Melanogenesis inhibitory pregnane glycosides from Cynanchum atratum.

Bioassay-guided fractionation of the methanolic extract from the roots of Cynanchum atratum has resulted in the isolation of three new pregnane glycosides (1-3) along with four known compounds (4-7). Their structures were identified by analysis of the spectroscopic data including extensive 2D NMR. All of the isolates were evaluated for their potential to inhibit the melanin production in α-melanocyte stimulating hormone (α-MSH)-activated B16 melanoma cells. Of these, compounds 4-7 dose-dependently inhibited the melanin production with the IC50 values ranging from 4 µM to 33 µM.

Wnt9b-dependent FGF signaling is crucial for outgrowth of the nasal and maxillary processes during upper jaw and lip development.

Outgrowth and fusion of the lateral and medial nasal processes and of the maxillary process of the first branchial arch are integral to lip and primary palate development. Wnt9b mutations are associated with cleft lip and cleft palate in mice; however, the cause of these defects remains unknown. Here, we report that Wnt9b(-/-) mice show significantly retarded outgrowth of the nasal and maxillary processes due to reduced proliferation of mesenchymal cells, which subsequently results in a failure of physical contact between the facial processes that leads to cleft lip and cleft palate. These cellular defects in Wnt9b(-/-) mice are mainly caused by reduced FGF family gene expression and FGF signaling activity resulting from compromised canonical WNT/ß-catenin signaling. Our study has identified a previously unknown regulatory link between WNT9B and FGF signaling during lip and upper jaw development.

The canonical Wnt signaling activator, R-spondin2, regulates craniofacial patterning and morphogenesis within the branchial arch through ectodermal-mesenchymal interaction.

R-spondins are a recently characterized family of secreted proteins that activate Wnt/ß-catenin signaling. Herein, we determine R-spondin2 (Rspo2) function in craniofacial development in mice. Mice lacking a functional Rspo2 gene exhibit craniofacial abnormalities such as mandibular hypoplasia, maxillary and mandibular skeletal deformation, and cleft palate. We found that loss of the mouse Rspo2 gene significantly disrupted Wnt/ß-catenin signaling and gene expression within the first branchial arch (BA1). Rspo2, which is normally expressed in BA1 mesenchymal cells, regulates gene expression through a unique ectoderm-mesenchyme interaction loop. The Rspo2 protein, potentially in combination with ectoderm-derived Wnt ligands, up-regulates Msx1 and Msx2 expression within mesenchymal cells. In contrast, Rspo2 regulates expression of the Dlx5, Dlx6, and Hand2 genes in mesenchymal cells via inducing expression of their upstream activator, Endothelin1 (Edn1), within ectodermal cells. Loss of Rspo2 also causes increased cell apoptosis, especially within the aboral (or caudal) domain of the BA1, resulting in hypoplasia of the BA1. Severely reduced expression of Fgf8, a survival factor for mesenchymal cells, in the ectoderm of Rspo2(-/-) embryos is likely responsible for increased cell apoptosis. Additionally, we found that the cleft palate in Rspo2(-/-) mice is not associated with defects intrinsic to the palatal shelves. A possible cause of cleft palate is a delay of proper palatal shelf elevation that may result from the small mandible and a failure of lowering the tongue. Thus, our study identifies Rspo2 as a mesenchyme-derived factor that plays critical roles in regulating BA1 patterning and morphogenesis through ectodermal-mesenchymal interaction and a novel genetic factor for cleft palate.

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis.

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/ß-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/ß-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/ß-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/ß-catenin signaling pathway.

Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-α.

Peroxisome proliferator-activated receptor-alpha (PPARα) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPARα agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPARα agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPARα. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P<0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPARα via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

Inhibitory constituents of Nardostachys chinensis on nitric oxide production in RAW 264.7 macrophages.

