Analysis of indications for pulmonary peripheral lesions diagnosed using radial endobronchial ultrasound-guided transbronchial lung biopsy.

The aim of the present study was to determine the indications for radial endobronchial ultrasound-guided transbronchial lung biopsy (rEBUS-D-TBLB) for the diagnosis of peripheral pulmonary lesions (PPL) located at the bronchopulmonary segments and subsegments. Data collected from 774 patients who underwent rEBUS-D-TBLB for suspected PPL, including clinical information, distribution of lesions, diagnostic spectrum and diagnostic rate, were collected and retrospectively reviewed. Additionally, the Wilcoxon signed-rank test was performed to analyze the diagnostic yield of lesions in bronchopulmonary subsegments under the lesion diameter limit of 3 cm. In total, 802 lesions were found in 774 patients. The diagnostic yield of rEBUS-D-TBLB for all lesions was 67.18%. Overall, 362 cases of malignant disease and 158 cases of benign disease were diagnosed, with sensitivities of 70.98 and 79.00% respectively. Lesions were distributed throughout the 18 bronchopulmonary segments of the lungs. The bronchopulmonary segments with >5% of the majority of the discovered lesions were LB1+2, LB3, LB6, LB10, RB1-4 and RB9. The diagnostic yield of rEBUS-D-TBLB was found to be >65% for lesions located at LB3, RB1-3 and RB9. Further rEBUS-D-TBLB examinations of the LB1+2a, LB6a and RB4b segments produced diagnostic yields of 81.25, 66.67 and 71.43% respectively. Finally, at segment RB4a, rEBUS-D-TBLB examination was more effective for lesions with diameters >3 cm compared with lesions with diameters <3 cm. The diagnostic yields for PPL distributed at LB1+2a, LB3, LB6a, RB1-3, RB4a (diameter >3 cm), RB4b, and RB9 using rEBUS-D-TBLB were higher compared with for other segments, providing a theoretical basis for the clinical application of rEBUS-D-TBLB for the diagnosis of PPL in patients.

Three new alkaloids isolated from the stem tuber of Pinellia pedatisecta.

The present study was designed to determine the chemical constituents of the stem tuber of Pinellia pedatisecta. The chemical constituents were isolated and purified by various chromatographic techniques, and their structures were elucidated on the basis of physicochemical properties and spectral data. Three new alkaloids (compounds 1, 2, and 3) were obtained and identified as 9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-amine (1), 4-(2-(2, 5-dioxopyrrolidin-1-yl)ethyl)phenyl acetate (2), and N-(9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-yl)acetamide (3). These compounds were evaluated for their cytotoxicity against human cervical cancer HeLa cells. Compounds 1 and 3 significantly inhibited the proliferation of HeLa cells with IC50 values being 3.02 ± 0.54 and 7.16 ± 0.62 µmol·L-1, respectively.

Pharmacokinetic Studies of Ganoderic Acids from the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), by LC-MS/MS.

Ganoderma lucidum is a famous medicinal mushroom that has been widely used in clinical practice and as a dietary supplementa. The triterpenoid ganoderic acids are the main constituents of G. lucidum. To determine the pharmacokinetic characteristics of ganoderic acids, we developed and validated a sensitive and selective liquid chromatography-tandem mass spectrometry method to determine simultaneously the concentration of 4 representative ganoderic acids in rat plasma after oral administration of the extract from G. lucidum. Because of the similarity of their chemical structures, the 4 components exhibited similar pharmacokinetic behaviors in some aspects. However, some of the pharmacokinetic parameters and the reabsorption peaks in the plasma concentration-time curves of ganoderic acids B and E after oral administration of the extract were different from those of ganoderic acids D and A because of the metabolic transformation among the ganoderic acids. These results increase our knowledge about the use of G. lucidum.

Anti-inflammatory diterpenoids from the root bark of Acanthopanax gracilistylus.

