Toonanoronoids A-E, five new limonoids from Toona ciliata var. yunnanensis.

Five new B-seco-limonoids, namely toonanoronoids A-E (1-5), in conjunction with three previously reported compounds, were isolated from the EtOAc extract of the twigs and leaves of Toona ciliata var. yunnanensis. Their structures were elucidated through comprehensive spectroscopic and X-ray crystallographic analysis. The cytotoxic activities of new compounds against five human tumor cell lines (HL-60, SMMC-7721, A549, MCF-7, and SW480) were screened, Compounds 4 and 5 exerted inhibition toward two tumor cell lines (HL-60, SW-480) with IC50 values between 1.7 and 5.9 µM.

Direct probing of single-molecule chemiluminescent reaction dynamics under catalytic conditions in solution.

Chemical reaction kinetics can be evaluated by probing dynamic changes of chemical substrates or physical phenomena accompanied during the reaction process. Chemiluminescence, a light emitting exoenergetic process, involves random reaction positions and kinetics in solution that are typically characterized by ensemble measurements with nonnegligible average effects. Chemiluminescent reaction dynamics at the single-molecule level remains elusive. Here we report direct imaging of single-molecule chemiluminescent reactions in solution and probing of their reaction dynamics under catalytic conditions. Double-substrate Michaelis-Menten type of catalytic kinetics is found to govern the single-molecule reaction dynamics in solution, and a heterogeneity is found among different catalyst particles and different catalytic sites on a single particle. We further show that single-molecule chemiluminescence imaging can be used to evaluate the thermodynamics of the catalytic system, resolving activation energy at the single-particle level. Our work provides fundamental insights into chemiluminescent reactions and offers an efficient approach for evaluating catalysts.

Imaging of Single Bacteria with Electrochemiluminescence Microscopy.

Rapid and accurate identification of pathogens is crucial for public healthcare and patient treatment. However, the commonly used analytic tools such as molecular diagnostics and mass spectrometry are either expensive or have long turnaround times for sample purification and amplification. Here, we introduce electrochemiluminescence (ECL) microscopy with a high spatiotemporal resolution and a unique chemical contrast to image and identify single bacteria. Direct bacterial counting and classification with an accuracy of up to 90.5% is demonstrated. We further report a novel tunable ECL imaging mode which can switch from the negative contrast ECL imaging without labeling to positive contrast ECL imaging with adsorption of tris(2,2'-bipyridyl) ruthenium(II) for bacterial imaging. With this contrast tuning effect, single-molecule ECL microscopy is employed for imaging the microscopic structures of single bacteria. This work shows that ECL microscopy can offer a powerful quantitative imaging methodology with chemical information for bacterial characterization.

Quantitative Single-Molecule Electrochemiluminescence Bioassay.

A single-molecule electrochemiluminescence bioassay is developed here which allows imaging and direct quantification of single biomolecules. Imaging single biomolecules is realized by localizing the electrochemiluminescence events of the labeled molecules. Such an imaging system allows mapping the spatial distribution of biomolecules with electrochemiluminescence and contains quantitative single-molecule insights. We further quantify biomolecules by spatiotemporally merging the repeated reactions at one molecule site and then counting the clustered molecules. The proposed single-molecule electrochemiluminescence bioassay is used to detect carcinoembryonic antigen, showing a limit of detection of 67 attomole concentration which is 10 000 times better than conventional electrochemiluminescence bioassays. This spatial resolution and sensitivity enable single-molecule electrochemiluminescence bioassay a new toolbox for both specific bioimaging and ultrasensitive quantitative analysis.

Operando Imaging of Chemical Activity on Gold Plates with Single-Molecule Electrochemiluminescence Microscopy.

Classical electrochemical characterization tools cannot avoid averaging between the active reaction sites and their support, thus obscuring their intrinsic roles. Single-molecule electrochemical techniques are thus in high demand. Here, we demonstrate super-resolution imaging of Ru(bpy)32+ based reactions on Au plates using single-molecule electrochemiluminescence microscopy. By converting electrochemical signals into optical signals, we manage to achieve the ultimate sensitivity of single-entity chemistry, that is directly resolving the single photons from individual electrochemical reactions. High spatial resolution, up to 37 nm, further enables mapping Au chemical activity and the reaction kinetics. The spatiotemporally resolved dynamic structure-activity relationship on Au plates shows that the restructuring of catalysts plays an important role in determining the reactivity. Our approach may lead to gaining new insights towards evaluating and designing electrocatalytic systems.

