Triterpene Glucosides from the Leaves of Aralia elata and Their Cytotoxic Activities

Three new triterpene glucosides, named congmuyenosides C–E ( 1 – 3 , resp.), along with four known ones, were isolated from an EtOH extract of Aralia elata (Miq .) Seem . leaves. The structures of the new compounds were identified as 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}caulophyllogenin ( 1 ), 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}hederagenin 28‐O‐β‐D ‐glucopyranosyl ester ( 2 ), 3‐O‐{β‐D ‐glucopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)]‐β‐D ‐glucopyranosyl}echinocystic acid 28‐O‐β‐D ‐glucopyranosyl ester ( 3 ) on the basis of spectral analyses, including MS, ¹H‐NMR, ¹³C‐NMR, DEPT, HSQC, HMBC, NOESY, and HSQC‐TOCSY experiments. All isolates obtained were evaluated for their cytotoxic activities against three human tumor cell lines (HepG2, SKOV3, and A549). Compound 3 showed significant cytotoxicity against A549 cell line (IC₅₀ 9.9±1.5 μM ).     

Formation of adducts in the reaction of glyoxal with 2'-deoxyguanosine and with calf thymus DNA

The reactions of glyoxal with 2′-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, ¹H and ¹³C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx₂-dG) and N²-(carboxymethyl)-9-(2′-deoxy-β-d-erythro-pentofuranosyl)-purin-6(9H)-one (Gx₁-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx₂-dG were found to be unstable and partly transformed to Gx₁-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2′-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2′-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx₁-dG was the only stable adduct. The transformation of Gx-dG to Gx₁-dG seemed to take place in single-stranded DNA and therefore, Gx₁-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound.     

Inhibition of NO Production by Grindelia argentina and Isolation of Three New Cytotoxic Saponins

A bioassay‐guided phytochemical analysis of the ethanolic extract of Grindelia argentina Deble & Oliveira ‐Deble (Asteraceae) allowed the isolation of a known flavone, hispidulin, and three new oleanane‐type saponins, 3‐O‐β‐D ‐xylopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐2β,3β,16α,23‐tetrahydroxyolean‐12‐en‐28‐oic acid 28‐O‐β‐D ‐xylopyranosyl‐(1→2)‐β‐D ‐apiofuranosyl‐(1→3)‐β‐D ‐xylopyranosyl‐(1→3)‐α‐L ‐rhamnopyranosyl‐(1→2)‐α‐L ‐arabinopyranosyl ester ( 2 ), 3‐O‐β‐D ‐glucopyranosyl‐2β,3β,23‐trihydroxyolean‐12‐en‐28‐oic acid 28‐O‐β‐D ‐xylopyranosyl‐(1→2)‐β‐D ‐apiofuranosyl‐(1→3)‐β‐D ‐xylopyranosyl‐(1→3)‐α‐L ‐rhamnopyranosyl‐(1→2)‐α‐L ‐arabinopyranosyl ester, ( 3 ) and 3‐O‐β‐D ‐xylopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐2β,3β,23‐trihydroxyolean‐12‐en‐28‐oic acid 28‐O‐β‐D ‐xylopyranosyl‐(1→2)‐β‐D ‐apiofuranosyl‐(1→3)‐β‐D ‐xylopyranosyl‐(1→3)‐α‐L ‐rhamnopyranosyl‐(1→2)‐α‐L ‐arabinopyranosyl ester ( 4 ), named grindeliosides A–C, respectively. Their structures were determined by extensive 1D‐ and 2D‐NMR experiments along with mass spectrometry and chemical evidence. The isolated compounds were evaluated for their inhibitory activities against LPS/IFN‐γ‐induced NO production in RAW 264.7 macrophages and for their cytotoxic activities against the human leukemic cell line CCRF‐CEM and MRC‐5 lung fibroblasts. Hispidulin markedly reduced LPS/IFN‐γ‐induced NO production (IC₅₀ 51.4 μM ), while grindeliosides A–C were found to be cytotoxic, with grindelioside C being the most active against both CCRF‐CEM (IC₅₀ 4.2±0.1 μM ) and MRC‐5 (IC₅₀ 4.5±0.1 μM ) cell lines.     

Synthesis and Immunostimulating Properties of Novel Adamant‐1‐yl Tripeptides

The aim of this work was to prepare L ‐ and D ‐(adamant‐1‐yl)‐Gly‐L ‐Ala‐D ‐isoGln peptides in order to study their adjuvant (immunostimulating) activities. Adjuvant activity of adamant‐1‐yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM; β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala) and structurally related adamant‐2‐yl tripeptides.     

