U46619, but not to phorbol-12,13-dibutyrate (PDBu). 5. Magnolol (3.0 or 10 μmol/L) decreased RhoA activation, as well as the phosphorylation of MLC₂₀ , MYPT1(Thr855) and CPI-17(Thr38) induced by either 8.0 mmol/L NaF or 30 nmol/L U46619. In contrast, magnolol did not affect PDBu (0.1 μmol/L)-induced phosphorylation of CPI-17(Thr38) . 6. In conclusion, magnolol reduces vascular contraction by inhibiting the RhoA/Rho kinase pathway in endothelium-denuded rat aorta.

Objectives Honokiol is a small-molecule polyphenol isolated from the species Magnolia obovata. We hypothesized that honokiol attenuated vascular contractions through the inhibition of the RhoA/Rho-kinase signalling pathway. Methods Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. Phosphorylation of 20 kDa myosin light chains (MLC20), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase (MLCP) of 17 kDa (CPI17) were examined by immunoblot. We also measured the amount of guanosine triphosphate RhoA as a marker for RhoA activation. Key findings Pretreatment with honokiol dose-dependently inhibited the concentration–response curves in response to sodium fluoride (NaF) or thromboxane A2 agonist U46619. Honokiol decreased the phosphorylation levels of MLC20, MYPT1Thr855 and CPI17Thr38 as well as the activation of RhoA induced by 8.0 mm NaF or 30 nm U46619. Conclusions These results demonstrated that honokiol attenuated vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway.

Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a β-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related β-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and Runx2 pathways. Our findings suggest that harmine has bone anabolic effects and may be useful for the treatment of bone-decreasing diseases and bone regeneration as a lead compound.

Citrus depressa Hayata (commonly known as shiikuwasa) is cultivated in the northern areas of Okinawa, Japan, and used as a juice. In this study, we examined the anti-obesity effects and mechanism of action of shiikuwasa peel extract (SE) using high-fat diet (HFD)-induced obese mice. Mice were fed a low-fat diet (LFD), HFD or HFD containing 1% or 1.5% (w/w) SE (HFD+1 SE and HFD+1.5 SE, respectively) for 5 weeks. The body weight gain and white adipose tissue weight were significantly decreased in the HFD+1.5 SE group compared with the HFD group. The plasma triglyceride and leptin levels were also significantly reduced in the HFD+1.5 SE group compared with the HFD group. Histological examinations showed that the sizes of the adipocytes were significantly smaller in the HFD+1.5 SE group than in the HFD group. The HFD+1.5 SE group also showed significantly lower mRNA levels of lipogenesis-related genes, such as activating protein 2, stearoyl-CoA desaturase 1, acetyl-CoA-carboxylase 1, fatty acid transport protein and diacylglycerol acyltransferase 1, than the HFD group. These results suggest that the anti-obesity effects of SE may be elicited by regulating the expressions of lipogenesis-related genes in white adipose tissue.

Osteogenic activity of six diarylheptanoids, acerogenin A (1), (R)-acerogenin B (2), aceroside I (3), aceroside B(1) (4), aceroside III (5) and (-)-centrolobol (6) and two phenolic compounds; (+)-rhododendrol (7) and (+)-cathechin (8), isolated from the stem bark of Acer nikoense (Nikko maple) was evaluated using alkaline phosphatase (ALP) activity as a marker for early osteoblast differentiation. We found that the diphenyl ether-type cyclic diarylheptanoids 1-5 promoted ALP activity in mouse preosteoblastic MC3T3-E1 cells without affecting cell proliferation, but linear-type diarylheptanoid 6 and phenolic compounds 7 and 8 did not. Diphenyl ether-type cyclic diarylheptanoids 1-4 also increased protein production of osteocalcin, a late stage maker for osteoblast differentiation, and induced osteoblastic mineralization. Structure-activity relationships of these compounds demonstrated that the stimulative efficacy of aglycones was higher than that of its glycosides. Taken together, diphenyl ether-type cyclic diarylheptanoids promote early- and late-stage osteoblastogenesis, which may open the possibility for the development of novel osteogenic agents.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ plays an important role in adipocyte differentiation. Its ligands, including thiazolidinediones, improve insulin sensitivity in type 2 diabetes. We investigated the effects of artepillin C, an ingredient of Baccharis dracunculifolia, on adipogenesis and glucose uptake using 3T3-L1 cells. In PPARγ ligand-binding assays, artepillin C exhibited binding affinity toward PPARγ. Artepillin C dose-dependently enhanced adipocyte differentiation of 3T3-L1 cells. As a result of the artepillin C-induced adipocyte differentiation, the gene expression of PPARγ and its target genes, such as aP2, adiponectin and glucose transporter (GLUT) 4, was increased. These increases were abolished by cotreatment with GW9662, a PPARγ antagonist. In mature 3T3-L1 adipocytes, artepillin C significantly enhanced the basal and insulin-stimulated glucose uptake. These effects were decreased by cotreatment with a PI3K inhibitor. Although artepillin C had no effects on the insulin signaling cascade, artepillin C enhanced the expression and plasma membrane translocation of GLUT1 and GLUT4 in mature adipocytes. In conclusion, these findings suggest that artepillin C promotes adipocyte differentiation and glucose uptake in part by direct binding to PPARγ, which could be the basis of the pharmacological benefits of green propolis intake in reducing the risk of type 2 diabetes.

Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10 μM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.

N,N′ -diphenethylurea isolated from Okinawan ascidian Didemnum molle enhances adipocyte differentiation in 3T3-L1 cells

Osteoclasts, bone-resorbing multinucleated cells, are differentiated from hemopoietic progenitors of the monocyte/macrophage lineage. Bone resorption by osteoclasts is considered a potential therapeutic target to the treatment of erosive bone diseases, including osteoporosis, rheumatoid arthritis, and periodontitis. In the present study, we found that alisol-B, a phyto-steroid from Alisma orientale Juzepczuk, exhibited inhibitory effects on osteoclastogenesis both in vitro and in vivo. Although RT-PCR analysis showed that alisol-B did not affect the 1alpha,25(OH)(2)D(3)-induced expressions of RANKL, OPG and M-CSF mRNAs in osteoblasts, addition of alisol-B to co-cultures of mouse bone marrow cells and primary osteoblasts with 10(-8)M 1alpha,25(OH)(2)D(3) caused significant inhibition of osteoclastogenesis. We further examined the direct effects of alisol-B on osteoclast precursors. Alisol-B strongly inhibited RANKL-induced osteoclast formation when added during the early stage of cultures, suggesting that alisol-B acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, alisol-B inhibited the phosphorylation of JNK, which are upregulated in response to RANKL in bone marrow macrophages, alisol-B also inhibited RANKL-induced expression of NFATc1 and c-Fos, which are key transcription factors for osteoclastogenesis. In addition, alisol-B suppressed the pit-forming activity and disrupted the actin ring formation of mature osteoclasts. In a hypercalcemic mouse model induced by 2-methylene-19-nor-(20S)-1alpha,25(OH)(2)D(3) (2MD), an analog of 1alpha,25(OH)(2)D(3), administration of alisol-B significantly suppressed 2MD-induced hypercalcemia as resulting from the inhibition of osteoclastogenesis. Taken together, these findings suggest that alisol-B may be a potential novel therapeutic molecule for bone disorders by targeting the differentiation of osteoclasts as well as their functions.

The present study examined the anti-obesity effect and mechanism of action of Korean white ginseng extracts (KGE) using high-fat diet (HFD)-induced obese mice. Mice were fed a low-fat diet (LFD), HFD or HFD containing 0.8 and 1.6% (w/w) KGE diet (HFD + 0.8KGE and HFD + 1.6KGE) for 8 weeks. We also examined the effects of KGE on plasma triglyceride (TG) elevation in mice administrated with oral lipid emulsion. Body weight gain and white adipose tissue (WAT) weight were significantly decreased in the HFD + 1.6KGE group, compared with the HFD group. The plasma TG levels were also significantly reduced in both HFD + 0.8KGE and HFD + 1.6KGE groups, while leptin levels were significantly decreased in only the HFD + 1.6KGE group, compared with the HFD group. The HFD + 1.6KGE group showed significantly lower mRNA levels of lipogenesis-related genes, including peroxisome proliferator-activated receptorγ2 (PPARγ2), sterol regulatory element binding protein-1c (SREBP-1c), lipoprotein lipase (LPL), fatty acid synthase (FAS) and diacylglycerol acyltransferase 1 (DGAT1), compared with the HFD group. In addition, a dose of 1000 mg/kg KGE inhibited the elevation of plasma TG levels compared with mice given the lipid emulsion alone. These results suggest that the anti-obesity effects of KGE may be elicited by regulating expression of lipogenesis-related genes in WAT and by delaying intestinal fat absorption.

Compactin (mevastatin), which inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, and thus biosynthesis of cholesterol and the prenylation of proteins, inhibits osteoclastic bone resorption. Although it has been suggested that compactin inhibits bone resorption by inducing apoptosis of osteoclasts, the pathway by which compactin inhibits resorption has not been established. We investigated the effect of compactin on the differentiation of osteoclasts and the relationship between the morphological changes elicited by compactin and its inhibitory effect on bone resorption. Compactin inhibited the differentiation of osteoclasts, interfering with the fusion process by which prefusion osteoclasts (pOCs) develop into multinucleated osteoclast-like cells (OCLs), and also disrupted the actin ring of OCLs. The potency of compactin to inhibit fusion of pOCs and to disrupt the actin ring of OCLs corresponded to that of compactin to inhibit bone resorption. The effects of compactin were prevented by the addition of MVA lactone or its downstream products farnesylpyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) but not by squalene. Apoptosis of OCLs was not induced by the concentration of compactin that inhibited fusion of pOCs and disrupted the actin ring. The normal process of pOC fusion and the integrity of the actin ring were restored by the withdrawal of compactin from the cultures after they had been treated with compactin for 24 h, but they were not restored by the addition of zVAD-fmk, a caspase inhibitor. Compactin also reversibly inhibited interleukin-1beta (IL-1beta)-, 1alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3)-, and parathyroid hormone (PTH)-stimulated 45Ca release in bone organ cultures. Our results indicate that the inhibitory effects of compactin on bone resorption result from the inhibition of fusion of pOCs into OCLs and disruption of actin ring in OCLs and that apoptosis of OCLs is not necessary for these inhibitory effects of compactin. These effects of compactin are likely to be a consequence of the inhibition of prenylation of proteins that play an important role in the fusion of pOCs and in maintaining actin ring integrity in OCLs.