Antifungal activity of novel indole derivative from endophytic bacteria Pantoea ananatis 4G-9 against Mycosphaerella musicola

An endophytic bacterium isolated from banana G-9 (AAA genotype) leaves exhibited strong antagonistic activity against Mycosphaerella musicola. The isolate was identified as Pantoea ananatis 4G-9 by 16S rRNA sequence analysis. Secondary metabolite obtained from P. ananatis 4G-9 was found to have antifungal activity. The active compound was purified from crude extract using column chromatography. Purity of the active compound was assessed using high-performance liquid chromatography. Spectral analysis of compound using infrared, mass spectrometry and nuclear magnetic resonance indicated that the compound structure is an indole derivative. The compound showed strong and dose-dependent antifungal activity against M. musicola. This is the first report on P. ananatis isolated as an endophyte from banana leaves and its antifungal activity against M. musicola.     

Preparation and isolation of 2-methylamino-3-phenylpropanoic acid enantiomers using selective crystallization

First, (RS)-2-chloro-3-phenylpropanoic acid [(RS)-CPP] was optically resolved using ethyl (S)-phenylalaninate as a resolving agent, aiming at preparation of optically active 2-methylamino-3-phenylpropanoic acid (MPP). The (R)-CPP obtained as the sodium salt monohydrate was reacted with methylamine to give (S)-2-methylamino-3-phenylpropanoic acid [(S)-MPP]. Next, the optical resolution of (RS)-MPP was also attempted via molecular compound formation with optically active mandelic acid (MAN). The molecular compound of (R)-MPP with (S)-MAN [(R)-MPP (S)-MAN] was obtained as the less soluble diastereomeric compound, while the (S)-MPP (S)-MAN compound was found to be the more soluble one. Recrystallization of (R)-MPP (S)-MAN compound from water, followed by treatment with acetone, gave optically pure (R)-MPP in 79% yield, based on a half amount of the starting (RS)-MPP. The (S)-MPP obtained from (S)-MPP (S)-MAN compound was again subjected to formation of molecular compound with (R)-MAN to give optically pure (S,)-MPP in 66% yield. Chirality 9:386–389, 1997. © 1997 Wiley-Liss, Inc.     

Water-Soluble Constituents of Cudrania tricuspidata （Carr.） Bur.

In order to find new structural and biologically active compounds, the constituents of the bark of Cudrania tricuspidata （Carr.） Bur. were investigated and a new 6-p-hydroxybenzyltaxifolin glucoside, named tricusposide （compound 1）, together with 16 known compounds, was isolated by solvent partition, macroporous adsorption resin AB-8, silica gel, Sephadex LH-20 chromatography. Using spectroscopic methods, the structures of the compounds were elucidated as 6-p-hydroxybenzyl taxifolin-7-O-β-D-glucoside （compound 1）, dihydroquerctin-7-O-β-D-glucoside （compound 2）, dihydrokaempferol-3-O-β-D-glucoside （compound 3）, dihydroquercetin （compound 4）, peonoside （compound 5）, sphaerobioside （compound 6）, quercimeritrin （compound 7）, genistein （compound 8）, aromadendrin （compound 9）, kaempferol （compound 10）, genistin （compound 11）, 3,4-dihydroxystyryl alcohol （compound 12）, sucrose （compound 13）, 1,3,5,6-tetrahydroxyxanthone （compound 14）, gericudranin E （compound 15）, gericudranin C （compound 16）, and orobol （compound 17）. Compounds 2-6, 8, 9, 12-14, and 17 were isolated from this genus for the first time.     

Insecticidal compound isolated from Syzygium lineare Wall. (Myrtaceae) against Spodoptera litura (Lepidoptera: Noctuidae)

A new crystal compound 2,5-diacetoxy-2-benzyl-4,4,6,6-tetramethyl-1,3-cyclohexanedione was isolated from the leaves of Syzygium lineare. The insecticidal activity of the compound was assessed against fourth instar larvae of Spodoptera litura. Its activity was better than the positive control azadirachtin. The compound was responsible for growth inhibition on S. litura. It induced larval, pupal and adult deformities even at low concentration. The compound may be useful as a botanical pesticide.     