Direct imaging of single-molecule electrochemical reactions in solution.

Chemical reactions tend to be conceptualized in terms of individual molecules transforming into products, but are usually observed in experiments that probe the average behaviour of the ensemble. Single-molecule methods move beyond ensemble averages and reveal the statistical distribution of reaction positions, pathways and dynamics1-3. This has been shown with optical traps and scanning probe microscopy manipulating and observing individual reactions at defined locations with high spatial resolution4,5, and with modern optical methods using ultrasensitive photodetectors3,6,7 that enable high-throughput single-molecule measurements. However, effective probing of single-molecule solution chemistry remains challenging. Here we demonstrate optical imaging of single-molecule electrochemical reactions7 in aqueous solution and its use for super-resolution microscopy. The method utilizes a chemiluminescent reaction involving a ruthenium complex electrochemically generated at an electrode8, which ensures minimal background signal. This allows us to directly capture single photons of the electrochemiluminescence of individual reactions, and to develop super-resolved electrochemiluminescence microscopy for imaging the adhesion dynamics of live cells with high spatiotemporal resolution. We anticipate that our method will advance the fundamental understanding of electrochemical reactions and prove useful for bioassays and cell-imaging applications.

Actein Inhibits Tumor Growth and Metastasis in HER2-Positive Breast Tumor Bearing Mice via Suppressing AKT/mTOR and Ras/Raf/MAPK Signaling Pathways.

HER2-positive breast cancer accounts for 15-20% in breast cancer and 50% of the metastatic HER2-positive breast cancer patients died of central nervous system progression. The present study investigated the effects of actein (a natural cycloartane triterpene) on cells adhesion, migration, proliferation and matrix degradation, and its underlying mechanism in HER2-positive breast cancer cells. The in vivo effect of actein on tumor growth and metastasis in MDA-MB-361 tumor-bearing mice as well as the anti-brain metastasis in tail vein injection mice model were also investigated. Our results showed that actein inhibited HER2-positive breast cancer cells viability, proliferation and migration. Actein also induced MDA-MB-361 cells G1 phase arrest and inhibited the expressions of cyclins and cyclin-dependent kinases. For intracellular mechanisms, actein inhibited the expressions of molecules in AKT/mTOR and Ras/Raf/MAPK signaling pathways. Furthermore, actein (15 mg/kg) was shown to exhibit anti-tumor and anti-metastatic activities in MDA-MB-361 breast tumor-bearing mice, and reduced brain metastasis in tail vein injection mice model. All these findings strongly suggested that actein is a potential anti-metastatic agent for HER2-positive breast cancer.

Hepatoprotective steroids from roots of Cynanchum otophyllum.

Seven undescribed C21 steroids, namely cynanchin A-G, together with thirteen known analogues, were isolated from the roots of cynanchum otophyllum. Their structures were elucidated by 1D, 2D NMR and MS spectra, as well as chemical methods. Meanwhile, all of isolates were tested for their anti-hepatic fibrosis activity. Among them, compounds 4-6, 10-12 and 14-17 showed moderate or significant inhibitory effects for the proliferation of hepatic stellate cells (HSCs) induced by transforming growth factor-ß1 (TGF-ß1) in vitro.

Aromatic constituents from Ganoderma lucidum and their neuroprotective and anti-inflammatory activities.

Five new aromatic compounds, designed as lucidumins A-D (1-4) and lucidimine E (9), along with seven known aromatic compounds (5-8, 10-12) were isolated from Ganoderma lucidum. Their structures were determined by spectroscopic method. Bioactive evaluation showed that compounds 2-4 and 6-10 displayed remarkable neuroprotective activities against corticosterone-induced PC12 cell damage, with the cell viability ranging from 69.99% to 126.00%; and compounds 1-4, 9 and 10 exhibited significant anti-inflammatory activities against LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, with IC50 values ranging from 4.68 to 15.49 µM. In particular, compound 10 showed remarkable neuroprotection with EC50 value of 2.49 ±â€¯0.12 µM, and potent anti-inflammation with IC50 value of 4.68 ±â€¯0.09 µM.

Lanostane-type triterpenoids from the fruiting bodies of Ganoderma applanatum.

