Synthesis and biological activity of 17α-alkynylamide derivatives of estradiol

Seven estradiol (E₂) derivatives with an alkynylamide side chain at the 17α position were synthesized starting from ethynylestradiol (EE₂). The main chemical step was the coupling reaction of the acetylide ion of EE₂ with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3–6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lenghts were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vitro system. At low doses (1 and 3 μg), a 14–57% inhibition of E₂-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 μg doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-buty,N-methyl-8-[3′,17′β-dihydroxy estra-1′,3′,5′(10′)-trien-17′α-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE₂ possessing a five-methylene (CH₂) side chain (compound 39) possesses the best antiestrogenic activity (44 ± 7% in the CD-1 mouse uterus assay at the 3μg dose and 57 ± 4% at 0.1 nM in human ZR-75-1 cancer cells in culture).     

Dimerization of Bisphenol A by Hyper Lignin-Degrading Fungus Phanerochaete sordida YK-624 Under Ligninolytic Condition

Bisphenol A (BPA) was treated with hyper lignin-degrading fungus Phanerochaete sordida YK-624 under ligninolytic condition. After preculturing P. sordida YK-624 for 4 days, BPA (final concentration, 1 and 0.1 mM) was added to cultures. Both 1- and 0.1-mM BPA were effectively decreased within a 24-h treatment and two metabolites were detected. Two metabolites (5,5′-bis-[1-(4-hydroxy-phenyl)1-methyl-ethyl]-biphenyl-2,2′-diol and 4-(2-(4-hydroxy-phenyl) propan-2-yl)-2-(4-(2-(4-hydroxyphenyl) propan-2-yl) phenoxy)phenol) were identified by ESI–MS and NMR analysis. These results indicated that BPA was oxidized to BPA phenoxy radicals by ligninolytic enzymes and then dimerized at extracellular region.     

Up-regulation of cyclic electron flow and down-regulation of linear electron flow in antisense-<Emphasis Type="Italic">rca</Emphasis> mutant rice

To investigate how excess excitation energy is dissipated in a ribulose-1,5-bisphospate carboxylase/oxygenase activase antisense transgenic rice with net photosynthetic rate (P _N) half of that of wild type parent, we measured the response curve of P _N to intercellular CO₂ concentration (C _i), electron transport rate (ETR), quantum yield of open photosystem 2 (PS2) reaction centres under irradiation (F_v′/F_m′), efficiency of total PS2 centres (Φ_PS2), photochemical (q_P) and non-photochemical quenching (NPQ), post-irradiation transient increase in chlorophyll (Chl) fluorescence (PITICF), and P700⁺ re-reduction. Carboxylation efficiency dependence on C _i, ETR at saturation irradiance, and F_v′/F_m′, Φ_PS2, and q_P under the irradiation were significantly lower in the mutant. However, NPQ, energy-dependent quenching (q_E), PITICF, and P700⁺ re-reduction were significantly higher in the mutant. Hence the mutant down-regulates linear ETR and stimulates cyclic electron flow around PS1, which may generate the ΔpH to support NPQ and q_E for dissipation of excess excitation energy.     

<Superscript>1</Superscript>H NMR-based metabolic profiling and target analysis: a combined approach for the quality control of <Emphasis Type="Italic">Thymus vulgaris</Emphasis>