Five new ent-pimarane (1-3, 7, and 8) and three new ent-kaurane diterpenoids (4-6) and a new oleanane triterpene acid (9), together with 22 known compounds, were isolated from the root bark of the medicinal herb Acanthopanax gracilistylus. The structures of 1-9 were established based on the interpretation of high-resolution MS and 1D- and 2D-NMR data. The absolute configurations of 7 and 11 were determined by single-crystal X-ray diffraction and electronic circular dichroism analysis. Compounds 7 and 8 represent rare naturally occurring structures based on the devinyl ent-pimarane skeleton. Compounds 3, 10, 14, 16, and 17 exhibited potent inhibitory effects on the release of interleukin-1ß (IL-1ß), interleukin-8 (IL-8), and tumor necrosis factor (TNF-α) in lipopolysaccharide-stimulated peripheral blood mononuclear cells.

Activity-guided isolation of NF-κB inhibitors and PPARγ agonists from the root bark of Lycium chinense Miller.

ETHNOPHARMACOLOGICAL RELEVANCE: The root bark of Lycium chinense Miller, Lycii radicis cortex, has been used in traditional Chinese medicine (TCM) to treat different inflammation-related symptoms, such as diabetes mellitus. The pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) is a key regulator of inflammation, while the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) is a key modulator of genes involved in diabetes development. To identify putative active compound(s) from Lycii radicis cortex inhibiting NF-κB or activating PPARγ. MATERIAL AND METHODS: Using activity-guided fractionation, six extracts with different polarity, isolated fractions, and purified compounds from Lycii radicis cortex were tested for NF-κB inhibition and PPARγ activation in vitro. The structure of the purified compounds was elucidated by NMR and MS techniques. RESULTS: The ethyl acetate extract and the methanol extract of Lycii radicis cortex suppressed tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB, while the dichloromethane extract activated PPARγ. Nine phenolic amide analogues, including trans-N-(p-coumaroyl)tyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), dihydro-N-caffeoyltyramine (4), three neolignanamides (5-7), and two lignanamide (8, 9), were isolated and their inhibitory potential on NF-κB was determined (1-4 were also contained in water decoction). Two of the nine isolated phenolic amides inhibited TNF-α-induced NF-κB activation. Trans-N-caffeoyltyramine was verified as the key component responsible for the NF-κB inhibition with an IC50 of 18.4µM in our cell-based test system. Activation of PPARγ was attributed to a palmitic-acid enriched fraction which displayed concentration-dependent effect ablated upon co-treatment with the PPARγ antagonist T0070907. CONCLUSIONS: Phenolic amides were confirmed as main components from Lycii radicis cortex responsible for NF-κB inhibition. Fatty acids were identified as the major plant constituent responsible for the PPARγ activation. Structure-activity relationship analysis suggests that the NF-κB inhibitory activity of trans-N-caffeoyltyramine may be attributed to its Michael acceptor-type structure (α,ß-unsaturated carbonyl group). The data of this study contribute to a better understanding of the molecular mechanism of action of Lycii radicis cortex extracts in the context of inflammation.

Metabolite identification of crude extract from Ganoderma lucidum in rats using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

The metabolism of traditional Chinese medicine is very complicated and has been a great challenge. In the present paper, a new strategy was established to study the metabolism of crude extract from Ganoderma lucidum using the highly separative and sensitive ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. Based on the investigation of the metabolism of five representative single compounds (ganoderic acid), a total of 90 metabolites were identified from the bile sample after oral administration of the crude extract. Among them, 21 compounds were identified by comparison with the reference standards, the other unknown metabolites were tentatively characterized by interpretation of the high resolution low collision energy and high collision energy mass spectra using the fragmentation rules. The metabolic characteristics and "soft spots" of the ganoderic acids were revealed. After being absorbed, the ganoderic acids from the extract could undergo extensive phases I and II metabolism in rat before excreted into the bile. The main ganoderic acids could transform from one to another through reduction, oxidation, deacetylation and desaturation reactions. Other metabolic transformation included hydroxylation, sulfation and glucuronidation. The total tendency was that the low polar ganoderic acids were transformed into the high polar metabolites to eliminate from the organism. The metabolic "soft spots" of the ganoderic acids were 3,7,15,23-carbonyl groups (or hydroxyl groups), angular methyl groups, 20(22)-double bond, 12-acetoxyl group and 26-carboxylic acid moiety. These results are considered to be important for the further investigation of G. lucidum.