Simenoside A,a New Triterpenoid Saponin from Gypsophila simonii Hub.‐Mor.

Saponins are amphiphilic glycoconjugates which give soap‐like foams in H₂O. A new triterpenoid saponin, simenoside A ( 1 ), based on gypsogenin aglycone, and the known saponin 2 were isolated from Gypsophila simonii Hub.‐Mor. The structure of the new saponin was elucidated as 3‐O‐β‐D ‐galactopyranosyl‐(1→2)‐[β‐D ‐xylopyranosyl‐(1→3)]‐β‐D ‐glucuronopyranosylgypsogenin 28‐O‐β‐D ‐glucopyranosyl‐(1→3)‐[β‐D ‐glucopyranosyl‐(1→2)‐β‐D ‐xylopyranosyl‐(1→4)]‐α‐L ‐rhamnopyranosyl‐(1→2)‐β‐D ‐fucopyranosyl ester on the basis of extensive spectral analyses and chemical evidence. Saponins 1 and 2 were isolated from G. simonii for the first time.     

Characterization of a type of β‐bend ribbon spiral generated by the repeating (Xaa–Yaa–Aib–Pro) motif: The solution structure of harzianin HC IX,a 14‐residue peptaibol forming voltage‐dependent ion channels

The three‐dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two‐dimensional nmr spectroscopy combined with molecular modeling. This 14‐residue peptide, Ac Aib¹ Asn² Leu³ Aib⁴ Pro⁵ Ala⁶ Ile⁷ Aib⁸ Pro⁹ Iva¹⁰ Leu¹¹ Aib¹² Pro¹³ Leuol¹⁴ (Aib, α‐aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short‐sequence peptaibol class characterized by an acetylated N‐terminus, a C‐terminal amino alcohol, and the presence of three Aib‐L ‐Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20‐residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane‐perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage‐gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence‐specific ¹H‐ and ¹³C‐nmr assignments and conformational nmr parameters (³J_NHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty‐seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β‐turns twisted into a continuous spiral from Asn² to Leuol¹⁴ and forming a 26 Å long helix‐like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn² side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φ_i = −90°, Ψ_i = −27°; Φ_i+1 = −98°, Ψ_i+1 = −17°; Φ_i+2 = −49°, Ψ_i+2 = −50°; Φ_i+3 = −78°, Ψ_i+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)_n sequences, the (Xaa–Yaa–Aib–Pro)‐β‐bend ribbon spiral. It is stabilized by 4 → 1 intramolecular hydrogen bonds and differs from both the canonical 3₁₀‐helix made of a succession of type III β‐turns and from the β‐bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999     

Interferon‐γ suppresses Na+–H+ exchanger in cultured human endolymphatic sac epithelial cells

Adequate regulation of endolymphatic pH is essential for maintaining inner ear function. The Na⁺–H⁺ exchanger (NHE) is a major determinant of intracellular pH (pH_i), and facilitates Na⁺ and fluid absorption in various epithelia. We determined the functional and molecular expression of NHEs in cultured human endolymphatic sac (ES) epithelial cells and examined the effect of IFN‐γ on NHE function. Serial cultures of human ES epithelial cells were generated from tissue samples. The molecular expression of NHE1, ‐2, and ‐3 isoforms was determined by real‐time RT‐PCR. The functional activity of NHE isoforms was measured microfluorometrically using a pH‐sensitive fluorescent dye, 2′,7′‐bis(carbonylethyl)‐5(6)‐carboxyfluorescein (BCECF), and a NHE‐inhibitor, 3‐methylsulfonyl‐4‐piperidinobenzoyl guanidine methanesulfonate (HOE694). NHE1, ‐2, and ‐3 mRNAs were expressed in human ES epithelial cells. Functional activity of NHE1 and ‐2 was confirmed in the luminal membrane of ES epithelial cells by sequentially suppressing Na⁺‐dependent pH_i recovery from intracellular acidification using different concentrations of HOE694. Treatment with IFN‐γ (50 nM for 24 h) suppressed mRNA expression of NHE1 and ‐2. IFN‐γ also suppressed functional activity of both NHE1 and ‐2 in the luminal membrane of ES epithelial cells. This study shows that NHEs are expressed in cultured human ES epithelial cells and that treatment with IFN‐γ suppresses the expression and functional activity of NHE1 and ‐2. J. Cell. Biochem. 107: 965–972, 2009. © 2009 Wiley‐Liss, Inc.     