Isolation and identification of antimicrobial agent-producing bacterium from <Emphasis Type="Italic">Taxus baccata</Emphasis> rhizosphere antagonistic against clinically significant microbes

A bacterium identified as Pseudomonas fluorescence was isolated from Taxus baccata rhizosphere. Ethyl acetate extract from its culture filtrate yielded an active antimicrobial compound that was purified by TLC. The active metabolites were resolved by column chromatography on silica gel (60–120 mesh). The compound was further characterized on the basis of spectral data (UV, IR and ¹HNMR), which indicated the presence of an aromatic ring and phenolic functionality. The compound showed significant antimicrobial activity against two-gram positive bacteria (B. subtilis and S. aureus), four-gram negative bacteria (E. coli, K. pneumoniae, S. flexneri and P. aeruginosa), and one pathogenic fungus (Candida albicans). The minimum inhibitory concentration (MIC) of the compound ranged between 75μg to 250 μg/ml.     

Computational and in vitro insights on snake venom phospholipase A2 inhibitor of phytocompound ikshusterol3-O-glucoside of Clematis gouriana Roxb. ex DC.

Ikshusterol3-O-glucoside was isolated from Clematis gouriana Roxb. ex DC. root. A structure of the isolated compound was determined on the basis of various spectroscopic interpretations (UV, NMR, FTIR, and GC-MS-EI). This structure was submitted in the PubChem compound database (SID 249494133). SID 249494133 was carried out by density functional theory calculation to observe the chemical stability and electrostatic potential of this compound. The absorption, distribution, metabolism, and excretion property of this compound was predicted to evaluate the drug likeness and toxicity. In addition, molecular docking, quantum polarized ligand docking, prime MMGBSA calculation, and induced fit docking were performed to predict the binding status of SID 249494133 with the active site of phospholipase A₂ (PLA₂) (PDB ID: 1A3D). The stability of the compound in the active site of PLA₂ was carried out using molecular dynamics simulation. Further, the anti-venom activity of the compound was assessed using the PLA₂ assay against Naja naja (Indian cobra) crude venom. The results strongly show that Ikshusterol3-O-glucoside has a potent snake-venom neutralizing capacity and it might be a potential molecule for the therapeutic treatment for snakebites.     

A Diterpene Endoperoxide from Microtoena insuavis （Hance） Prain ex Dunn

A novel endoperoxlde diterpene, 7a-hydroxy-abieta-8（14）-en-18-oi 9α,13α-endoperoxide （compound 1）, was isolated from the stems of Microtoena insuavis （Hance） Prain ex Dunn, along with 4,4＇-dlhydroxytruxillic acid （compound 2）, gallic acid （compound 3）, ellaglc acid （compound 4）, 3-O-methylellaglc acid 3＇-O-α- rhamnopyranoslde （compound 5）, 3＂＇-O-methylcrenatoslde （compound 6）, crenatoslde （compound 7）, aptgenin （compound 8）, succinic acid （compound 9）, β-sitosterot （compound 10）, and β-daucosterol （compound 11）. The structures of these compounds were elucidated on the basis of spectral evidence.     

Isolation,structural identification and herbicidal activity of N-phenylpropanamide from Streptomyces sp. KA1-3

The secondary metabolites produced by Streptomyces sp. KA₁-3, cultured on starch casein broth, was extracted by ethyl acetate and concentrated. Purification of the compound by thin layer chromatography lead to isolation of N-phenylpropanamide from one polar fraction. The structure of the herbicidal compound was elucidated on the basis of UV, FT-IR, mass and H¹ NMR spectroscopy. The herbicidal activity of the isolate was tested against the weeds Cassia occidentalis L. and rhizome Cyperus rotundus L. by moist chamber technique and rolled towel paper assay method. Herbicidal activity of the bioactive compound N-phenylpropanamide was further evaluated under in vitro condition. The herbicidal compound showed 80% of seed germination inhibition in C. occidentalis L. and rhizome C. rotundus L. weed. The actinobacterium can be used as a source for bioherbicidal agent.     