Phenanthrenes, 9,10-dihydrophenanthrenes, bibenzyls with their derivatives, and malate or tartrate benzyl ester glucosides from tubers of Cremastra appendiculata.

Eleven previously unknown compounds and 23 known compounds, including 20 phenanthrene or 9,10-dihydrophenanthrene derivatives, five bibenzyls, seven malate or tartrate benzyl ester glucosides, adenosine and gastrodin were isolated from tubers of Cremastra appendiculata. Among the obtained compounds, two are the first isolated dimers with one phenanthrene or bibenzyl unit connected to C-3 of 2,3,4,5-tetrahydro-phenanthro[2,1-b]furan moiety. In addition, 33 of these compounds were evaluated in vitro for their cytotoxic activity against two cancer cell lines. Among the compounds examined, one compound showed moderate cytotoxic activity, while five showed weak cytotoxic activity against the A549 cell line.

Elution-extrusion counter-current chromatography separation of two new benzyl ester glucosides and three other high-polarity compounds from the tubers of Pleione bulbocodioides.

INTRODUCTION: The tubers of Pleione bulbocodioides (Franch.) Rolfe, with gastrodin and benzyl ester glucosides as main components, have been used in traditional Chinese medicine for the treatment of various cancers and bacterial infections. Up to now, their official quality control method is still inadequate, and the difficulty of obtaining these high-polarity compounds is one of the major reasons. OBJECTIVE: To develop a rapid and efficient method for preparative separation of the high-polarity compounds gastrodin and benzyl ester glucosides. METHODS: An optimised solvent system composed of n-butanol:ethanol:water (20:1:20, v/v/v) was applied for the elution-extrusion counter-current chromatography (EECCC) separation. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase at a flow rate of 1.5 mL/min, a rotation speed of 850 rpm and a temperature of 35°C. RESULTS: Five high-polarity glucosides, including two new compounds, (E)-4-ß-D-glucopyranosyloxycinnamic acid 9-(4-ß-D-glucopyranosyloxybenzyl) ester (4 mg) and (Z)-2-(2-methylpropyl)butenedioic acid bis(4-ß-D-glucopyranosyloxybenzyl) ester (9 mg), and three main components, gastrodin (87 mg), dactylorhin A (60 mg) and militarine (15 mg), with HPLC purities of 95.4%, 96.4%, 91.1%, 97.2% and 95.5% respectively, were yielded from 400 mg of the prepared sample. CONCLUSION: Elution-extrusion counter-current chromatography could be used as a useful tool for the separation of high-polarity compounds such as gastrodin and benzyl ester glucosides and the enrichment of the minor ones.

Pharmacokinetics of ganoderic acid D and its main metabolite by liquid chromatography-tandem mass spectrometry.

The present study aims to investigate the pharmacokinetics of ganoderic acid D (GD), a representative active triterpenoid from Ganoderma lucidum. A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of the concentrations of GD and its main metabolite (ganoderic acid B) in rat plasma. Following protein precipitation, the analytes were separated on a reversed-phase C18 column. Acetonitrile-water-acetic acid (40:60:0.01) was used at a flow-rate of 0.2ml/min. A triple quadrupole mass spectrometer equipped with an electrospray ionization source was used as the detector and was operated in the negative ion mode. Multiple reaction monitoring using the characteristic transitions was performed to quantify the analytes. The method had a lower limit of quantification of 8.19ng/ml for GD, and 8.59ng/ml for ganoderic acid B (GB). The calibration curves were demonstrated to be linear over the concentration range of 8.19-4096ng/ml and 8.59-2149ng/ml, respectively. Variations within- and between-batch were less than 6.4% and 4.6%, respectively. The extraction recovery rates ranged from 98.8 to 105.2% and 100.7 to 113.6%, respectively. The validated method was successfully applied to the quantification of GD and GB concentrations in rat plasma after oral administration (or intravenous administration) of GD preparations at a dose of 15mg/kg. The data showed that the absolute bioavailability increased from 22% to 70% after the GD suspension was changed to GD loaded solid lipid nanoparticles. In the meantime, the Cmax increased from 107.2 to 1555.6ng/ml; the tmax changed from 2.0h to 0.3h. These results are very helpful in the further studies.