The activity-guided fractionation of the MeOH extract of the rhizomes and roots of Nardostachys chinensis led to the isolation of two new sesquiterpenoids, narchinol B (8) and narchinol C (9), along with 10 known compounds, ursolic acid (1), nardosinone (2), pinoresinol (3), desoxo-narchinol A (4), kanshone B (5), epoxyconiferyl alcohol (6), debilon (7), 4α,5-dimethyl-1,3-dioxo-1,2,3,4,4α,5,6,7-octahydronaphthalene (10), p-coumaric acid (11), and isoferulic acid (12). Their structures were determined using spectroscopic techniques, which included 1D- and 2D-NMR. Among the isolates, compounds 2, 4, 5, 8 and 9 showed inhibitory activity against LPS-induced NO production with IC(50) values of 4.6-21.6 µM.

Antioxidative oligostilbenes from Caragana sinica.

Two new oligostilbenes, caragasinins A (5) and B (10), and eight known compounds, kobophenol A (1), (+)-α-viniferin (2), (+)-ampelopsin F (3), pallidol (4), (+)-isoampelopsin F (6), miyabenol C (7), carasinaurone (8) and caraphenol B (9) were isolated from the ethylacetate-soluble extract of the roots of Caragana sinica. The structures of the isolates were determined on the basis of extensive spectroscopic analysis including 1D, 2D NMR and HRESI-MS. These compounds were assessed for antioxidant activities. Caragasinin A (5), caraphenol B (9), and caragasinin B (10) showed moderate DPPH scavenging activity and lipid peroxidation inhibitory activities with IC(50) values ranging from 34.7±1.0 to 89.1±2.3µM.

Pyrrolidinone diterpenoid from Isodon excisus and inhibition of nitric oxide production in lipopolysaccharide-induced macrophage RAW264.7 cells.

A new pyrrolidinone diterpenoid, excisusin F (1), was isolated from the aerial parts of Isodon excisus (Lamiaceae), together with four known compounds, and their structures were determined mainly by NMR (1D and 2D) and mass spectrometry. Excisusin F (1) and inflexarabdonin E (3) showed potent inhibitory effects of LPS-induced nitric oxide production in RAW264.7 cells with the IC(50) value of 10.4 and 3.8 µM, respectively.

Dimeric ent-kaurane diterpenoids from Isodon excisus.

Five new dimeric ent-kauranoids, biexcisusins A-E (1-5), were isolated from the aerial parts of Isodon excisus. The structures and relative configurations of these compounds were determined on the basis of spectroscopic data interpretation. Of these, biexcisusins C-E (3-5) are dimeric ent-kaurane diterpenoids exhibiting an unprecedented linkage through a nine-membered lactone ring between two ent-kaurane subunits. Compounds 1-5 showed no inhibitory effects on the LPS-induced production of nitric oxide in murine macrophage RAW264.7 cells, up to a dose of 50 µM.

Phenanthrenes from Dendrobium nobile and their inhibition of the LPS-induced production of nitric oxide in macrophage RAW 264.7 cells.

Bioactivity-guided isolation of the methanol extract of the stems of Dendrobium nobile yielded a new phenanthrene together with nine known phenanthrenes and three known bibenzyls. Their structures were elucidated by analysis of the spectroscopic data including 2D-NMR. All of the isolates were evaluated for their potential to inhibit the LPS-induced production of nitric oxide in murine macrophage RAW 264.7 cells. Compounds 1-4, 7-13 inhibited nitric oxide production with the IC(50) values ranging from 9.6 microM to 35.7 microM.

Canonical WNT/ß-Catenin Signaling Activated by WNT9b and RSPO2 Cooperation Regulates Facial Morphogenesis in Mice.