Novel Potato Micro-Tuber-Inducing Compound, (3R,6S)-6-Hydroxylasiodiplodin,from a Strain of Lasiodiplodia theobromae

A novel potato micro-tuber-inducing compound was isolated from the culture broth of Lasiodiplodia theobromae Shimokita 2. The structure of the isolated compound was determined as (3R,6S)-6-hydroxylasiodiplodin by means of spectroscopic analyses, the modified Mosher method, and chemical conversion. The compound showed potato micro-tuber-inducing activity at a concentration of 10^?4 M, using the culture of single-node segments of potato stems in vitro.     

Novel compound from Trachyspermum ammi (Ajowan caraway) seeds with antibiofilm and antiadherence activities against Streptococcus mutans: a potential chemotherapeutic agent against dental caries

Aim: The objective of this study was to isolate and characterize the active compound from Trachyspermum ammi seeds, exhibiting antibiofilm activity against Streptococcus mutans, a major causal organism of dental caries. Methods and Results: Purification of the active compound from the seeds was performed by silica gel chromatography, and spectroscopic methods (FTIR, NMR and MS) were employed for its identification and structure determination. Antibiofilm and antiadherence activities of the active compound against S. mutans were analysed. Confocal microscopy was performed to visualize the effect of the compound on biofilm structure of S. mutans. Around 50% reduction was observed in adherence at 39·06 μg ml^?1 and in biofilm at 78·13 μg ml^?1. It was found effective against adherent cells of S. mutans, reduced water‐insoluble glucan synthesis and inhibited the reduction in pH. Confocal microscopy revealed scattered cells at sub‐MIC concentration of the compound, resulting in distorted biofilm architecture in contrast to clustered cells seen in control. Conclusion: This study revealed a novel compound, a naphthalene derivative, isolated first time from T. ammi seeds with antibiofilm activity against S. mutans. Significance and Impact of the Study: Trachyspermum ammi represents an interesting source of a novel compound, (4aS, 5R, 8aS) 5, 8a‐di‐1‐propyl‐octahydronaphthalen‐1‐(2H)‐one, with a great potential to be used as a therapeutic agent against dental caries.     

卡伍尔氏链霉菌NA4的Ⅱ型聚酮基因簇的靶向激活及其产物鉴定

【目的】以基因组信息为导向,定向激活海洋来源卡伍尔氏链霉菌（Streptomyces cavourensis） NA4中沉默的Ⅱ型聚酮类次级代谢产物生物合成基因簇,鉴定新产生的次级代谢产物的结构和抑菌活性。【方法】通过添加启动子和敲除负调控基因的方法激活实验室培养条件下沉默或低表达的生物合成基因簇,并完成目标化合物的分离与纯化,通过电喷雾质谱（electrospray ionization-mass spectrometry,ESI-MS）和核磁共振（nuclear magnetic resonance,NMR）数据分析鉴定目标化合物结构,对目标化合物进行抑菌活性鉴定,基于生物信息学信息推导化合物的生物合成途径。【结果】根据基因组生物信息学分析,从海洋来源链霉菌Streptomyces cavourensis NA4中选取一个编码PKSⅡ型次级代谢产物的生物合成基因簇开展研究,成功激活目标基因簇,从中分离到1个PKSⅡ型化合物,推导了其生物合成途径并进行了抑菌活性鉴定。【结论】基因组导向下的天然产物挖掘,可以目标明确地分离产物,充分挖掘链霉菌编码次级代谢产物的潜力。     

Preparation of 6-azafulleroid-6-deoxy-2,3-di-O-myristoylcellulose

6-Azafulleroid-6-deoxy-2,3-di-O-myristoylcellulose (3) was synthesized from 6-azido-6-deoxycellulose (1) by two reaction steps. The myristoylation of compound 1 with myristoyl chloride/pyridine proceeded smoothly to give 6-azido-6-deoxy-2,3-di-O-myristoylcellulose (2) in 97.0% yield. The reaction of compound 2 with fullerene (C₆₀) was carried out by microwave heating to afford compound 3 in high yield. It was found from FT-IR, ¹³C NMR, UV–vis, differential pulse voltammetry (DPV), SEC analyses that compound 3 was the expected C₆₀-containing polymer. Consequently, maximum degree of substitution of C₆₀ (DS_C60) of compound 3 was 0.33.     