Cytotoxic Oleanane‐Type Saponins from the Leaves of Albizia anthelmintica Brongn.

Two new oleanane‐type saponins: β‐d ‐xylopyranosyl‐(1 → 4)‐6‐deoxy‐α‐l ‐mannopyranosyl‐(1 → 2)‐1‐O‐{(3β)‐28‐oxo‐3‐[(2‐O‐β‐d ‐xylopyranosyl‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐yl}‐β‐d ‐glucopyranose ( 1 ) and 1‐O‐[(3β)‐28‐oxo‐3‐{[β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐arabinopyranosyl‐(1 → 6)‐2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐yl]β‐d ‐glucopyranose ( 2 ), along with two known saponins: (3β)‐3‐[(β‐d ‐Glucopyranosyl‐(1 → 2)‐β‐d ‐glucopyranosyl)oxy]olean‐12‐en‐28‐oic acid ( 3 ) and (3β)‐3‐{[α‐l ‐arabinopyranosyl‐(1 → 6)‐[β‐d ‐glucopyranosyl‐(1 → 2)]‐β‐d ‐glucopyranosyl]oxy}olean‐12‐en‐28‐oic acid ( 4 ) were isolated from the acetone‐insoluble fraction obtained from the 80% aqueous MeOH extract of Albizia anthelmintica Brongn . leaves. Their structures were identified using different NMR experiments including: ¹H‐ and ¹³C‐NMR, HSQC, HMBC and ¹H,¹H‐COSY, together with HR‐ESI‐MS/MS, as well as by acid hydrolysis. The four isolated saponins and the fractions of the extract exhibited cytotoxic activity against HepG‐2 and HCT‐116 cell lines. Compound 2 showed the most potent cytotoxic activity among the other tested compounds against the HepG2 cell line with an IC₅₀ value of 3.60μm . Whereas, compound 1 showed the most potent cytotoxic effect with an IC₅₀ value of 4.75μm on HCT‐116 cells.     

Enantiomeric and Diastereoisomeric (Mixed) L/ D‐Octaarginine Derivatives – A Simple Way of Modulating the Properties of Cell‐Penetrating Peptides

Cell‐penetrating peptides (CPPs) are promising vehicles for delivery of drugs, antibiotics, proteins, nucleic acid derivatives, etc. into eukaryotic and prokaryotic target cells. To prevent premature degradation, CPPs consisting of D ‐ or β‐amino acid residues have been used. We present simple models for the various modes of delivery of physiologically active cargoes by CPPs, depending on the nature of their conjugation (Fig. 1), and we describe the plasma stability of oligoarginines (OAs) 1 – 4 , the most common unnatural CPPs. Fluorescein‐labeled L ‐octaarginine 1 was found to have a half‐life (t_1/2) of <0.5 min, the D ‐enantiomer ( 2 ) of >7 d (Fig. 2). For possible medicinal applications, the former type of derivative would be too unstable, and the latter one undesirably persistent. Thus, seven of the 256 possible ‘mixed’ Flua‐L /D ‐octaarginine amides, 4a – 4g , were synthesized and shown to have half‐lives in heparine‐stabilized human plasma between 8 min and 5.5 h (Figs. 3 and 4). The cell penetration of the new OAs was investigated with ‘healthy’ and with apoptotic HEK cells (Figs. 5–8), and their interactions with phospholipid bilayers were studied, using anionic lipid vesicles (Figs. 9 and 10). There are surprisingly large differences in the rates of cell penetration and binding to vesicle walls between the various stereoisomeric octaarginine derivatives 1, 2 , and 4a – 4g (Figs. 5 and 7). – The role of D ‐amino acids and D ‐peptides in nature and in drug design is briefly discussed and referenced.     

Synthesis of 2‐amino‐3‐cyanopyridine derivatives and investigation of their carbonic anhydrase inhibition effects

The conversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻) to each other is very important for living metabolism. Carbonic anhydrase (CA, E.C.4.2.1.1), a metalloenzyme familly, catalyzes the interconversion of these ions (CO₂ and HCO₃⁻) and are very common in living organisms. In this study, a series of novel 2‐amino‐3‐cyanopyridines supported with some functional groups was synthesized and tested as potential inhibition effects against both cytosolic human CA I and II isoenzymes (hCA I and II) using by Sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity chromatography. The structural elucidations of novel 2‐amino‐3‐cyanopyridines were achieved by NMR, IR, and elemental analyses. K _i values of the novel synthesized compounds were found in range of 2.84–112.44 μM against hCA I and 2.56–31.17 μM against hCA II isoenzyme. While compound 7d showed the best inhibition activity against hCA I (K _i: 2.84 μM), the compound 7b demonstrated the best inhibition profile against hCA II isoenzyme (K _i: 2.56 μM).     

