Plant-derived secondary metabolites as the main source of efflux pump inhibitors and methods for identification / 药物分析学报

The upsurge of multiple drug resistance (MDR) bacteria substantially diminishes the effectiveness of antibiotic arsenal and therefore intensifies the rate of therapeutic failure. The major factor in MDR is efflux pump-mediated resistance. A unique pump can make bacteria withstand a wide range of struc-turally diverse compounds. Therefore, their inhibition is a promising route to eliminate resistance phenomenon in bacteria. Phytochemicals are excellent alternatives as resistance-modifying agents. They can directly kill bacteria or interact with the crucial events of pathogenicity, thereby decreasing the ability of bacteria to develop resistance. Numerous botanicals display noteworthy efflux pumps inhibi-tory activities. Edible plants are of growing interest. Likewise, some plant families would be excellent sources of efflux pump inhibitors (EPIs) including Apocynaceae, Berberidaceae, Convolvulaceae, Cucur-bitaceae, Fabaceae, Lamiaceae, and Zingiberaceae. Easily applicable methods for screening plant-derived EPIs include checkerboard synergy test, berberine uptake assay and ethidium bromide test. In silico high-throughput virtual detection can be evaluated as a criterion of excluding compounds with efflux substrate-like characteristics, thereby improving the selection process and extending the identification of EPIs. To ascertain the efflux activity inhibition, real-time PCR and quantitative mass spectrometry can be applied. This review emphasizes on efflux pumps and their roles in transmitting bacterial resistance and an update plant-derived EPIs and strategies for identification.

Stress and defense responses in plant secondary metabolites production

In the growth condition(s) of plants, numerous secondary metabolites (SMs) are produced by them to serve variety of cellular functions essential for physiological processes, and recent increasing evidences have implicated stress and defense response signaling in their production. The type and concentration(s) of secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage and environmental factors during growth. This suggests the physiological adaptive responses employed by various plant taxonomic groups in coping with the stress and defensive stimuli. The past recent decades had witnessed renewed interest to study abiotic factors that influence secondary metabolism during in vitro and in vivo growth of plants. Application of molecular biology tools and techniques are facilitating understanding the signaling processes and pathways involved in the SMs production at subcellular, cellular, organ and whole plant systems during in vivo and in vitro growth, with application in metabolic engineering of biosynthetic pathways intermediates.

Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

A survey of Senecio spp. affecting livestock in Uruguay and their associated pyrrolizidine alkaloid content / Senecio spp. Afetando a pecuária no Uruguai e sua associação com alcaloides pirrolizidínicos

ABSTRACT: In Eastern Uruguay there has been a significant increase of seneciosis in grazing livestock with most affected localities related to counties neighboring the Brazilian border. A survey in 28 farms associated with poisoning outbreaks in grazing cattle in Eastern Uruguay was carried out. Fifty populations of Senecio plants were collected for alkaloid analysis and species identification. Four species were identified: S. oxyphyllus DC, S. madagascariensis Poir, S. brasiliensis (Spreng.) Less., and S. selloi DC. Alkaloids were identified by a combination of GC-MS and HPLC-MS analysis and included: retrorsine in S. oxyphyllus; retrorsine, usaramine, and senecivernine/senecionine in S. selloi; retrorsine, senecivernine/senecionine, integerrimine, and usaramine in S. madagascariensis; and integerrimine, retrorsine and senecionine in S. brasiliensis. Total mean alkaloid concentration was reported to be highest in S. brasiliensis (17.6mg/g) followed by S. oxyphyllus (6.2mg/g), S. selloi (1.8mg/g) and S. madagascariensis (0.6mg/g). Alkaloid concentrations were also reported to be higher in 2015 vs. 2016 probably due to a common environmental factor. The species S. oxyphyllus and S. madagascariensis were not previously recognized as toxic plants in Eastern Uruguay. Particularly, S. oxyphyllus was present in 82% of the farms surveyed and occurred in high density with relative high concentrations of pyrrolizidine alkaloids suggesting S. oxyphyllus may be the main species involved in the reported outbreaks of seneciosis.

