New phenylbutenoids and terpene glycosides from Ginkgo biloba leaves.

Our continued works on the chemical constituents of Ginkgo biloba (G. biloba) leaves has led to the isolation of two novel phenylbutenoids (1, 2), along with five previously unidentified terpene glycosides (3-7). Among them, compounds 1 and 2 represent unique (Z)-phenylbutenoids, 3-6 are megastigmane glycosides, and 7 is identified as a rare bilobanone glycoside (Fig. 1). This study marks the first reported isolation of phenylbutenoid and bilobanone glycoside from G. biloba. The chemical structures of these compounds were elucidated through extensive spectroscopic analysis, including HR-ESI-MS and various 1D and 2D NMR experiments. Furthermore, the absolute configurations of these molecules were determined using Mosher's method, ECD experiments, and Cu-Kα X-ray crystallographic analyses.

Gerberdriasins A-F, six undescribed coumarin derivatives from Gerbera anandria (Linn) Sch-Bip and their protective effects on scopolamine-induced injury in PC12 cells.

A chemical investigation on the herb Gerbera anandria (Linn) Sch-Bip led to the isolation and identification of six previously undescribed coumarin derivatives, named Gerberdriasins A-F (1-6). Structurally, their chemical structures and absolute configurations were determined by nuclear magnetic resonance (1D and 2D NMR), high resolution electrospray ionization mass spectroscopy (HR-ESI-MS), experimental and quantum mechanical nuclear magnetic resonance (QM-NMR) methods, Mosher's method and calculated electronic circular dichroism (ECD) experiments. The biological activity of the obtained compounds showed that they displayed significant neuroprotective effects against scopolamine-induced injury in PC12 cells at the concentrations 12.5, 25.0 and 50.0 nM. Further study demonstrated that 1 could inhibit cell apoptosis, decrease malondialdehyde (MDA) levels and increase superoxide dismutase (SOD) activity in scopolamine-treated PC12 cells.

Vlasouliodes A-D, four new C30 dimeric sesquiterpenes exhibiting potential inhibition of MCF-7 cells from Vladimiria souliei.

As our ongoing interest to search bioactive dimeric sesquiterpenes from the genus Vladimiria (Asteraceae), the plant of Vladimiria souliei was studied. Based on the repetitive chromatographic fractionation, a chemical investigation on the roots of Vladimiria souliei led to the isolation and the identification of four previously undescribed sesquiterpene dimers, vlasouliodes A-D (1-4). Their chemical structures were elucidated by comprehensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR spectroscopic data. The absolute configurations of them were unambiguously established by the experimental and calculated ECD data. In the in vitro biological activity evaluation, 1 and 3 displayed pronounced inhibitory activity against human breast adenocarcinoma cell lines (MCF-7) with IC50 values of 17.12 ± 0.42 µM and 13.12 ± 0.10 µM, respectively. Additionally, treatment with 1 and 3 induced cell apoptosis in MCF-7 cells, down-regulated the expression of Caspase-3 and up-regulated the expression of Cleaved-caspase-3.

Coumarin-monoterpenes from Gerbera anandria (Linn.) Sch.-Bip and their neuroprotective activity.

Thirty-two undescribed coumarin-monoterpenes, including the first report of six pairs of enantiomeric and twenty congeners, were isolated from the petroleum ether extract of the stems of Gerbera anandria (Linn.) Sch.-Bip. Structurally, these compounds represented C3-substituted 5-methyl-4-hydroxycoumarin-monoterpenes. Among them, 1-7 and 10-24 were rare 5-methylcoumarin-monoterpenes formed through a furan ring. Their chemical structures and absolute configurations were determined by comprehensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR spectroscopic data, Mosher's method, ECD calculations and single crystal X-ray diffraction. Furthermore, biological studies revealed that compounds 1-3, 3a, 5, 5a, 11-12, 21-22 and 26 had the neuroprotective effects on scopolamine-induced injury in PC12 cells. Notably, 3 exhibited the strongest neuroprotective activity with the cell viability values of 77.24%. Meanwhile, pretreatment with 3 significantly downregulate apoptosis and reactive oxygen species (ROS) production, as well as strengthen antioxidant enzyme activities (MDA and SOD). Moreover, pretreatment with 3 also could attenuate the down-regulation of HO-1 and Nrf2 induced by scopolamine. In conclusion, these results demonstrated that these compounds possessed the protective effects on scopolamine-injured PC12 cells through anti-apoptotic and anti-oxidant activities.

New ent-kaurane diterpenoid acids from Nouelia insignis Franch and their anti-inflammatory activity.