Simultaneous determination of 24 constituents in Cortex Lycii using high-performance liquid chromatography-triple quadrupole mass spectrometry.

A fast high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry method was developed to determine 24 components including 11 phenolic compounds, 9 phenolic amides, and 4 cyclic peptides in Cortex Lycii. The analytes were quantified by a triple quadrupole instrument in multiple reaction monitoring (MRM) mode. The fragmentation patterns of phenolic amides and cyclic peptides using ESI and collision-induced dissociation (CID) techniques are reported. This assay method was validated with respect to linearity (r(2)>0.9920), precision, repeatability, and accuracy (recovery rate between 93.0 and 105.9% with RSD<4.4%). The analytical results of 28 batches of Cortex Lycii indicated that cyclic peptides and phenolic amides were not only the abundant constituents, but also the characteristic components for Cortex Lycii to distinguish from the adulterants. Principle component analysis (PCA) was used to discriminate samples from different geographical regions of China, and cyclic peptides were considered to be the chemical markers responsible for the classification. The systematic and integrated assessment of Cortex Lycii provides sufficient evidence for the establishment of the quality standard.

Neolignanamides, lignanamides, and other phenolic compounds from the root bark of Lycium chinense.

Seven new neolignanamides (1-7), including two pairs of cis- and trans-isomers, and a new lignanamide (8) were isolated from the EtOAc-soluble fraction of an EtOH extract of the root bark of Lycium chinense, together with 22 known phenolic compounds (9-30), four of which were obtained from the genus Lycium for the first time. Compounds 5, 6, and 7 are unusual dimers having a rare connection mode between the two cinnamic acid amide units, while compounds 6, 7, and 8 are the first naturally occurring dimers derived from two dissimilar cinnamic acid amides. The cinnamic acid amides, neolignanamides, and lignanamides possess moderate radical-scavenging activity against the DPPH (2,2-diphenyl-1-picrylhydrazyl) and superoxide radicals.

Inhibition of epithelial-mesenchymal transition in A549 cell by transfected Napsin A.

BACKGROUND: Epithelial-mesenchymal transition is a cellular process characterized by the loss of cell adhesion, inhibition of E-cadherin expression, and increased cell mobility. Cells without Napsin A are susceptible to transition. Further studies are required to investigate whether this transition can be reversed by restoration of Napsin A. METHODS: A Napsin A expression vector PLJM1-Napsin A plasmid was constructed and then transfected into the epithelial cell line A549 by lentivirus transfection to obtain A549-PLJM1-Napsin A cell line. Cell proliferation was assayed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide and cell cycle was measured by flow cytometry. The E-cadherin, type I collagen, and focal adhesion kinase mRNA level was detected by reverse transcription-polymerase chain reaction. The Napsin A, E-cadherin, type I collagen, and focal adhesion kinase protein level in A549 cells was detected by Western blotting. RESULTS: Transforming growth factor-b1 induced epithelial-mesenchymal transition in A549 cells, as demonstrated by significant reduction of E-cadherin mRNA and protein levels (P < 0.01) as well as up-regulation of type I collagen (P < 0.01). Transfection of Napsin A in A549 cells can partially block the transforming growth factor-b1-regulated expression of E-cadherin and type I collagen (P < 0.01). In addition, transforming growth factor-b1-induced cell proliferation was inhibited by Napsin A (P < 0.01). Further study demonstrated that Napsin A caused G(0)/G(1) arrest and inhibited the expression of focal adhesion kinase (P < 0.01), a key protein in the integrin signaling pathway, in the in vitro epithelial-mesenchymal transition model. CONCLUSIONS: Sustained Napsin A expression in A549 cells can inhibit the transforming growth factor-b1-induced epithelial-mesenchymal transition. This may be due to the Napsin A-mediated inhibition of focal adhesion kinase expression and integrin signaling pathway.