RESUMO: No leste do Uruguai houve um aumento significativo da seneciosis no gado de pastagem com a maioria das localidades afetadas em municípios vizinhos à fronteira brasileira. Aplicou-se um questionário em 28 fazendas associadas a surtos de intoxicação em bovinos de pastagem na fronteira leste do Uruguai. Coletaram-se cinquenta populações de Senecio para análise de alcalóides e identificação de espécies. Identificaram-se quatro espécies: S. oxyphyllus DC, S. madagascariensis Poir, S. brasiliensis (Spreng.) Less. e S. selloi DC. Identificaram-se os seguintes alcalóides mediante análise combinado de GC-MS e HPLC-MS: retrorsina em S. oxyphyllus; retrorsina, usaramina e senecivernina/senecionina em S. selloi; retrorsina, senecivernina/senecionina, integerimina e usaramina em S. madagascariensis; e integerrimina, retrorsina e senecionina em S. brasiliensis. A concentração média total de alcalóides foi mais elevada em S. brasiliensis (17,6mg/g) seguido de S. oxyphyllus (6,2mg/g), S. selloi (1,8mg/g) e S. madagascariensis (0,6mg/g). As concentrações de alcaloides foram maiores em 2015 em vista à 2016, provavelmente, devido a um fator ambiental comum. As espécies S. oxyphyllus e S. madagascariensis não foram previamente reconhecidas como plantas tóxicas no leste do Uruguai. Particularmente, S. oxyphyllus esteve presente em 82% das fazendas pesquisadas e ocorreu em alta densidade com concentrações relativamente elevadas de alcalóides de pirrolizidina sugerindo que S. oxyphyllus pode ser a principal espécie envolvida nos surtos relatados de seneciosis.

Anti-proliferative effect of terpenes on human prostate cancer cells: Natural sources and their potential role as chemopreventive agents / Efecto anti-proliferativo de los terpenos en células de cáncer de próstata humana: Fuentes naturales y su rol potencial como agentes quimiopreventivos

ABSTRACT Prostate cancer is one of the neoplastic diseases with the highest morbidity and mortality in the world. The diversity of available treatments and side effects related to therapeutic treatments are severe and affect a patient's quality of life. Thus, to creating new therapeutic alternatives to reduce morbidity and creating safe and effective therapies is a constant challenge. Recently, the use of traditional medicine and chemoprevention has gained importance. Several clinical and epidemiological studies suggest that a high-terpenoid compound-based diet is associated with a reduced risk of prostate cancer. This review is focused on the anti-proliferative effects of different terpenoids isolated from natural sources on human prostate cancer cells, with the aim of setting the basis to use these compounds as phytotherapeutic, nutraceutical and functional ingredients.

RESUMEN El cáncer de próstata es una de las neoplasias que produce gran morbilidad y mortalidad en el mundo. Los tratamientos y los efectos secundarios de la terapia son severos y afectan la calidad de vida del paciente. Por esto, es necesario crear nuevas alternativas terapéuticas para reducir la morbilidad y generar terapias seguras y efectivas es un desafío permanente. En los últimos años, el uso de la medicina tradicional y agentes quimiopreventivos han adquirido importancia. Estudios clínicos y epidemiológicos sugieren que una dieta basada en altas dosis de terpenoides se asocia a una reducción en el riesgo de presentar cáncer de próstata. Esta revisión se centra en los efectos anti-proliferativos sobre las células de cáncer de próstata en humanos de diferentes terpenoides aislados de fuentes naturales, con el objetivo de establecer las bases para utilizar estos compuestos como ingredientes fitoterapéuticos, nutracéuticos y funcionales.

