Synthesis of a Library of Terpenoid Alkaloid-Like Compounds Containing Medium-Sized Rings via Reconstruction of the Humulene Skeleton.

The construction of a chemical library based on natural products is a promising method for the synthesis of natural product-like compounds. In this study, we synthesized a terpenoid alkaloid-like compound library based on the humulene skeleton. Our strategy, which enables access to diverse ring systems such as 11-membered monocyclic, oxabicyclic, and medium-sized aza ring-containing scaffolds, involves the introduction of a nitrogen atom, an intermolecular C-O bond formation via Lewis acid-mediated epoxide-opening transannulation, and a ring-reconstruction strategy based on olefin metathesis. A cheminformatics analysis based on their structural and physicochemical properties revealed that the synthesized compounds have high three-dimensionality and high natural product likeness scores but with structural novelty. The usefulness of the terpenoid alkaloid-like compound library for drug discovery and the accessibility to structure-activity relationship studies were validated by performing an assay for osteoclast-specific tartrate-resistant acid phosphatase activity, resulting in the identification of a seed compound for bone-resorptive diseases such as osteoporosis.

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.

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.

Construction of a Meroterpenoid-Like Compounds Library Based on Diversity-Enhanced Extracts.

The structural diversity of natural products and their derivatives have long contributed to the development of new drugs. However, the difficulty in obtaining compounds bearing skeletally novel structures has recently led to a decline of pharmaceutical research into natural products. This paper reports the construction of a meroterpenoid-like library containing 25 compounds with diverse molecular scaffolds obtained from diversity-enhanced extracts. This method constitutes an approach for increasing the chemical diversity of natural-product-like compounds by combining natural product chemistry and diversity-oriented synthesis. Extensive pharmacological screening of the library revealed promising compounds for anti-osteoporotic and anti-lymphoma/leukemia drugs. This result indicates that the use of diversity-enhanced extracts is an effective methodology for producing chemical libraries for the purpose of drug discovery.

Observation and quantitative analysis of dislocations in steel using electron channeling contrast imaging method with precise control of electron beam incident direction.

Electron channeling contrast imaging (ECCI) was applied by precisely controlling the primary electron beam incident direction of the crystal plane in scanning electron microscope (SEM), and the dislocation contrast in steel materials was investigated in detail via SEM/ECCI. The dislocation contrast was observed near a channeling condition, where the incident electron beam direction of the crystal plane varied, and the backscattered electron intensity reached a local minimum. Comparing the dislocation contrasts in the visualized electron channeling contrast (ECC) images and transmission electron microscope (TEM) images, the positions of all dislocation lines were coincident. During the SEM/ECCI observation, the dislocation contrast varied depending on the incident electron beam direction of the crystal plane and accelerating voltages, and optimal conditions existed. When the diffraction condition g and the Burgers vector b of dislocation satisfied the condition g⸱b = 0, the screw dislocation contrast in the ECC image disappeared. An edge dislocation line was wider than a screw dislocation line. Thus, the SEM/ECCI method can be used for dislocation characterization and the strain field evaluation around dislocation, like the TEM method. The depth information of SEM/ECCI, where the channeling condition is strictly satisfied, can be obtained from dislocation contrast deeper than 5ξg, typically used for depth of SEM/ECCI.

Biselyngbyaside, isolated from marine cyanobacteria, inhibits osteoclastogenesis and induces apoptosis in mature osteoclasts.

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.

Detection of adiponectin and monocyte chemoattractant protein-1 using a calixcrown derivatives-coated protein chip.

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.

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action.

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling.

Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a ß-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 ß-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.

Osteogenic activity of diphenyl ether-type cyclic diarylheptanoids derived from Acer nikoense.

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.

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.

Identification of oxytetracycline as a chondrogenic compound using a cell-based screening system.

To effectively treat degenerative joint diseases including osteoarthritis (OA), small chemical compounds need to be developed that can potently induce chondrogenic differentiation without promoting terminal differentiation. For this purpose, we screened natural and synthetic compound libraries using a Col2GFP-ATDC5 system and identified oxytetracycline (Oxy) as a chondrogenic compound. Oxy induced cartilaginous matrix synthesis and mRNA expressions of chondrocyte markers in ATDC5 cells. In addition, Oxy suppressed mineralization and mRNA expressions of terminal chondrocyte differentiation markers in ATDC5 cells, primary chondrocytes, and cultured metatarsal bones. Oxy's induction of Col2 mRNA expression was decreased by the addition of Noggin and was increased by the addition of BMP2. Furthermore, Oxy increased mRNA expression of Id1, Bmp2, Bmp4, and Bmp6. These data suggest that Oxy induces chondrogenic differentiation in a BMP-dependent manner and suppresses terminal differentiation. Oxy may be useful for treatment of OA and also for regeneration of cartilage tissue.