The characterization of T. vulgaris plant material for quality control purposes was performed by NMR-based methods. Direct extraction of 141 T. vulgaris samples with DMSO-d ₆ enabled the obtainment of crude extracts with a representative composition in terms of both volatile and non-volatile constituents. The acquisition of 600 MHz ¹H NMR spectra resulted in a dataset which was analyzed by a combination of metabolic profiling and target analysis approaches. Preliminary analysis of the ¹H NMR spectra was performed by principal component analysis, which revealed sample discrimination on a chemotype basis (thymol, carvacrol and linalool chemotypes). Further minor discriminative constituents were identified as p-cymene, γ-terpinene, rosmarinic acid, and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl. Metabolite identification was accomplished by 1D and 2D NMR techniques and supported by spiking experiments. Fast dereplication of constituents not available as reference compounds was performed by HPLC–SPE–NMR experiments. A targeted approach based on qHNMR was validated for quantification of the identified secondary metabolites. Validation was performed in terms of precision (intra-day RSD ≤ 4.51%, inter-day RSD ≤ 4.18%), repeatability (RSD ≤ 2.30%), accuracy (recovery rates within 93.4 and 103.4%), linearity (correlation coefficients ≥ 0.9990), robustness, and stability. The amount of the dominant monoterpene in thymol, carvacrol, and linalool chemotypes was respectively found to be within 0.4–2.6, 0.7–2.3, and 1.1–3.6% (w/w). Variable amounts of the precursors p-cymene and γ-terpinene were found in thymol and carvacrol chemotypes. The highest amount of rosmarinic acid and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl in the analyzed samples was respectively 4.6 and 0.4% (w/w). Since quantification is performed on a weight basis, the essential oil content can be estimated based on the sum of the quantified monoterpenes. The NMR-based analysis of T. vulgaris represents a more comprehensive approach in comparison to traditional chromatographic methods such as GC and LC, respectively employed for the analysis of volatile and non-volatile constituents. Further advantages lie in the simple sample preparation, rapidity and reproducibility of the NMR analysis.     

Characterization of structure and activity of garlic peroxidase (POX<Subscript>1B</Subscript>)

Structural characterization and study of the activity of new POX_1B protein from garlic which has a high peroxidase activity and can be used as a biosensor for the detection of hydrogen peroxide and phenolic compounds were performed and compared with the findings for other heme peroxidases. The structure–function relationship was investigated by analysis of the spectroscopic properties and correlated to the structure determined by a new generation of high-performance hybrid mass spectrometers. The reactivity of the enzyme was analyzed by studies of the redox activity toward various ligands and the reactivity with various substrates. We demonstrated that, in the case of garlic peroxidase, the heme group is pentacoordinated, and has an histidine as a proximal ligand. POX_1B exhibited a high affinity for hydrogen peroxide as well as various reducing cosubstrates. In addition, high enzyme specificity was demonstrated. The k _cat and K _M values were 411 and 400 mM⁻¹ s⁻¹ for 3,3′,5,5′-tetramethylbenzidine and 2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), respectively. Furthermore, the reduction of nitro compounds in the presence of POX_1B was demonstrated by iron(II) nitrosoalkane complex assay. In addition, POX_1B showed a great potential for application for drug metabolism since its ability to react with 1-nitrohexane in the presence of sodium dithionite was demonstrated by the appearance of a characteristic Soret band at 411 nm. The high catalytic efficiency obtained in the case of the new garlic peroxidase (POX_1B) is suitable for the monitoring of different analytes and biocatalysis.     

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth

Summary The effect of 17β-estradiol (E₂) on growth of GH₄C₁ rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E₂ responsive only when growth was retarded by reducing the T₃ concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E₂ to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E₂ to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors. This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc.     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. II. The ligninolytic enzymes of the coal-humic-acid-depolymerizing fungus Nematoloma frowardii b19

The production of ligninolytic enzymes was studied in surface cultures of the South American white-rot fungus Nematoloma frowardii b19 and four other strains of this ecophysiological group (Clitocybula dusenii b11, Auricularia sp. m37a, wood isolates u39 and u45), which are able to depolymerize low-rank-coal-derived humic acids with the formation of fulvic-acid-like compounds. The fungi produced the three crucial enzymes of lignin degradation – lignin peroxidase, manganese peroxidase and laccase. In the case of N. frowardii b19, laccase and the two peroxidases could be stimulated by veratryl alcohol. Manganese (II) ions (Mn²⁺) caused a rapid increase of Mn peroxidase activity accompanied by the complete repression of lignin peroxidase. Under nitrogen-limited conditions the growth as well as the production of ligninolytic enzymes was partly repressed. During the depolymerization process of coal humic acids using solid agar media, gradients of ligninolytic enzyme activities toward 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate) and syringaldazine were detectable inside the agar medium. Received: 5 August 1996 / Received revision: 13 November 1996 / Accepted: 15 November 1996     

DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l⁻¹). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l⁻¹. Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as “dye-decolorizing peroxidases”. Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn²⁺) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.     