BIOCHEMICAL COMPOSITION ON CACALIA HASTATA LINNA / Монголын эм зүй, эм судлал

The purpose of the present study was to determine plant first metabolites: is dry matter, general acidity, an ash, protein, oil, pectin, cellulose, lignin and starch, ascorbic acid, citrine, carotene, tocopherol and enzymes: catalase, polyphenoloxidase and extractive substances, plant secondary metabolites: is tannin, coumarin, total amount of flavonoids, saponin, total amount of alkaloids, alkaloid hastacine in leaves and stem of Cacalia hastata L. Key words: plant first metabolites, plant secondary metabolites, Cacalia hastata, Introduction Cacalia hastata is a perennial and herbaceous plant with thin thread, short downy stemrhizous. It grows up to 40-150 cm in height and deltoid – sword like leaves are from 6 to 20 cm long, 20 cm wide. This species was only one in Mongolia. It distributed to Khuvsgul, Khentey, Khangai, Mongol Daguur, and Khyangan in Mongolia (Ligaa et al.,2006, Grubov., 2008). C.hastata is a medicinal plant of Mongolia (Bayarsukh, 2008) [ Mong..Ilden iguushin Engel: Hastate Cacalia ]. It is also a medicinal herb of China [Chin.:Shan jianzi] ( Roeder, 2008). Distributrion: Central Mongolia and Central China. Use: Leaves are used against gastric and stomach ulcer, respiratory infection, inflammation of the stomach, inflammation of mouth cavity. Extract has anti-inflammatory activity: is antibacterical, spasmolytic, choleretic, antipyretic, and antihaemorrhagic. Contains the alkaloids platyphylline hastacine alkaloid, sesquiterpenes (Hayashi et al.,1972, Ligaa, 2006), Triterpenes, carotenoids (Olennikov et al, 2005, 2004), water soluble polysaccharide: is glucoarabinogalactane (cacalane A) and two arabinogalactane (cacalans B, C), pectines are in leaves of C. hastate included in class of high etherify pectin. Also, hemicellulosical derivative group A as soon as xyloglucofructan ; group B- xylona, di-and tricarbonic acid, photosynthesis pigment, vitamin C, phenolic compounds are coumarin and tannin, vitamin thiamine (B1) riboflavine (B2) and chemical elements (Olennikov et al., 2004, 2005; Ligaa, et al., 2006). Above ground parts of C.hastate used for pneumonitis, festering wound, leaves, flower recovered to ulcer and haemorrhagic. (Ligaa et al., 2006) Mongolian, Nepalese and Tibetan plant containing pyrrolizidine alkaloids (Roeder and Wiedenfeld, 2009). Researchers 1968 (Australian, Moscow, USSR) were isolated alkaloids platyphylline, hastacine, (neoplatyphylline, platynecine hastanecine). Analyses were made by the Australian Microanalytical Service, Melbourne. The solvent used in paper chromatography was the upper phase resulting from shaking n-butanol with an equal volume of 5% acetic acid. Neoplatyphylline (C18H27NO5), was isolated from mother liquors resulting from the preparation of platyphylline from Seneco rhombifolius. (Willd.) Alkaline and acid hydrolyses were said to give the same products as from platyphylline, which is an ester of platynecine with senecic acid. Hastacine, also C18H27NO5, was obtained from Cacalia hastata, another species of the tribe Senecioneae, family Compositae (Asteraceae). It gave on hydrolysis a saturated diol, hastanecine, isomeric with platynecine, and a dicarboxylic acid, C10H16NO5 , with properties similar to those later observed for integerrinecic acid. The structures of these two alkaloids have now been clarified in conjunction with a paralel study on the stereochemistry of the saturated pyrrolizidine diols (Culvenor et al., 1968). Altanchimeg (2001) was isolated alkaloids platyphylline and hastacine from above ground parts of Cacalia hastata, cultivated in Mongolia. The researches of Mongolia have already took a new form of drug and stability for pressing drug with capsul of Cacalia hastata (Jambaninj, 2012., Jambaninj et al., 2013). Some researches isolated a new sesquiterpene from roots of Cacalia delphiniifolia Steb. et Zucc. (Japanese name “Momijigasa”). They isolated 8-oxocacalol and stereochemisrtry of cacalone epimers. The isolation was performed by repeated column chromatography with silica gel [Rf 0.55, as compared with Rf 0.53 of cacalol on TLC (SiO2, benzene ) (Kanji Omura et al.,1978 )].