Eleven undescribed ent-kaurane-type diterpenoid acids, namely noueinsiancins A-K (1-11), together with sixteen related known analogs (12-27) were isolated from Nouelia insignis Franch. The chemical structures and absolute configurations of the new compounds were confirmed by the extensive spectroscopic data, electronic circular dichroism (ECD) data analysis and single crystal X-ray diffraction. Additionally, the anti-inflammatory assay was applied to estimate the nitric oxide (NO) inhibitory activities of all compounds by using lipopolysaccharide (LPS)-induced RAW 264.7 cells in vitro. The results revealed that 4-7 and 13-17 significantly inhibited NO production at the concentrations of 2.5 µM, 5.0 µM and 10.0 µM. Meanwhile, compounds 6 and 7 were found to down-regulate the protein expression levels of IL-6 and TNF-α in RAW 264.7 cells induced by LPS in a dose-dependent manner. In conclusion, these findings provided the reference values for exploring the new chemicals with biological activities from this genus.

Vlasoulides A and B, a pair of neuroprotective C32 dimeric sesquiterpenes with a hexacyclic 5/7/5/5/(5)/7 carbon skeleton from the roots of Vladimiria souliei.

Vlasoulides A and B (1 and 2), a pair of epimeric C32 sesquiterpene lactone dimers, featuring a 5/7/5/5/(5)/7 ring system were isolated from the roots of Vladimiria souliei. Their chemical structures were determined by comprehensive analysis of spectroscopic data, including HRESIMS and 1D and 2D NMR spectroscopic data. Their absolute configurations were established by Mosher's method and ECD experiments. Furthermore, biological studies showed that compound 1 showed prominent neuroprotective effects against glutamate-induced neurotoxicity in PC-12 cells, with EC50 values of 13.54 ± 0.33 µM, while, the EC50 value of compound 2 is greater than 30 µM.

A new bilobalide isomer and two cis-coumaroylated flavonol glycosides from Ginkgo biloba leaves.

A new bilobalide isomer (1), together with two flavonol glycosides (2, 3), have been isolated and elucidated from the extract of Ginkgo biloba leaves. Significantly, 1 was a new sesquiterpene lactone with two lactone ring groups, both 2 and 3 were two flavonol glycosides with a same cis-coumaroylated fragment. Their chemical structures were elucidated by NMR and MS spectroscopic date and the absolute configuration of 1 was specific established by Cu-Kα X-ray crystallographic analyses. However, 1-3 showed no obvious anti-platelet aggregation activity.

Lineariifolianoids M-O, three rare sesquiterpene lactone dimers inhibiting NO production from Inula lineariifolia.

Three rare squiterpene lactone dimers lineariifolianoids M-O (1-3) were isolated from Inula lineariifolia for the first time. Their structures and absolute configuration were established on the basis of by NMR and MS spectroscopic data and X-ray crystallography. Furthermore, those three compounds exhibited significant inhibitory activity against LPS-induced NO production in RAW 264.7 macrophages with IC50 values of 1.421, 1.087 and 1.243 µM, respectively.

Bilobetin induces kidney injury by influencing cGMP-mediated AQP-2 trafficking and podocyte cell cycle arrest.

BACKGROUND: Ginkgo biloba (Gb) extracts have been used as a traditional Chinese medicine. Gb contains flavonoids, which are considered to be its active ingredients and have been used in the treatment of a variety of diseases. However, few scientific research studies on the side effects of flavonoid in Gb have been reported. PURPOSE: The present study aimed to investigate the effect of bilobetin on the kidney of Sprague-Dawley (SD) rats. STUDY DESIGN AND RESULT: In this study, rats were injected with 50 mg/kg of bilobetin, a biflavone isolated from Gb, for 7 days and aristolochic acid was used as positive controls. The results showed that the body weight and urine output of the rats were dramatically decreased, and urinary protein increased after the intraperitoneal injection of bilobetin compared with the control group. Bilobetin treatment showed vacuolar degeneration in the renal tubular epithelium, glomerular atrophy by histostaining, and podocyte fusion by electron microscopy. This study further showed that bilobetin promoted the trafficking of aquaporin 2 (AQP-2) onto the plasma membrane to achieve the function of urine concentration by in vivo study in rats and in vitro study in IMCD-3 cells. The redistribution of AQP-2 is due to increased expression of cGMP in IMCD-3 cells, which in turn promoted the phosphorylation of AQP-2 at site Ser-256. The proteinuria caused by bilobetin may be attributed to podocyte cell cycle arrest at G2/M transition, which is may associated with AKT and MAPK signaling. CONCLUSIONS: The current study showed that bilobetin has some side effects on kidneys at a dose of 50 mg/kg in SD rats and provides insight into the potential detrimental effects of monomeric ingredients in Gb.