Glyoxal and malonaldehyde formation by ultraviolet irradiation of aqueous formaldehyde

Irradiation of dilute aqueous formaldehyde (5 × 10^?2–10^?3M) in the absence of oxygen by ultaviolet light from high- or low-pressure mercury lamps resulted in the formation of glyoxal and of malonaldehyde. The concentration of malonaldehyde reached a maximum after several hours and then declined. This maximal malonaldehyde concentration was proportional to the initial formaldehyde concentration. At initially 0.05 M formaldehyde (pH 9.4 and 36°C) malonaldehyde reached maximally 3.4 × 10^?5 M. In the range of pH 8.0–11.6, the maximal malonaldehyde concentration was reached at pH 9.4. Quantum yields of glyoxal and malonaldehyde after irradiation of 0.01 M formaldehyde (in 0.01 M NaHCO₃, 27°C, at 254 nm, under argon, for 195 min) were 7 × 10^?3 and 1.5 × 10^?3, respectively. In the presence of acetone (0.01 M), the chemical and quantum yields of glyoxal were enhanced, while those of malonaldehyde decreased. The known reaction of malonaldehyde with urea to form pyrimidines may be a model of a prebiotic synthesis of pyrimidines.     

Synthesis and Anti‐HIV Activity of [ddN]‐[ddN] Dimers and Benzimidazole Nucleoside Dimers

In an attempt to combine the HIV‐inhibitory capacity of different 2′,3′‐dideoxynucleoside (ddN) analogs, we have designed and synthesized several dimers of [AZT]‐[AZT] and [AZT]‐[d4T]. In addition, we also synthesized the dimers of 1‐(1H‐benzimidazol‐1‐yl)‐1‐deoxy‐β‐D ‐ribofuranose. The in vitro anti‐HIV activity of these compounds on a pseudotype virus, pNL4‐3.Luc.R‐E‐, in the 293T cells has been determined. Among these compounds, 2,2′‐(propane‐1,3‐diyl)bis[1‐(β‐D ‐ribofuranosyl)‐1H‐benzimidazole] ( 3 ) showed the highest anti‐HIV activity with similar effect as AZT.     

β‐sheet breaker peptide inhibitor of Alzheimer's amyloidogenesis with increased blood–brain barrier permeability and resistance to proteolytic degradation in plasma

Short synthetic peptides homologous to the central region of Aβ but bearing proline residues as β‐sheet blockers have been shown in vitro to bind to Aβ with high affinity, partially inhibit Aβ fibrillogenesis, and redissolve preformed fibrils. While short peptides have been used extensively as therapeutic drugs in medicine, two important problems associated with their use in central nervous system diseases have to be addressed: (a) rapid proteolytic degradation in plasma, and (b) poor blood–brain barrier (BBB) permeability. Recently, we have demonstrated that the covalent modification of proteins with the naturally occurring polyamines significantly increases their permeability at the BBB. We have extended this technology to iAβ11, an 11‐residue β‐sheet breaker peptide that inhibits Aβ fibrillogenesis, by covalently modifying this peptide with the polyamine, putrescine (PUT), and evaluating its plasma pharmacokinetics and BBB permeability. After a single intravenous bolus injection in rats, both ¹²⁵I‐YiAβ11 and ¹²⁵I‐PUT‐YiAβ11 showed rapid degradation in plasma as determined by trichloroacetic acid (TCA) precipitation and paper chromatography. By switching to the all d ‐enantiomers of YiAβ11 and PUT‐YiAβ11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively. The permeability coefficient × surface area product at the BBB was five‐ sevenfold higher in the cortex and hippocampus for the ¹²⁵I‐PUT‐d ‐YiAβ11 compared to the ¹²⁵I‐d ‐YiAβ11, with no significant difference in the residual plasma volume. In vitro assays showed that PUT‐d ‐YiAβ11 retains its ability to partially inhibit Aβ fibrillogenesis and dissolve preformed amyloid fibrils. Because of its five‐ to sevenfold increase in permeability at the BBB and its resistance to proteolysis in the plasma, this polyamine‐modified β‐sheet breaker peptide may prove to be an effective inhibitor of amyloidogenesis in vivo and, hence, an important therapy for Alzheimer's disease. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 371–382, 1999     