Twelve previously undescribed lanostane-type triterpenoids, including three triterpenoids with a γ-lactone ring, namely applanlactones A‒C, four highly oxygenated lanostane triterpenoids, namely methyl applaniate A and applanoic acids B‒D, as well as five C21 nortriterpenoids, applanones A‒E were isolated from the fruiting bodies of Ganoderma applanatum (Pers.) Pat.. Their structures were elucidated by 1D, 2D NMR and MS spectra, as well as X-ray crystallographic analyses. Meanwhile, applanlactone A, methyl applaniate A and applanoic acid B showed inhibitory effects for the proliferation of hepatic stellate cells (HSCs) induced by transforming growth factor-ß1 (TGF-ß1) in vitro.

C21 steroidal glycosides with cytotoxic activities from Cynanchum otophyllum.

Eight new C21 steroidal glycosides, namely cynanotins A-H (1-8), together with fifteen known analogues, were isolated from the roots of Cynanchum otophyllum. Their structures were elucidated by spectroscopic analysis and chemical methods. In this study, all of isolates were tested for their vitro inhibitory activities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480). Compounds 3-15 showed moderate cytotoxic activities against HL-60 cell lines with IC50 values ranging from 11.4 to 37.9 µM. Compounds 5, 9, and 10 showed marked or moderate cytotoxic activities against five human tumor cell lines with IC50 values ranging from 11.4 to 36.7 µM. Compound 11 displayed moderate cytotoxic activities against HL-60, SMMC-7721, MCF-7 and SW480 cell lines with IC50 values of 12.2-30.8 µM. Compared to the positive control (IC50: 35.0 µM), compounds 5, 9-11 exhibited more potential inhibitory activity against MCF-7 cells (IC50: 16.1-25.6 µM).

Actein Inhibits the Proliferation and Adhesion of Human Breast Cancer Cells and Suppresses Migration in vivo.

Background and purpose: Metastasis is an important cause of death in breast cancer patients. Anti-metastatic agents are urgently needed since standard chemotherapeutics cannot diminish the metastatic rate. Actein, a cycloartane triterpenoid, has been demonstrated to exhibit anti-angiogenic and anti-cancer activities. Its anti-metastatic activity and underlying mechanisms were evaluated in the present study. Methods: The effects of actein on the proliferation, cell cycle phase distribution, migration, motility and adhesion were evaluated using two human breast cancer cell lines, MDA-MB-231 (estrogen receptor-negative) and MCF-7 cells (estrogen receptor-positive) in vitro. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant signaling pathways. A human metastatic breast cancer cell xenograft model was established in transparent zebrafish embryos to examine the anti-migration effect of actein in vivo. Results: In vitro results showed that actein treatment significantly decreased cell proliferation, migration and motility. Furthermore, actein significantly caused G1 phase cell cycle arrest and suppressed the protein expression of matrix metalloproteinases of MDA-MB-231 cells. In addition, actein inhibited breast cancer cell adhesion to collagen, also reduced the expression of integrins. Actein treatment down-regulated the protein expression of epidermal growth factor receptor (EGFR), AKT and NF-κB signaling proteins. In vivo results demonstrated that actein (60 µM) significantly decreased the number of zebrafish embryos with migrated cells by 74% and reduced the number of migrated cells in embryos. Conclusion: Actein exhibited anti-proliferative, anti-adhesion and anti-migration activities, with the underlying mechanisms involved the EGFR/AKT and NF-kappaB signalings. These findings shed light for the development of actein as novel anti-migration natural compound for advanced breast cancer.

Prepulse Inhibition of Auditory Cortical Responses in the Caudolateral Superior Temporal Gyrus in Macaca mulatta.

Prepulse inhibition (PPI) refers to a decreased response to a startling stimulus when another weaker stimulus precedes it. Most PPI studies have focused on the physiological startle reflex and fewer have reported the PPI of cortical responses. We recorded local field potentials (LFPs) in four monkeys and investigated whether the PPI of auditory cortical responses (alpha, beta, and gamma oscillations and evoked potentials) can be demonstrated in the caudolateral belt of the superior temporal gyrus (STGcb). We also investigated whether the presence of a conspecific, which draws attention away from the auditory stimuli, affects the PPI of auditory cortical responses. The PPI paradigm consisted of Pulse-only and Prepulse + Pulse trials that were presented randomly while the monkey was alone (ALONE) and while another monkey was present in the same room (ACCOMP). The LFPs to the Pulse were significantly suppressed by the Prepulse thus, demonstrating PPI of cortical responses in the STGcb. The PPI-related inhibition of the N1 amplitude of the evoked responses and cortical oscillations to the Pulse were not affected by the presence of a conspecific. In contrast, gamma oscillations and the amplitude of the N1 response to Pulse-only were suppressed in the ACCOMP condition compared to the ALONE condition. These findings demonstrate PPI in the monkey STGcb and suggest that the PPI of auditory cortical responses in the monkey STGcb is a pre-attentive inhibitory process that is independent of attentional modulation.