Isolation, structure elucidation and biological activity of metabolites from Sch-642305-producing endophytic fungus Phomopsis sp. CMU-LMA

Eight polyketide compounds were isolated from the cultivation broth of Phomopsis sp. CMU-LMA. We have recently described LMA-P1, a bicyclic 10-membered macrolide, obtained as a bioconversion derivative of Sch-642305, the major compound isolated in this study. Benquinol is the ethyl ester derivative of the 13-dihydroxytetradeca-2,4,8-trienoic acid produced by Valsa ambiens. This compound is concomitantly produced with the 6,13-dihydroxytetradeca-2,4,8-trienoic acid (DHTTA) previously isolated from Mycosphaerellarubella. The absolute configuration of the new compound, (2R,3R,4S,5R)-3-hydroxy-2,4-dimethyl-5-[(S,Z)-3-methylpentenyl]-tetrahydro-pyranone LMA-P2 was confirmed by X-ray crystallography. The δ-lactone 2,3-dihydroxytetradecan-5-olide (DHTO) was previously isolated from Seiridium unicorne. This compound may form through the cyclization of the methyl-2,3,5-trihydroxytridecanoate LMA-P3, a new linear polyketide isolated in this study. Benquoine, a new 14-membered lactone generated from the cyclization of benquinol, is proposed as the key precursor for the biosynthesis of Sch-642305. Antimicrobial activity and cancer cell viability inhibition by the new compounds were investigated. Benquoine exhibits antimicrobial activity against Gram positive bacteria, and cytotoxicity against HCT-116 cancer cell line.     

An improved technique for isolating codominant compound microsatellite markers

An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the constructed DNA library using compound SSR primer (AC)₆(AG)₅ or (TC)₆(AC)₅ and an adaptor primer for the suppression-PCR. A locus-specific primer was designed from the sequence flanking the compound SSR. The primer pairs of the locus-specific and compound SSR primers were used as a compound SSR marker. Because only one locus-specific primer was needed for design of each marker and only a common compound SSR primer was needed as the fluorescence-labeled primer for analyzing all the compound SSR markers, this approach substantially reduced the cost of developing codominant markers and analyzing their polymorphism. We have demonstrated this technique for Dendropanax trifidus and easily developed 11 codominant markers with high polymorphism for D. trifidus. Use of the technique for successful isolation of codominant compound SSR markers for several other plant species is currently in progress.     

Self-excreted mediator from <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 for electron transfer to carbon electrodes

Escherichia coli K-12 was cultured under anaerobic conditions to form biofilm on carbon fiber electrodes in glucose-containing medium. The anodic current increased with the development of the biofilm and depended on the glucose concentration. Cyclic voltammetric results support the presence of a redox compound(s) excreted from E. coli cells in the biofilm. The compound remained in the film under conditions of continuous flow and gave a couple of oxidation and reduction waves, which may be assigned to a menaquinone-like compound based on the mid-point potential (−0.22 V vs Ag|AgCl at pH 7.1) and its pH dependence. The catalytic current started to increase around the anodic peak potential of the redox compound and also increased by the permeabilization of the E. coli cell membranes with ethylenediamine tetraacetic acid-treatment. The results indicate that the E. coli-excreted redox compound works as a mediator for the electron transfer from the E. coli cells to the electrode as the final electron acceptor. The activity of the redox compound in the E. coli-biofilm as a mediator with some mobility was also verified for diaphorase-catalyzed electrochemical oxidation of NADH.     

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine in normal human urine

Summary A compound, which had the same mobility on a high-voltage paper electrophoretogram and the sameR _F value on a thin-layer chromatogram as those ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (I), was partially purified from human urine by ion-exchange column chromatography. The compound gave a signal at m/z 260 on its FAB mass spectrum, which was assigned as MH⁺ of compound I. These results suggest that the urinary compound is compound I and it is a physiological precursor of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid [Kinuta et al., (1991) Biochem J 275: 617–621].     