Dodecyl α‐L‐Rhamnopyranosyl‐(1→2)‐β‐D‐fucopyranoside Derivatives from the Glandular Trichome Exudate of Erodium pelargoniflorum

Chemical investigation of the glandular trichome exudate of Erodium pelargoniflorum (Geraniaceae) led to the isolation of two dodecyl disaccharide derivatives, named pelargoside A1 and pelargoside B1 ( 1 and 2 , resp.). The structures of 1 and 2 were determined as dodecyl 4‐O‐acetyl‐α‐L ‐rhamnopyranosyl‐(1→2)‐4‐O‐acetyl‐β‐D ‐fucopyranoside and dodecyl 3,4‐di‐O‐acetyl‐α‐L ‐rhamnopyranosyl‐(1→2)‐4‐O‐acetyl‐β‐D ‐fucopyranoside, respectively, by spectroscopic studies, including 2D‐NMR, and chemical transformations. In addition, undecyl, tridecyl, and tetradecyl homologs of 1 and 2 , named pelargosides A2–A4 and pelargosides B2–B4, were also characterized as minor constituents of the exudate.     

Triterpene Saponins from the Roots of Ilex asprella

Six new triterpene saponins, ilexasprellanosides A–F ( 1 – 6 , resp.), together with eleven known compounds were isolated from the roots of Ilex asprella. The new saponins were characterized as ursa‐12,18‐dien‐28‐oic acid 3‐O‐β‐D ‐xylopyranoside ( 1 ), 19α‐hydroxyursolic acid 3‐O‐β‐D ‐(2′‐O‐acetylxylopyranoside) ( 2 ), 19α‐hydroxyursolic acid 3‐O‐β‐D ‐glucuronopyranoside ( 3 ), 3β,19α‐dihydroxyolean‐12‐en‐23,28‐dioic acid 28‐O‐β‐D ‐glucopyranoside ( 4 ), 19α‐hydroxyoleanolic acid 3‐O‐β‐D ‐(2′‐O‐acetylxylopyranoside) ( 5 ), 19α‐hydroxyoleanolic acid 3‐O‐β‐D ‐glucuronopyranoside ( 6 ). The structures of the new compounds were elucidated by analysis of their spectroscopic data and chemical degradation. Compounds 2, 4 , oleanolic acid 3‐O‐β‐D ‐glucuronopyranoside, 3‐β‐acetoxy‐28‐hydroxyurs‐12‐ene, and pomolic acid showed significant cytotoxic activities against human tumor cell line A549 (IC₅₀ values of 1.87, 2.51, 1.41, 3.24, and 5.63 μM , resp.).     

Six New Polyhydroxysteroidal Glycosides,Anthenosides S1 – S6, from the Starfish Anthenea sibogae

Six new polyhydroxysteroidal glycosides, anthenosides S1  –  S6 ( 1  –  6 ), along with a mixture of two previously known related glycosides, 7 and 8 , were isolated from the methanolic extract of the starfish Anthenea sibogae. The structures of 1  –  6 were established by NMR and HR‐ESI‐MS techniques as well as by chemical transformations. All new compounds have a 5α‐cholest‐8(14)‐ene‐3α,6β,7β,16α‐tetrahydroxysteroidal nucleus and differ from majority of starfish glycosides in positions of carbohydrate moieties at C(7) and C(16) ( 1  –  4 , 6 ) or only at C(16) ( 5 ). The 4‐O‐methyl‐β‐d ‐glucopyranose residue ( 2 ) and Δ²⁴‐cholestane side chain ( 3 ) have not been found earlier in the starfish steroidal glycosides. The mixture of 7 and 8 slightly inhibited the proliferation of human breast cancer T‐47D cells and decreased the colony size in the colony formation assay.     

Effect of β-amyloid (25-35) on mitochondrial function and expression of mitochondrial permeability transition pore proteins in rat hippocampal neurons