Biotransformation of gambogenic acid by Chaetomium globosum CICC 2445.

Microbial transformation of gambogenic acid (1), a caged polyprenylated xanthone isolated from the resin of Garcinia hanburyi, was carried out with Chaetomium globosum CICC 2445, after screening forty-six strains of filamentous fungi. A new caged polyprenylated xanthone, 16,17-dihydroxygambogenic acid (2), was specifically obtained, as a result of hydroxylation at C-16, and C-17. Its structure was elucidated on the basis of spectroscopic methods. The cytotoxicity of compounds 1 and 2 against HeLa tumor cell line was evaluated, with both of them being modestly active.

Simultaneous determination of atractylenolide II and atractylenolide III by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of Atractylodes Macrocephala Rhizoma extract.

Atractylenolide II (AII) and atractylenolide III (AIII) are the major active components in Atractylodes Macrocephala Rhizoma (AMR). In this study, a sensitive, rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of AII and AIII in rat plasma using loliolide as internal standard (IS). After protein precipitation with ethyl acetate, the analytes were injected into an LC-MS/MS system for quantification. Chromatography was performed using a C(18) column, eluting with water and acetonitrile (45:55, v/v) at 0.2 mL/min. All analytes including IS were monitored under positive ionization conditions by multiple reaction monitoring with an electrospray ionization source. The validated method was successfully applied to the pharmacokinetic study of AII and AIII in rat plasma after oral administration of AMR extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine.

Proteomic studies on protective effects of salvianolic acids, notoginsengnosides and combination of salvianolic acids and notoginsengnosides against cardiac ischemic-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza and Panax notoginseng are popularly used traditional Chinese medicine for cardiovascular disorders and they are often used in the form of combination. However, mechanisms of their cardioprotective effects were still not clear. In the present study, the protective effects of salvianolic acids (SA), notoginsengnosides (NG) and combination of SA and NG (CSN) against rat cardiac ischemia-reperfusion injury were checked and the protein expression profiles of heart tissues were examined to search their possible protein targets. MATERIALS AND METHODS: The cardioprotective effects of SA, NG and CSN were checked in a rat model of ischemia-reperfusion (IR) by temporarily occluding coronary artery for 20 min followed by reperfusion. Rats were grouped into sham-operation group, IR group, IR+SA group, IR+NG group and IR+CSN group. The plasma creatine kinase (CK) activities were measured using commercial kit and the percentages of infarcted area in total ventricle tissue were calculated after nitroblue-tetrazolium (N-BT) staining of heart tissue slices. Two-dimensional protein electrophoresis (2-DE) was used to check the protein expression profiles of heart tissues. Then, proteins differentially expressed between IR group and sham-operation group were identified using matrix assisted laser desorption ionization-time of flight-mass spectrometry/mass spectrometry (MALDI-TOF MS/MS). The regulative effects of SA, NG and CSN on these IR-related proteins were analyzed. RESULTS: Treatments including SA, NG and CSN all showed cardioprotective effects against ischemia-reperfusion injury and CSN exhibited to be the best. Eighteen proteins involved in IR injury were found. These proteins are involved in pathways including energy metabolism, lipid metabolism, muscle contraction, heat shock stress, cell survival and proliferation. The regulation of these proteins by SA, NG or CSN suggested possible protein targets in their cardioprotective effects. CONCLUSIONS: SA and NG showed both similarity and difference in their protein targets involved in cardioprotective effects. The capability of CSN to regulate both protein targets of SA and NG might be the basis of CSN to show cardioprotective effects better than that of SA or NG.

[Roles of interleukin-10 differentiated dendritic cell of allergic asthma patients in T-lymphocyte proliferation in vitro].