Photobiochemistry of folates: A photochemical reduction of folic acid

Exposure of deaerated folic acid solutions containing an electron donor to UV radiation (310–390 nm, I = 0.4 W m⁻²) induced formation of dihydrofolic acid (DHFA), a photoexcitation which gave tetrahydrofolic acid (THFA). Only DHFA was formed in the presence of EDTA (E′_o = +0.40 V), while the presence of stronger reductants—NADH (E′_o = −0.32 V) and boron hydride (E′_o = −0.48 V)—induced photoreduction to THFA. It was demonstrated that UV radiation had no effect on the THFA formylation, giving the coenzyme 5,10-methenyltetrahydrofolic acid and its transformation into another coenzyme, 5-formyltetrahydrofolic acid.     

Purification and characterisation of lignin peroxidase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> MTCC-137

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K _m, k _cat and k _cat/K _m values of the enzyme using veratryl alcohol and H₂O₂ as the substrate were 61 M, 2.13 s⁻¹, 3.5 × 10⁴ M⁻¹s⁻¹ and 71 M, 2.13 s⁻¹, 3.0 × 10⁴ M⁻¹ s⁻¹ respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25°C.     

Stimulation of artemisinin synthesis by combined cerebroside and nitric oxide elicitation in <Emphasis Type="Italic">Artemisia annua</Emphasis> hairy roots

This work examined the accumulation of artemisinin and related secondary metabolism pathways in hairy root cultures of Artemisia annua L. induced by a fungal-derived cerebroside (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine. The presence of the cerebroside induced nitric oxide (NO) burst and artemisinin biosynthesis in the hairy roots. The endogenous NO generation was examined to be involved in the cerebroside-induced biosynthesis of artemisinin by using NO inhibitors, N _ω-nitro-l-arginine methyl ester and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The gene expression and activity of 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate synthase were stimulated by the cerebroside, but more strongly by the potentiation of NO. While the mevalonate pathway inhibitor, mevinolin, only partially inhibited the induced artemisinin accumulation, the plastidic 2-C-methyl-d-erythritol 4-phosphate pathway inhibitor, fosmidomycin, nearly arrested artemisinin accumulation induced by cerebroside and the combination elicitation with an NO donor, sodium nitroprusside (SNP). With the potentiation by SNP at 10 μM, the cerebroside elicitor stimulated artemisinin production in 20-day-old hairy root cultures up to 22.4 mg/l, a 2.3-fold increase over the control. These results suggest that cerebroside plays as a novel elicitor and the involvement of NO in the signaling pathway of the elicitor activity for artemisinin biosynthesis.     

Benzothiazole bipyridine complexes of ruthenium(II) with cytotoxic activity

A series of benzothiazole-substituted trisbipyridine ruthenium(II) analogues {[Ru(bpy)₂(4,5′-bbtb)]²⁺, [Ru(bpy)₂(5,5′-bbtb)]²⁺ and [Ru(bpy)₂(5-mbtb)]²⁺ [bpy is 2,2′-bipyridine, bbtb is bis(benzothiazol-2-yl)-2,2′-bipyridine, 5-mbtb is 5-(benzothiazol-2-yl),5′-methyl-2,2′-bipyridine]} have been prepared and compared with the complex [Ru(bpy)₂(4,4′-bbtb)]²⁺ reported previously. From the UV–vis spectral studies, substitution at the 5-position of the bpy causes the ligand-centred transitions to occur at considerably lower energy than for those with the functionality at the 4-position, while at the same time causing the emission to be effectively quenched. However, substitution at the 4-position causes the metal-to-ligand charge transfer to occur at lower energies. Fluorescent intercalator displacement studies indicate that the doubly substituted complexes displace ethidium bromide from a range of oligonucleotides, with the greater preference shown for bulge and hairpin sequences by the Λ enantiomer. Since the complexes only show small variation in the UV–vis spectra on the introduction of calf thymus DNA and a small increase in fluorescence they do not appear to be intercalators, but appear to associate within one of the grooves. All of the reported bisbenzothiazole complexes show reasonable cytotoxicity against a range of human cancer cell lines. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High constitutive peroxidase activity and constitutive thermotolerance in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