The R-spondin (RSPO) family of proteins potentiate canonical WNT/ß-catenin signaling and may provide a mechanism to fine-tune the strength of canonical WNT signaling. Although several in vitro studies have clearly demonstrated the potentiation of canonical WNT signaling by RSPOs, whether this potentiation actually occurs in normal development and tissue function in vivo still remains poorly understood. Here, we provide clear evidence of the potentiation of canonical WNT signaling by RSPO during mouse facial development by analyzing compound Wnt9b and Rspo2 gene knockout mice and utilizing ex vivo facial explants. Wnt9b;Rspo2 double mutant mice display facial defects and dysregulated gene expression pattern that are significantly more severe than and different from those of Wnt9b or Rspo2 null mutant mice. Furthermore, we found suggestive evidence that the LGR4/5/6 family of the RSPO receptors may play less critical roles in WNT9b:RSPO2 cooperation. Our results suggest that RSPO-induced cooperation is a key mechanism for fine-tuning canonical WNT/ß-catenin signaling in mouse facial development.

Prenylated and benzylated flavonoids from the fruits of Cudrania tricuspidata.

Three new prenylated isoflavones, 5,7-dihydroxy-6-(2''-hydroxy-3''-methylbut-3''-enyl)-4'-methoxylisoflavone (1), 5,4'-dihydroxy-6-(3''-methylbut-2''-enyl)-2'''-(4'''-hydroxy-4'''-methylethyl)-3'''-methoxydihydrofurano-[4''',5''';7,8]isoflavone (2), and 5,4'-dihydroxy-8-(3''-methylbut-2''-enyl)-2'''-(4'''-hydroxy-4'''-methylethyl)furano-[4''',5''';6,7]isoflavone (3), a benzylated dihydroflavonol, 5,7,4'-trihydroxy-8-p-hydroxybenzyldihydroflavonol (4), and eight known flavonoids (5-12) were isolated from the fruits of Cudrania tricuspidata. The structures of these compounds were determined on the basis of MS and (1)H and (13)C NMR spectroscopic data, including 2D NMR experiments. Compounds 2, 3, 6, 7, 8, 10, 11, and 12 inhibited LPS-induced nitric oxide production, with IC(50) values of 11.8-41.8 microM.

Catalponol enhances dopamine biosynthesis and protects against L-DOPA-induced cytotoxicity in PC12 cells.

The effects of catalponol (1) on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalponol at concentration ranges of 1-5 microM increased the intracellular levels of dopamine at 12-48 h. Catalponol at concentrations of up to 10 microM did not alter cell viability. Tyrosine hydroxylase (TH) activity was enhanced by 1 at 3 microM in a time-dependent manner, but aromatic L-amino acid decarboxylase activity was not. Catalponol also increased the intracellular levels of cyclic AMP and TH phosphorylation. In addition, catalponol at 3 microM associated with L-DOPA (20-50 microM) further enhanced the increases in dopamine levels induced by L-DOPA (50-100 microM) at 24 h. Catalponol at 2-5 microM inhibited L-DOPA (100-200 microM)-induced cytotoxicity at 48 h. These results suggest that 1 enhanced dopamine biosynthesis by inducing TH activity and protected against L-DOPA-induced cytotoxicity in PC12 cells, which was mediated by the increased levels of cyclic AMP.

Hesperetin, a bioflavonoid, inhibits rat aortic vascular smooth muscle cells proliferation by arresting cell cycle.