OBJECTIVE: To explore the roles of interleukin (IL)-10 differentiated peripheral blood monocyte-derived dendritic cell (DC-10) of allergic asthma patients in T-lymphocytes proliferation in vitro. METHODS: From January to June 2011, 10 subjects with dust mite allergic asthma treated at Third Affiliated Hospital of Soochow University were enrolled. Their peripheral blood monocytes were isolated by Ficoll-Hypaque solution density gradient centrifugation and adherent method. And the adherent monocytes were routinely cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF)+interleukin-4 (IL-4)+tumor necrosis factor-alpha (TNF-α), stimulated with/without interleukin-10 (IL-10), pulsed with dust mite allergen and finally harvested. The cell surface molecules including CD80, CD83, CD86, human leukocyte antigen (HLA)-DR and immunoglobulin-like transcript 2 (ILT2) were detected by immunofluorescent labeling and flow cytometry. And cellular functions were estimated by detecting the capacities of DC uptake antigens with fluorescein isothiocyanate (FITC)-dextran capturing assay. The IL-10 differentiation DC (DC-10) were cultured with autologous peripheral T cells (DC-10 group), either alone (DC-TNF group) or together (combined group) with autologous immunostimulatory DC (DC-TNF). And the impact of this treatment on T-cell responses was assessed for each donor by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) assay. The production of interferon-γ (IFN-γ), IL-4, interleukin-5 (IL-5) and interleukin-13 (IL-13) were measured with the quantification of enzyme linked immunosorbent assay (ELISA) kits. RESULTS: In DC-10, the levels of some mature DC's markers (CD80, CD83, CD86 & HLA-DR) decreased, ILT2 increased and there were the higher capacities of up-taking FITC-dextran particle (72.32%±2.93% vs 54.41%±2.95%, P<0.01). Compared with the DC-TNF group (1.74±0.15), the T cell proliferation of the DC-10 group (1.06±0.18) and that of the combined group (1.34±0.16) were significantly inhibited (P<0.01). The secretion levels of IFN-γ, IL-4, IL-5 and IL-10 were (2998±141), (157±17), (2608±254) and (55±11) ng/L in the DC-10 group versus (3223±203), (149±19), (2465±183) and (88±10) ng/L respectively in the combined group. They were all significantly reduced versus (3639±209), (173±16), (2771±183) and (127±11) ng/L in the DC-TNF group (all P<0.01). CONCLUSIONS: The IL-10-treated human DC may express a tolerogenic phenotype and induce the allergen tolerance of T cells. And the induction of DC-10 represents a promising target for therapeutic intervention of effectively managing the clinical outcomes of allergic asthma.

Chemical constituents of Spatholobus suberectus.

AIM: To investigate chemical constituents of Spatholobus suberectus Dunn. METHODS: Isolation and purification were carried out by column chromatographic methods. Compounds were characterized based on their physical characteristics and spectra data. RESULTS: Seventeen compounds were isolated from ethanol extract of S. suberectus. The structures were elucidated as prestegane B (1), (2R, 3R)-buteaspermanol (2), (+)-medioresinol (3), (2R, 3R)-3,7-dihydroxyflavanone (4), benzeneethanol (5), 4, 7, 2'-trihydroxy-4'-methoxyisoflavanol (6), naringenin (7), blumenol A (8), protocatechuic acid ethyl ester (9), liquiritigenin (10), 7, 4'-dihydroxy-8-methoxy-isoflavone (11), 3, 5, 7, 3', 5'-pentahydroxyflavanone (12), protocatechuic acid (13), glycyroside (14), 8-methylretusin-7-O-ß-D-glucopyranoside (15), 3, 3', 4', 5, 6, 7, 8-heptahydroxyflavan (16), and dulcisflavan (17). CONCLUSION: All compounds are firstly isolated from the title plant and compounds 1, 3 were isolated from the Spatholobus genus for the first time.

Macromolecular and small-molecule modulation of intracellular Aß42 aggregation and associated toxicity.