Vlasoulamine A, a Neuroprotective [3.2.2]Cyclazine Sesquiterpene Lactone Dimer from the Roots of Vladimiria souliei.

Vlasoulamine A (1), an unprecedented sesquiterpene lactone dimer featuring a fully hydrogenated pyrrolo[2,1,5- cd]indolizine core, and vlasoulones A and B (2 and 3), a pair of epimeric dimers formed from a proposed Diels-Alder [4 + 2] cycloaddition between a germacrane sesquiterpene lactone and a eudesmane sesquiterpene, were isolated from the roots of Vladimiria souliei. Their structures and absolute configurations were established by NMR, MS, and single-crystal X-ray spectroscopic analysis. Moreover, 1 exhibited neuroprotective activity when evaluated for glutamate-induced cytotoxicity, nuclear Hoechst 33258 staining, and measuring intracellular reactive oxygen species levels, using a rat pheochromocytoma PC12 cell-based model system.

Five rare dimeric sesquiterpenes exhibiting potential neuroprotection activity from Vladimiria souliei.

Two rare C32 sesquiterpene lactone dimers, vlasouliolides J-K (1-2), together with three C30 sesquiterpene dimers, vlasoulioliones A-C (3-5), were isolated from Vladimiria souliei. Their chemical structures were elucidated by NMR and MS spectroscopic data. Among them, compounds 1, 3 and 5 were unambiguously established by Cu-Kα X-ray crystallographic analysis. Furthermore, compound 1 showed prominent neuroprotective effects against glutamate-induced neurotoxicity in PC-12 cells, with EC50 value of 2.11 ±â€¯0.35 µM. And the leakage of LDH from glutamate-induced PC-12 was significantly reduced by compound 1 at concentration of 10 µM.

Five rare C32 sesquiterpene lactone dimers with anti-inflammation activity from Vladimiria souliei.

Five rare sesquiterpene lactone dimers, vlasouliolides E-I, were isolated from Vladimiria souliei. Their chemical structures were elucidated by spectroscopic analysis. Furthermore, 2 and 4 were unambiguously confirmed by Cu-Kα X-ray crystallographic analysis. Compounds 1, 2, 4 and 5 exhibited significant anti-inflammatory activity against LPS-induced NO production in RAW 264.7 cells with IC50 values of 1.88, 4.89, 7.24 and 2.46µM, respectively. Additionally, compounds 1 and 2 were revealed with potent inhibitory activity of the phosphorylation progress of NF-κB P65.

Protein Arginine Methyltransferase 6 (Prmt6) Is Essential for Early Zebrafish Development through the Direct Suppression of gadd45αa Stress Sensor Gene.

Histone lysine methylation is important in early zebrafish development; however, the role of histone arginine methylation in this process remains unclear. H3R2me2a, generated by protein arginine methyltransferase 6 (Prmt6), is a repressive mark. To explore the role of Prmt6 and H3R2me2a during zebrafish embryogenesis, we identified the maternal characteristic of prmt6 and designed two prmt6-specific morpholino-oligos (MOs) to study its importance in early development, application of which led to early epiboly defects and significantly reduced the level of H3R2me2a marks. prmt6 mRNA could rescue the epiboly defects and the H3R2me2a reduction in the prmt6 morphants. Functionally, microarray data demonstrated that growth arrest and DNA damage-inducible, α, a (gadd45αa) was a significantly up-regulated gene in MO-treated embryos, the activity of which was linked to the activation of the p38/JNK pathway and apoptosis. Importantly, gadd45αa MO and p38/JNK inhibitors could partially rescue the defect of prmt6 morphants, the downstream targets of Prmt6, and the apoptosis ratios of the prmt6 morphants. Moreover, the results of ChIP quantitative real time PCR and luciferase reporter assay indicated that gadd45αa is a repressive target of Prmt6. Taken together, these results suggest that maternal Prmt6 is essential to early zebrafish development by directly repressing gadd45αa.

Splice blocking of zygotic sox31 leads to developmental arrest shortly after Mid-Blastula Transition and induces apoptosis in zebrafish.