 During the isolation of mutations in the heat-inducible hsp70-1 gene of Neurospora crassa by RIP (repeat-induced point mutations), several transformants were generated by electroporation of conidia with a plasmid harboring an incomplete copy of this gene. One isolate, designated E-45, containing ectopically integrated hsp70-1 DNA, exhibited a slow growth rate, low-temperature sensitivity, constitutive thermotolerance (without prior heat shock), and high constitutive peroxidase activity. The constitutive form of peroxidase (CP) was distinguishable from the heat-inducible form (HIP) by immunoinactivation employing polyclonal antiserum against the latter enzyme and by electrophoretic resolution in nondenaturing polyacrylamide gels. This enzyme was purified to near homogeneity and some of its properties examined. The relative molecular mass of native CP was in the range of 118–136 kDa, as estimated by gel filtration analysis on size exclusion matrices, whereas SDS-PAGE analysis yielded a size of ∼37 kDa for the polypeptide. Substrate saturation kinetics studies were conducted using ABTS [2,2′-azino-bis (3-ethylbenzthiazole-6-sulfonic acid)] and H₂O₂ as substrates: K _m, V _max, and K _cat values for H₂O₂ were ∼22 μM, ∼447 nmol mg⁻¹, and 0.33 s⁻¹, respectively, and those for ABTS were ∼55 μM, ∼453 nmol mg⁻¹, and 0.3 s⁻¹, respectively. Guaiacol was not used as a substrate by this enzyme. CP peroxidase was shown to be a heme-containing enzyme, stable at temperatures up to 58°C. Received: August 5, 2002 / Accepted: January 22, 2003 Acknowledgments This work was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to M.K.). The financial support provided to A. M. in the form of a graduate studentship award by the AHFMR (Alberta Heritage Foundation for Medical Research) and of a graduate teaching assistantship to A. S. by the Department of Biological Sciences, University of Calgary, is gratefully acknowledged. Correspondence to:M. Kapoor     

Three new neolignan glucosides from the stems of Dendrobium aurantiacum var. denneanum

Three new neolignan glucosides (1–3), together with four known analogs (4–7), have been isolated from the stems of Dendrobium aurantiacum var. denneanum. Structures of the new compounds including the absolute configurations were determined by spectroscopic and chemical methods as (−)-(8R,7′E)-4-hydroxy-3,3′,5,5′-tetramethoxy-8,4′-oxyneolign-7′-ene-9,9′-diol 4,9-bis-O-β-d-glucopyranoside (1), (−)-(8S,7′E)-4-hydroxy-3,3′,5,5′-tetramethoxy-8,4′-oxyneolign-7′-ene-9,9′-diol 4,9-bis-O-β-d-glucopyranoside (2), and (−)-(8R,7′E)-4-hydroxy-3,3′,5,5′,9′-pentamethoxy-8,4′-oxyneolign-7′-ene-9-ol 4,9-bis-O-β-d-glucopyranoside (3), respectively.     

Effects of different irradiances on the photosynthetic process during ex-vitro acclimation of Anoectochilus plantlets

Six months old in vitro-grown Anoectochilus formosanus plantlets were transferred to ex-vitro acclimation under low irradiance, LI [60 μmol(photon) m⁻² s⁻¹], intermediate irradiance, II [180 μmol(photon) m⁻² s⁻¹], and high irradiance, HI [300 μmol(photon) m⁻² s⁻¹] for 30 d. Imposition of II led to a significant increase of chlorophyll (Chl) b content, rates of net photosynthesis (P _N) and transpiration (E), stomatal conductance (g _s), electron transfer rate (ETR), quantum yield of electron transport from water through photosystem 2 (Φ_PS2), and activity of ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBPCO, EC 4.1.1.39). This indicates that Anoectochilus was better acclimated at II compared to LI treatment. On the other hand, HI acclimation led to a significant reduction of Chl a and b, P _N, E, g _s, photochemical quenching, dark-adapted quantum efficiency of open PS2 centres (F_v/F_m), probability of an absorbed photon reaching an open PS2 reaction centre (F_v′/F_m′), ETR, Φ_PS2, and energy efficiency of CO₂ fixation (Φ_CO2/Φ_PS2). This indicates that HI treatment considerably exceeded the photo-protective capacity and Anoectochilus suffered HI induced damage to the photosynthetic apparatus. Imposition of HI significantly increased the contents of antheraxanthin and zeaxanthin (ZEA), non-photochemical quenching, and conversion of violaxanthin to ZEA. Thus Anoectochilus modifies its system to dissipate excess excitation energy and to protect the photosynthetic machinery.     