Aß (amyloid ß-peptide) has a central role in AD (Alzheimer's disease) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aß aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aß peptide. In the present study we establish a mammalian cell system for the direct visualization of Aß formation by expression of an Aß(42)-EGFP (enhanced green fluorescent protein) fusion protein in the human embryonic kidney cell line T-REx293, and use this to identify both macromolecules and small molecules that reduce aggregation and associated cell toxicity. Thus a molecular shield protein AavLEA1 [Aphelenchus avenae LEA (late embryogenesis abundant) protein 1], which limits aggregation of proteins with expanded poly(Q) repeats, is also effective against Aß(42)-EGFP when co-expressed in T-REx293 cells. A screen of polysaccharide and small organic molecules from medicinal plants and fungi reveals one candidate in each category, PS5 (polysaccharide 5) and ganoderic acid DM respectively, with activity against Aß. Both PS5 and ganoderic acid DM probably promote Aß aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aß on cell physiology and to identify reagents that counteract those effects.

Simultaneous determination of four sesquiterpenoids in Atractylodes Macrocephala Rhizoma by GC-FID: optimisation of an ultrasound-assisted extraction by central composite design.

INTRODUCTION: Atractylodes Macrocephala Rhizoma (AMR) is a traditional Chinese medicine containing several sesquiterpenoids with a series of effects. These bioactive compounds may be used as chemical markers for the quality control of AMR. It is necessary to optimise the extraction method and conditions in order to improve extraction productivity. OBJECTIVE: To develop a simple and effective method for the extraction of sesquiterpenoids from AMR and then to simultaneously determine four sesquiterpenoids, selina-4 (14), 7(11)-dien-8-one (SA), atractylenolide II (AII), atractylenolide III (AIII) and atractylenolide VII (AVII), in AMR. METHODOLOGY: Ultrasound-assisted extraction (UAE) was optimised by central composite design (CCD) to obtain the maximum efficiency. The gas chromatography method was validated and applied for the quantification of four sesquiterpenoids. RESULTS: The optimum values of factors were: particle size (120 mesh), extraction time (26 min), extraction temperature (39°C) and 31 mL of chloroform. The selectivity, linear range, limits of detection (LOD) and quantification (LOQ), accuracy, precision and repeatability of the method developed indicated its validity. The application of the method showed that the contents of four sesquiterpenoids in AMR were rather variable. CONCLUSION: The results indicated that the described GC method could be used for the quality control of AMR and its related preparations. Meanwhile, this research revealed that UAE under optimum conditions could be considered as a powerful tool for the extraction of phytochemicals from plants.

Paraptosis accompanied by autophagy and apoptosis was induced by celastrol, a natural compound with influence on proteasome, ER stress and Hsp90.

In the present study, we found that celastrol, a natural compound with well-known apoptosis-inducing effect, could also induce paraptosis-like cytoplasmic vacuolization in cancer cell lines including HeLa cells, A549 cells and PC-3 cells derived from cervix, lung and prostate, respectively. Further study using HeLa cells indicated that the vacuoles induced by celastrol might be derived from dilation of endoplasmic reticulum. And, in celastrol-treated cells, markers of autophagy such as transformation of microtubule-associated protein 1 light chain 3 (LC3)I to LC3II and LC3 punctates formation were identified. Interestingly, autophagy inhibitors could not interrupt but enhance the induction of cytoplasmic vacuolization. Furthermore, MAPK pathways were activated by celastrol and inhibitors of MEK and p38 pathways could prevent the formation of cytoplasmic vacuolization. Celastrol treatment also induced G2/M cell cycle arrest and apoptosis in HeLa cells. In conclusion, celastrol induced a kind of paraptosis accompanied by autophagy and apoptosis in cancer cells. The coincidence of apoptosis and autophagy together with paraptosis might contribute to the unique characteristics of paraptosis in celastrol-treated cells such as the dependence of paraptosis on MAPK pathways and dynamic change of LC3 proteins. Both paraptosis and apoptosis could contribute to the cell death induced by celastrol while autophagy might serve as a kind of survival mechanism. The potency of celastrol to induce paraptosis, apoptosis and autophagy at the same dose might be related to its capability to affect a variety of pathways including proteasome, ER stress and Hsp90.