Here we report that splice blocking morpholinos (Sb MO) against zebrafish sox31 elicit developmental arrest, likely through creating a series of dominant negative splicing variants. Embryos injected with the Sb MO develop normally before the Mid-Blastula Transition (MBT); however, they do not initiate epiboly. Microarray analysis of mRNAs collected at the dome stage revealed that the Sb MO impairs activation of a large set of zygotic genes and reduces degradation of maternal mRNA during MBT. Furthermore, an apoptotic response occurs in Sb morphants at about 6hpf. SoxB1 family genes including sox31 thus play an essential role for early embryos traversing the transitional stage.

Caprin-2 enhances canonical Wnt signaling through regulating LRP5/6 phosphorylation.

The low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Frizzled and transmit signals from the plasma membrane to the cytosol. However, the mechanism for LRP5/6 signal transmission remains undefined. Here, we identify cytoplasmic activation/proliferation-associated protein 2 (Caprin-2) as a LRP5/6-binding protein. Our data show that Caprin-2 stabilizes cytosolic beta-catenin and enhances lymphoid enhancer-binding factor 1/T cell factor-dependent reporter gene activity as well as the expression of Wnt target genes in mammalian cells. Morpholino-mediated knockdown of Caprin-2 in zebrafish embryos inhibits Wnt/beta-catenin signaling and results in a dorsalized phenotype. Moreover, Caprin-2 facilitates LRP5/6 phosphorylation by glycogen synthase kinase 3, and thus enhances the interaction between Axin and LRP5/6. Therefore, Caprin-2 promotes activation of the canonical Wnt signaling pathway by regulating LRP5/6 phosphorylation.

Nuclear Dvl, c-Jun, beta-catenin, and TCF form a complex leading to stabilization of beta-catenin-TCF interaction.

In canonical Wnt signaling, Dishevelled (Dvl) is a critical cytoplasmic regulator that releases beta-catenin from degradation. Here, we find that Dvl and c-Jun form a complex with beta-catenin-T-cell factor 4 (TCF-4) on the promoter of Wnt target genes and regulate gene transcription. The complex forms via two interactions of nuclear Dvl with c-Jun and beta-catenin, respectively, both of which bind to TCF. Disrupting the interaction of Dvl with either c-Jun or beta-catenin suppresses canonical Wnt signaling-stimulated transcription, and the reduction of Dvl diminished beta-catenin-TCF-4 association on Wnt target gene promoters in vivo. Expression of a TCF-Dvl fusion protein largely rescued the c-Jun knockdown Wnt signaling deficiency in mammalian cells and zebrafish. Thus, we confirm that c-Jun functions in canonical Wnt signaling and show that c-Jun functions as a scaffold in the beta-catenin-TCFs transcription complex bridging Dvl to TCF. Our results reveal a mechanism by which nuclear Dvl cooperates with c-Jun to regulate gene transcription stimulated by the canonical Wnt signaling pathway.

Developmental expression of cyclin H and Cdk7 in zebrafish: the essential role of cyclin H during early embryo development.

Cyclin-dependent kinase 7 (Cdk7) is the catalytic subunit of the metazoan Cdk-activating kinase (CAK). Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H complex has been implicated in the regulation of RNA polymerase in several species, the precise function of their orthologs in zebrafish has not been fully elucidated. In this study, we isolated from zebrafish blastula embryos two cDNAs encoding the orthologs of human Cyclin H and Cdk7, and examined the role of Cdk7/Cyclin H in zebrafish embryogenesis. Sequence analysis showed that the zebrafish Cyclin H and Cdk7 cDNAs encode proteins with 65% and 86% identity to the respective human orthologs. RT-PCR and whole-mount in situ hybridization analyses of their expression in unfertilized eggs, embryos and organs of adult fish suggested that Cyclin H and Cdk7 messages are maternally loaded. Our data also showed that their transcripts were detected throughout development. Distribution of Cyclin H transcripts was found to be ubiquitous during early stages of development and become restricted to the anterior neural tube, brain, eyes, procreate tissues, liver and heart by 5 days post-fertilization. Expression of a dominant-negative form of Cyclin H delayed the onset of zygotic transcription in the early embryo, resulting in apoptosis at 5 hours post-fertilization and leading to sever defects in tissues normally exhibiting high levels of Cyclin H expression. These results implicate Cyclin H in the regulation of the transcriptional machinery during midblastula transition and suggest that it is an essential gene in early zebrafish larval development.

Delay of ZGA initiation occurred in 2-cell blocked mouse embryos.

One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which 2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos, which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is often assumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in cultured embryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of [35S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strain were compared in their development both in vitro and in vivo. The detection of TRC expression, a marker of ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even in the 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as compared with normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG, TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo. But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA, but to a delay of ZGA.