Palmatine attenuates osteoclast differentiation and function through inhibition of receptor activator of nuclear factor-κb ligand expression in osteoblast cells.

Osteoclasts are the only cell type capable of resorbing mineralized bone, and they act under the control of numerous cytokines produced by supporting cells such as osteoblasts and stromal cells. Among cytokines, receptor activator of nuclear factor-κB ligand (RANKL) was found to be a key osteoclastogenetic molecule that directly binds to its cognate receptor, RANK, on osteoclast precursor cells. In turn, RANKL, which is an essential factor for differentiation and activation of osteoclasts, is one of the major targets of anti-resorptive agents. In this study, we found that palmatine, an isoquinoline alkaloid originally isolated from Coptis chinensis, had an inhibitory effect on osteoclast differentiation and function in vitro. Palmatine inhibited osteoclast formation in the co-culture system with mouse bone marrow cells (BMC) and osteoblasts in the presence of 10 nM 1α,25-(OH)(2)D(3). Palmatine did not affect osteoclast formation induced by RANKL in the BMC cultures. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that palmatine significantly inhibited the expression of 1α,25-(OH)(2)D(3)-induced expression of RANKL mRNAs in stromal cells without loss of cell viability. Moreover, palmatine suppressed resorption pit formation by mature osteoclasts on dentin slices and induced disruption of actin ring formation in mature osteoclasts with an impact on cell viability. Taken together, these results suggest that palmatine attenuates osteoclast differentiation through inhibition of RANKL expression in osteoblast cells, and its inhibitory effect on bone resorption is due to its disruptive effect on actin rings in mature osteoclasts. Therefore, palmatine might be an ideal candidate as an anti-resorptive agent for the prevention and treatment of bone disorders such as osteoporosis.

Honokiol inhibits osteoclast differentiation and function in vitro.

Honokiol, a neolignan, is a physiologically active component of kouboku (Magnolia obovata), a herb used in traditional Chinese medicine. This study investigated the effects of honokiol on the differentiation and function of osteoclasts induced by receptor activator of nuclear factor-kappaB ligand (RANKL). Honokiol markedly inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of TRAP-positive multinucleated cells in both bone marrow-derived monocytes and RAW264 cells. In experiments to elucidate its mechanism of action, honokiol was found to suppress RANKL-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The RANKL-induced expressions of c-Fos and nuclear factor of activated T cells-c1 (NFATc1), which are crucial transcriptional factors for osteoclastogenesis, were also reduced by treatment with honokiol. Furthermore, honokiol induced disruption of the actin rings in mature osteoclasts (mOCs) without affecting the cell viability and suppressed osteoclastic pit formation on dentin slices. Taken together, these results suggest that honokiol inhibits osteoclast differentiation by suppressing the activation of MAPKs (p38 MAPK, ERK and JNK), decreasing the expressions of c-Fos and NFATc1, and attenuates bone resorption by disrupting the actin rings in mOCs. Therefore, honokiol could prove useful for the treatment of bone diseases associated with excessive bone resorption.

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.

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.

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.

Development of high-throughput screening system for osteogenic drugs using a cell-based sensor.

To effectively treat osteoporosis and other bone-loss disorders, small compounds that potently induce bone formation are needed. The present study initially attempted to establish a monitoring system that could detect osteogenic differentiation easily, precisely, and noninvasively. For this purpose, we established pre-osteoblastic MC3T3E1 cells stably transfected with the GFP reporter gene driven by a 2.3 kb fragment of rat type I collagen promoter (Col1a1GFP-MC3T3E1). Among these cells, we selected a clone that fluoresced upon osteogenic stimulation by BMP2. The GFP fluorescence intensity corresponded well to the intensity of alkaline phosphatase (ALP) staining and to the level of osteocalcin (Oc) mRNA. Using this system, we screened natural and synthetic compound libraries and thus identified an isoflavone derivative, glabrisoflavone (GI). GI induced ALP staining and Oc mRNA in a dose-dependent manner. The Col1a1GFP-MC3T3E1 system may be useful for identifying novel osteogenic drugs.

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.