Diet can be one of the most important factors that influence risks for cardiovascular diseases. Hesperetin, a flavonoid present in grapefruits and oranges, is one candidate that may benefit the cardiovascular system. In this study, we have investigated the effect of hesperetin on the platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic vascular smooth muscle cells (VSMCs). Hesperetin significantly inhibited 50 ng/ml PDGF-BB-induced rat aortic VSMCs proliferation and [(3)H]-thymidine incorporation into DNA at concentrations of 5, 25, 50, and 100 microM. In accordance with these findings, hesperetin revealed blocking of the PDGF-BB-inducible progression through G(0)/G(1) to S phase of the cell cycle in synchronized cells. Western blot showed that hesperetin inhibited not only phosphorylation of retinoblastoma protein (pRb) and expressions of cyclin A, cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2) as well as proliferating cell nuclear antigen (PCNA) protein, but also downregulation of cyclin-dependent kinase inhibitor (CKI) p27(kip1), while did not affect CKI p21(cip1), p16(INK4), p53, and CDK4 expressions as well as early signaling transductions such as PDGF beta-receptor, extracellular signal-regulated kinase (ERK) 1/2, Akt, p38, and JNK phosphorylation. These results suggest that hesperetin inhibits PDGF-BB-induced rat aortic VSMCs proliferation via G(0)/G(1) arrest in association with modulation of the expression or activation of cell-cycle regulatory proteins, which may contribute to the beneficial effect of grapefruits and oranges on cardiovascular system.

ent-Kaurane diterpenoids from Isodon japonicus.

Five ent-kaurane diterpenoids, 6beta,7beta,14beta-trihydroxy-1alpha,19-diacetoxy-7alpha,20-epoxy- ent-kaur-16-en-15-one (1), 1alpha,6beta,7beta-trihydroxy-11alpha,19-diacetoxy-7alpha,20-epoxy-ent-kaur-16-en-15-one (2), 6-hydroxy-1alpha,19-diacetoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide (3), 19-hydroxy-1alpha,6-diacetoxy-6,7-seco- ent-kaur-16-en-15-one-7,20-olide (4), and 6-aldehyde-1alpha,19-diacetoxy-6,7-seco- ent-kaur-16-en-15-one-7,20-olide (5), along with 10 known ent-kaurane diterpenoids, pseurata C (6), longikaurin C (7), effusanin C (8), longikaurin B (9), longikaurin D (10), effusanin D (11), excisanin B (12), lasiokaurin (13), megathyrin A (14), and loxothyrin A (15), were isolated from the aerial parts of Isodon japonicus. Their structures were determined on the basis of spectroscopic (1D-, 2D-NMR and MS) and chemical evidence. The isolates were evaluated for their inhibitory effects on LPS-induced production of nitric oxide in murine macrophage RAW264.7 cells.

Methylpiperate derivatives from Piper longum and their inhibition of monoamine oxidase.

We have previously reported that piperine, a known piperidine alkaloid from Piper longum, competitively inhibited mouse brain MAO-A and MAO-B activities. Piperine also showed in vivo antidepressant-like activity against the tail suspension test. In the present study, we further expanded on the identification of MAO inhibitors from the fruit of P. longum. Activity-guided fractionation of a methylene chloride soluble extract led to the isolation of three known piperine-related compounds, methylpiperate (1), guineensine (2), and piperlonguminine (3). Of these, methylpiperate (1) and guineensine (2) showed significant MAO inhibitory activities with IC50 values of 3.6 and 139.2 microM, respectively. Furthermore, methylpiperate (1) exhibited a selective inhibitory effect against MAO-B (IC50 value: 1.6 microM) than MAO-A (IC50 value: 27.1 microM). The kinetic study using the Lineweaver-Burk plots analysis suggested that methylpiperate (1) competitively inhibits MAO-A and MAO-B activities with the Ki values of 23.5 and 1.3 microM, respectively.

A new abietane diterpenoid from Isodon inflexus.

A new ent-abietane diterpenoid, 3alpha,6beta-dihydroxy-7,17-dioxo-ent-abieta-15(16)-ene (1), and three known ent-kaurane diterpenids, kamebacetal A (2), kamebakaurin (3), and excisanin A (4), and a known triterpenoid, ursolic acid (5), were isolated from the aerial parts of Isodon inflexus. Their chemical structures were determined by extensive analysis of spectroscopic data including 1D-and 2D-NMR experiments. All isolates (1-5) were evaluated for their potential to inhibit LPS-induced nitric oxide production in RAW264.7 cells. Of these, compounds 1-4 inhibited the production of NO with IC(50) values ranging from 1.0 to 26.5 microM.

Effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells.

The effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalpalactone at 5-30µM decreased intracellular dopamine content with the IC(50) value of 22.1µM. Catalpalactone at 5-20µM, but not 30µM, did not alter cell viability. Catalpalactone at 20µM inhibited tyrosine hydroxylase (TH) and aromatic-l-amino acid decarboxylase (AADC) activities. Catalpalactone also decreased cyclic AMP levels and inhibited TH phosphorylation. In addition, catalpalactone at 20µM reduced the increases in dopamine levels induced by L-DOPA (20-50µM). Catalpalactone (5-30µM) associated with L-DOPA (50-100µM) enhanced L-DOPA-induced cytotoxicity at 48h, which was prevented by N-acetyl-l-cysteine. These results suggest that catalpalactone inhibited dopamine biosynthesis by reducing TH and AADC activities and enhanced L-DOPA-induced cytotoxiciy in PC12 cells.

Antithrombotic and antiplatelet activities of Korean red ginseng extract.

The antithrombotic and antiplatelet activities of Korean red ginseng extract (KRGE) were examined on rat carotid artery thrombosis in vivo and platelet aggregation in vitro and ex vivo. The KRGE significantly prevented rat carotid arterial thrombosis in vivo in a dose-dependent manner. Administration of the KRGE to rats significantly inhibited adenosine diphosphate (ADP)- and collagen-induced platelet aggregation ex vivo, although it failed to prolong coagulation times such as activated partial thromboplastin and prothrombin time indicating that the antithrombotic effect of the red ginseng may be due to its antiplatelet aggregation rather than anticoagulation effect. In line with the above observations, the red ginseng inhibited the U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregations in vitro in a concentration-dependent manner, with IC(50) values of 390 +/- 15, 485 +/- 19, 387 +/- 11 and 335 +/- 15 microg/ml, respectively. Consistently, serotonin secretion was also inhibited by ginseng in the same pattern. These results suggest that the red ginseng has a potent antithrombotic effect in vivo, which may be due to the antiplatelet rather than the anticoagulation activity, and the red ginseng intake may be beneficial for individuals with high risks of thrombotic and cardiovascular diseases.

Monoamine oxidase inhibitory components from Cayratia japonica.

Seven flavonoids were isolated from the whole plants and fruits of Cayratia japonica through the activity-guided isolation of a methanol extract using a monoamine oxidase (MAO) inhibition assay as a monitor. The chemical structures of the isolates were assigned as apigenin-7-O-beta-D-glucuronopyranoside (1), apigenin (2), luteolin (3), luteolin-7-O-beta-D-glucopyranoside (4), (+)-dihydroquercetin (taxifolin) (5), (+)-dihydrokaempferol (aromadendrin) (6) and quercetin (7). Among the isolated compounds, flavones such as apigenin (2) and luteolin (3), as well as the flavonol, quercetin (7) showed potent inhibitory effects against the MAO activity with IC50 values of 6.5, 22.6, and 31.6 microM, respectively. However, the flavone glycosides, apigenin-7-O-beta-D-glucuronopyranoside (1) and luteolin-7-O-beta-D-glucopyranoside (4), showed mild MAO inhibition (IC50 values: 81.7 and 118.6 microM, respectively). The flavanonol derivatives, taxifolin (5) and aromadendrin (6), also showed weak inhibition (IC50 values: 154.7 and 153.1 microM, respectively). Furthermore, quercetin (7) had a more potent inhibitory effect on MAO-A (IC50 value: 2.8 microM) than MAO-B (IC50 value: 90.0 microM). Apigenin (2) and luteolin (3) also preferentially inhibited MAO-A (IC50 values: 1.7 and 4.9 microM, respectively) compared with MAO-B (IC50 values: 12.8 and 59.7 microM, respectively).