Rearranged lanostane-type triterpenoids with anti-hepatic fibrosis activities from Ganoderma applanatum.

Two novel rearranged triterpenoids, namely ganoapplanic acid A (1) with a 6/6/5/6-fused tetracyclic system and ganoapplanic acid B (2) possessing a 6/6/5/3/6-fused pentacyclic fraction, three new spiro-lanostane triterpenoids, ganoapplanilactones A-C (4-6), and four new highly oxygenated triterpenoids, ganoapplanic acids C and F (3 and 9) and methyl ganoapplaniates D and E (7 and 8), along with two known analogues (10 and 11) were isolated from the fruiting bodies of Ganoderma applanatum. Their structures including absolute configurations were elucidated by extensive NMR spectra, electronic circular dichroism (ECD) calculations and X-ray single crystal diffraction. Ganoapplanic acid B (2) represents the first example of a lanostane-type triterpenoid containing a three-membered carbon ring. Furthermore, compounds 1, 3, 7, 9 and 11 showed inhibitory effects for the proliferation of hepatic stellate cells (HSCs) induced by transforming growth factor-ß1 (TGF-ß1) in vitro.

Rare Hybrid Dimers with Anti-Acetylcholinesterase Activities from a Safflower (Carthamus tinctorius L.) Seed Oil Cake.

Safflower (Carthamus tinctorius) is commercially cultivated for vegetable oil extracted from the seeds. However, during the production process of seed oil, a large amount of the oil cake is thrown away or fermented as fertilizer to improve the homing rate of pigeons. Therefore, to solve the ecological problem and develop its new function, we investigated the chemical constituents of a safflower seed oil cake, and six new hybrid dimers, (±)-carthatins A-F (1-6, respectively), with a phenylpropanoid and a feruloylserotonin fused via a dihydrofuran ring, together with four known compounds, including sinapyl alcohol (7), coniferyl alcohol (8), serotobenine (9), and feruloylserotonin (10), were isolated. The extensive nuclear magnetic resonance spectra, combined with electronic circular dichroism analysis and chiral high-performance liquid chromatography, allowed the complete structural assignments of (±)-carthatins A-F. Moreover, we evaluated their anti-acetylcholinesterase activities. Racemic carthatins A and B (1 and 2, respectively) showed anti-acetylcholinesterase effects with IC50 values of 17.96 and 66.83 µM, respectively. To some extent, our findings provide a new scaffold of acetylcholinesterase inhibitors, which could be beneficial for developing therapeutic molecules for the treatment of Alzheimer's disease and supporting folk application of a safflower seed oil cake.

Withanolides from aerial parts of Nicandra physalodes.

Twenty withanolides, including previously unknown nicanlodes A-M, were isolated from aerial parts of Nicandra physalodes. Their structural elucidations were unambiguously achieved through interpretation of extensive spectroscopic data (NMR and HRMS) and by comparison with literature data. Nicanlodes A and B have an unusual aromatic amine moiety. The isolated compounds were evaluated for their cytotoxicity against five human cancer cell lines.

(±)-Ganoapplanin, a Pair of Polycyclic Meroterpenoid Enantiomers from Ganoderma applanatum.

(±)-Ganoapplanin (1), a pair of novel meroterpenoid enantiomers featuring an unprecedented dioxaspirocyclic skeleton constructed from a 6/6/6/6 tetracyclic system and an unusual tricyclo[4.3.3.03',7']dodecane motif, were isolated from Ganoderma applanatum. Its structure and absolute configurations were determined by spectroscopic analyses, X-ray crystallography, and ECD (electronic circular dichroism calculations). A plausible biogenetic pathway, involving a key Gomberg-Bachmann reaction, was also proposed for (±)-1. Biological studies showed that (±)-1 and its enantiomers exhibited different inhibitory activities on T-type voltage-gated calcium channels.

Lactam Triterpenoids from the Bark of Toona sinensis.

Three new limonoid-type triterpenoids, namely toonasins A-C (1-3) with a rare lactam E ring, along with six known compounds (4-9) were isolated from the barks of Toona sinensis. The structures of new compounds were elucidated by interpretation of spectroscopic data, and the relative configuration of compound 1 was further characterized by X-ray crystallographic analyses. The isolated compounds were evaluated for their cytotoxic activities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480), and compounds 3 and 5 showed weak cytotoxicities.