Biological Control of Phytophthora Root Rot of Pepper Using Trichoderma harzianum and Streptomyces rochei in Combination

A combination of two compatible micro‐organisms, Trichoderma harzianum and Streptomyces rochei, both antagonistic to the pathogen Phytophthora capsici, was used to control root rot in pepper. The population of the pathogen in soil was reduced by 75% as a result. Vegetative growth of the mycelium of P. capsici was inhibited in vitro on the second day after P. capsici and T. harzianum were placed on the opposite sides of the same Petri plate. Trichoderma harzianum was capable of not only arresting the spread of the pathogen from a distance, but also after invading the whole surface of the pathogen colony, sporulating over it. Scanning electron microscopy showed the hyphae of P. capsici surrounded by those of T. harzianum, their subsequent disintegration, and the eventual suppression of the pathogen's growth. Streptomyces rochei produced a zone of inhibition, from which was obtained a compound with antioomycete property secreted by the bacteria. When purified by high‐pressure liquid chromatography, this compound was identified as 1‐propanone, 1‐(4‐chlorophenyl), which seems to be one of the principal compounds involved in the antagonism. A formulation was prepared that maintained the compound's capacity to inhibit growth of the pathogen for up to 2 years when stored at room temperature in the laboratory on a mixture of plantation soil and vermiculite. The two antagonists, added as a compound formulation, were effective at pH from 3.5 to 5.6 at 23–30°C. The optimal dose of the antagonists in the compound formulation was 3.5 × 10⁸ spores/ml of T. harzianum and 1.0 × 10⁹ FCU/ml of S. rochei. This is the first report of a compound biocontrol formulation of these two antagonists with a potential to control root rot caused by P. capsici.     

Isolation and structural elucidation of a growth stimulant for arbuscular mycorrhizal fungus from Laminaria japonica Areschoug

Arbuscular Mycorrhizal (AM) fungi enhance terrestrial plant growth by forming a symbiotic relationship with the roots of its host plant. A growth stimulant for AM fungi was isolated from a brown alga Laminaria japonica Areschoug. The active substance for in vitro AM hyphal growth was isolated from 75% methanol extracts of L. japonica using a succession of chromatographic procedures, including flash chromatography equipped with an octa decyl silane (ODS) column, gel filtration chromatography and HPLC using an ODS column. Spores of Gigaspora margarita Becker & Hall, an AM fungus, were exposed to the compound in vitro, and hyphal growth of G. margarita was measured after two weeks of incubation. At 40 mg L⁻¹, the compound significantly stimulated the in vitro hyphal growth of G. margarita, compared to the control. This compound was elucidated as 5′-deoxy-5′-methylamino-adenosine by EIMS and NMR spectrum.     

N1, N14-diferuloylspermine as an antioxidative phytochemical contained in leaves of Cardamine fauriei

Most Brassicaceae vegetables are ideal dietary sources of antioxidants beneficial for human health. Cardamine fauriei (Ezo-wasabi in Japanese) is a wild, edible Brassicaceae herb native to Hokkaido, Japan. To clarify the main antioxidative phytochemical, an 80% methanol extraction from the leaves was fractionated with Diaion® HP-20, Sephadex® LH-20, and Sep-Pak® C18 cartridges, and the fraction with strong antioxidant activity depending on DPPH method was purified by HPLC. Based on the analyses using HRESIMS and MS/MS, the compound might be N¹, N¹⁴-diferuloylspermine. This rare phenol compound was chemically synthesized, whose data on HPLC, MS and ¹H NMR were compared with those of naturally derived compound from C. fauriei. All results indicated they were the same compound. The radical-scavenging properties of diferuloylspermine were evaluated by ORAC and ESR spin trapping methods, with the diferuloylspermine showing high scavenging activities of the ROO^·, O₂^·?, and HO^· radicals as was those of conventional antioxidants.     

Development of the Gateway Recycling Cloning System for Multiple Linking of Expression Cassettes in a Defined Order,and Direction on Gateway Compatible Binary Vectors

A new antimicrobial compound was isolated from Scorodocarpus borneensis Becc. The chemical structure was determined to be methylthiomethyl (methylsulfonyl)methyl disulfide on the basis of its spectroscopic data. This compound exhibited considerably strong antimicrobial activities against all bacteria and fungi tested, except for Pseudomonas aeruginosa, with 12.5–25 μg/ml of MIC.