The aim of this study was to assess the effect of the β‐amyloid fragment Aβ_25–35 on mitochondrial structure and function and on the expression of proteins associated with the mitochondrial permeability transition pore (MPTP) in rat hippocampal neurons. Ninety clean‐grade Sprague–Dawley rats were randomly assigned to six groups (n = 15 per group). Aβ_25–35 (1, 5, or 10 µg/rat) was injected into hippocampal area CA1. Normal saline was injected as a control. The effect of Aβ_25–35 injection on hippocampal structure was assessed by transmission electron microscopy. Ca²⁺‐ATPase activity, [Ca²⁺]_i, and mitochondrial membrane potential were measured. The expression of genes associated with the MPTP, including the voltage‐dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (Cyp‐D), were evaluated. Results showed that Aβ_25–35 injection damaged the mitochondrial structure of hippocampal neurons, decreased Ca²⁺‐ATPase activity and mitochondrial membrane potential, and increased [Ca²⁺]_i. The expression levels for VDAC, ANT, and Cyp‐D in all groups were significantly (P < 0.05) higher than those in the normal control group after Aβ_25–35 injection. These results indicate that Aβ_25–35 damages mitochondria in rat hippocampal neurons and effects mitochondrial dysfunction, as well as increasing the expression of genes associated with the MPTP. Mitochondrial dysfunction may result in increased MPTP gene expression, leading to neurodegenerative effects. J. Cell. Biochem. 112: 1450–1457, 2011. © 2011 Wiley‐Liss, Inc.     

Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Cytotoxic Steroidal Saponins from the Rhizomes of Tacca integrifolia

Three new steroid saponins (3β,25R)‐spirost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[β‐D ‐glucopyranosyl‐(1→4)‐6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 1 ), (3β,22R,25R)‐26‐(β‐D ‐glucopyranosyloxy)‐22‐hydroxyfurost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 3 ), and (3β,22R,25R)‐26‐(β‐D ‐glucopyranosyloxy)‐22‐hydroxyfurost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[β‐D ‐glucopyranosyl‐(1→4)‐6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 5 ), as well as the new pregnane glycoside (3β,16β)‐3‐{[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranosyl]oxy}‐20‐oxopregn‐5‐en‐16‐yl (4R)‐5‐(β‐D ‐glucopyranosyloxy)‐4‐methylpentanoate ( 6 ), were isolated from the rhizomes of Tacca integrifolia together with two known (25R) configurated steroid saponins (3β,25R)‐spirost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 2 ) and (3β,22R,25R)‐26‐(β‐D ‐glucopyranosyloxy)‐22‐methoxyfurost‐5‐en‐3‐yl 6‐deoxy‐α‐L ‐mannopyranosyl‐(1→2)‐[6‐deoxy‐α‐L ‐mannopyranosyl‐(1→3)]‐β‐D ‐glucopyranoside ( 4 ). The cytotoxic activity of the isolated compounds was evaluated in HeLa cells and showed the highest cytotoxicity value for compound 2 with an IC₅₀ of 1.2±0.4 μM . Intriguingly, while compounds 1 – 5 exhibited similar cytotoxic properties between 1.2±0.4 ( 2 ) and 4.0±0.6 μM ( 5 ), only compound 2 showed a significant microtubule‐stabilizing activity in vitro.     

Four New Steroidal Glycosides,Protolinckiosides A – D,from the Starfish Protoreaster lincki

Four new steroidal glycosides, protolinckiosides A – D ( 1 – 4 , resp.), were isolated along with four previously known glycosides, 5 – 8 , from the MeOH/EtOH extract of the starfish Protoreaster lincki. The structures of 1 – 4 were elucidated by extensive NMR and ESI‐MS techniques as (3β,4β,5α,6β,7α,15α,16β,25S)‐4,6,7,8,15,16,26‐heptahydroxycholestan‐3‐yl 2‐O‐methyl‐β‐d ‐xylopyranoside ( 1 ), (3β,5α,6β,15α,24S)‐3,5,6,8,15‐pentahydroxycholestan‐24‐yl α‐l ‐arabinofuranoside ( 2 ), sodium (3β,6β,15α,16β,24R)‐29‐(β‐d ‐galactofuranosyloxy)‐6,8,16‐trihydroxy‐3‐[(2‐O‐methyl‐β‐d ‐xylopyranosyl)oxy]stigmast‐4‐en‐15‐yl sulfate ( 3 ), and sodium (3β,6β,15α,16β,22E,24R)‐28‐(β‐d ‐galactofuranosyloxy)‐6,8,16‐trihydroxy‐3‐[(2‐O‐methyl‐β‐d ‐xylopyranosyl)oxy]ergosta‐4,22‐dien‐15‐yl sulfate ( 4 ). The unsubstituted β‐d ‐galactofuranose residue at C(28) or C(29) of the side chains was found in starfish steroidal glycosides for the first time. Compounds 1 – 4 significantly decreased the intracellular reactive oxygen species (ROS) content in RAW 264.7 murine macrophages at induction by proinflammatory endotoxic lipopolysaccharide (LPS) from E. coli.