Biological activity of enantiomeric complexes [PtCl2L2] (L2 is aromatic bisphosphanes and aromatic diamines)

Enantiomeric complexes of formula [PtCl₂L₂] [L₂ is (R)-(+)-BINAP and (S)-(−)-BINAP, where BINAP is 2,2′-bis(diphenylphosphane)-1,1′-binaphthyl, and (R)-(+)-DABN and (S)-(−)-DABN, where DABN is 1,1′-binaphthyl-2,2′-diamine], were tested for their cytotoxic activity against three cancer cell lines and for their ability to bind to the human telomeric sequence folded in the G-quadruplex structure. Similar experiments were carried out on prototypal complexes cisplatin and cis-[PtCl₂(PPh₃)₂] for comparison. Platinum complexes containing phosphanes proved less cytotoxic to cancer cell lines and less likely to interact with the nucleobases of the G-quadruplex than those containing amines; in both cases the S-(−) isomer was more active than the R-(+) counterpart. More specifically, whereas all the platinum complexes were able to platinate the G-quadruplex structure from the human telomeric repeat, the extent and sites of platination depended on the nature of the ligands. Complexes containing (bulky) phosphanes interacted only with the adenines of the loops, whereas those containing the less sterically demanding amines interacted with adenines and some guanines of the G-quartet.     

Isolation and characterization of a novel bacterium, <Emphasis Type="Italic">Sphingomonas bisphenolicum</Emphasis> strain AO1, that degrades bisphenol A

Bisphenol A (2,2-bis(4-hydroxyphenyl) propane, BPA), which is used as a synthetic resin material or a plasticizer, is a pollutant that␣possesses endocrine-disrupting activity. Bioremediation of BPA is used to decrease its polluting effects, and here we report a novel bacterial strain AO1, which is able to degrade BPA. This strain was isolated using enrichment cultivation from a soil sample from a vegetable-growing field; the sample was one of 500 soil samples collected across Japan. Strain AO1 degraded 100 mg/l BPA to an undetectable level within 6 h in MYPG medium (containing malt extract, yeast extract, peptone, and glucose) and within 48 h in minimum medium containing 1% glucose at 30°C. Strain AO1 can utilize BPA as a sole source of carbon and as an energy source under aerobic conditions. The estrogenic activity of BPA in MYPG medium was ultimately reduced by strain AO1, although the activity initially increased. Taxonomical analysis showed that strain␣AO1 is closely related to Sphingomonas chlorophenolicum and S. herbicidovorans, neither of which have a capacity for BPA degradation. DNA–DNA hybridization showed that strain AO1 is a novel species of the Sphingomonas genus, and we designated AO1 as S. bisphenolicum.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

A new acylated flavonol diglycoside from <Emphasis Type="Italic">Atriplex littoralis</Emphasis>

A new acetylated flavonol glycoside: patuletin 3-O-[5′″-O-feruloyl-β-D-apiofuransyl (1′″→2′′)-β-D-glucopyranoside] (2), together with a known patuletin 3-O-β-D-glucopyranoside (1) were isolated from the aerial part of Artiplex littoralis L. (Chenopodiacease). Their structures were elcidated by acid hydrolysis and spectroscopic methods including UV, ¹H, ¹³C NMR and ESI-MS for both compounds, additionally 2D-NMR, HSQC, HMBC experiments were performed for 2.     

Laccase activities of Penicillium chrysogenum in relation to lignin degradation

 An extracellular laccase capable of oxidizing ABTS (the diammonium salt of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) was detected in ligninolytic cultures of Penicillium chrysogenum. By contrast, no lignin peroxidase, manganese-dependent peroxidase or aryl-alcohol oxidase was detected at any time during culturing. Both ABTS laccase activity and mineralization of dehydrogenative polymerizate of coniferyl alcohol were regulated by the C/N ratio in the medium and partially inhibited in the presence of thioglycolic acid, suggesting that both events are associated. In the presence of several known laccase inducers neither ABTS laccase activity nor mineralization rates were enhanced. However, a new laccase was detected in P. chrysogenum, able to oxidize 2,6-dimethoxyphenol but not involved in lignin mineralization. Studies with the known ligninolytic basidiomycete Trametes villosa suggest that lignin degradation by this fungus also involves the action of laccase. Received: 6 July 1995/Received revision: 28 October 1995/Accepted: 6 November 1995