Atividade antimicrobiana in vitro de extratos de plantas do bioma caatinga em isolados de Escherichia coli de suínos / Antimicrobial activity in vitro extracts from caatinga plants in isolated Escherichia coli from pigs

RESUMO A suinocultura é uma atividade pecuária bem consolidada no Brasil. Por outro lado a colibacilose neonatal, cujo patógeno é Escherichia coli, pode diminuir a produtividade nas granjas e causar prejuízos aos produtores. O tratamento baseia-se na utilização de drogas antimicrobianas. Todavia, o uso indiscriminado dessas substâncias tem levado a seleção de cepas resistentes. Diante disso, a busca por alternativas terapêuticas, como as plantas medicinais, tem se tornado cada vez mais comum. Dessa maneira, objetivou-se determinar a atividade antimicrobiana de cinco extratos etanólicos de plantas do bioma caatinga: Amburana cearensis (Fr. Allem) A.C. Smith, Encholirium spectabile Mart., Hymenaea courbaril L, Neoglaziovia variegata Mez e Selaginella convoluta Spring frente a 43 isolados de Eschericha coli coletados de suínos. Para o teste de sensibilidade in vitro foi realizada a técnica da Concentração Bactericida Mínima (CBM) pelo método da microdiluição em microplaca. Os extratos apresentaram atividade antimicrobiana nas seguintes médias 138,75 175,28, 128,36, 127,71 e 129,33 μg/mL, respectivamente. Essa atividade antibacteriana pode estar relacionada a ação de metabólitos secundários presentes nos extratos dessas plantas. Dessa forma, nosso estudo pode contribuir para o desenvolvimento de alternativas terapêuticas no tratamento de infecções, como a colibacilose neonatal em suíno, bem como para o conhecimento acerca das plantas medicinais da Caatinga.

ABSTRACT Swine production is a well-established livestock activity in Brazil. On the other hand, the Neonatal Colibacillosis, whose pathogen is Escherichiacoli, can decrease the productivity on farms and cause losses to producers. The treatment of the disease is based on the use of antimicrobial drugs. However, the free use of these substances has led to the selection of resistant strains. Thus, the search for alternative therapies such as medicinal plants has become becoming increasingly common. In this context, we aimed to determine the antimicrobial activity of ethanol extracts of five plants from the caatinga biome: A. cearensis (Fr. Allem) AC Smith, Encholirium spectabile Mart, Hymenaea courbaril L, Neoglaziovia variegata Mez and Selaginella convoluta Spring in face of isolates of Eschericha coli collected from pigs. For the in vitro susceptibility testing, the method of Minimum Bactericidal Concentration (MBC) was chosen The extracts showed antimicrobial activity in the following averages 138.75 175.28, 128.36, 127.71 and 129.33 mg / mL, respectively. This antibacterial activity could be related to the action of secondary metabolites in the extracts of these plants. Thus, the current study can contribute to the development of alternative therapies for the treatment of infections such as swine Colibacillosis Neonatal, as well as to the knowledge of Caatinga medicinal plants.

Interaction of plant secondary metabolites and organic carbon substrates affected on biodegradation of polychlorinated biphenyl.

Using plant secondary metabolites (PSM) as an inducer to stimulate biodegradation of polychlorinated biphenyl (PCB) is considered a promising strategy to remove PCB from the environment. In this study, the effects of PSM including naringin, salicylic acid or biphenyl on PCB biodegradation using soil isolates were tested in mineral salt medium using either soil organic carbon (MSMS) or glucose (MSMG). In addition, the effects of surfactant hydroxypropyl-b-cyclodextrin (HP-b-CD) were examined. The results indicated that PCB removal was higher in medium with glucose as carbon substrate than in medium with SOC, and further enhanced with biphenyl amendment. However, interactions between salicylic acid and glucose limited PCB removal in treatments using both chemicals as carbon substrate while PCB removal was sustained in treatment using salicylic acid and SOC. Removal of tetra-chlorinated PCB was improved by adding HP-b-CD, suggesting increased bioavailability due to surfactant. These results suggest that interaction of organic carbon substrates could influence PCB degradation in contaminated environments.