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Effects of essential oils from medicinal plants used in Brazil against epec and etec
Duarte, M. C. T.; Leme;Delarmelina, C.; Figueira, G. M.; Sartoratto, A.; Rehder, V. L. G.
CPQBA/UNICAMP (Research Center for Chemistry, Biology and Agriculture), CP 6171, CEP 13083-970, Campinas
(SP), Brazil.E-mail: mduarte@cpqba.unicamp.br

ABSTRACT: Effects of essential oils from medicinal plants used in brazil against epec and etec escherichia coli .
Essential oils obtained from leaves of 28 medicinal plants commonly used in Brazil were screened against anti-enteropathogenic (EPEC) and anti-enterotoxigenic (ETEC) Escherichia coli. The oils were obtained by water-distillationusing a Clevenger-type system and their Minimal Inhibitory Concentration (MIC) was determined. Among the plantsstudied, Cymbopogon martinii exhibited a more reach inhibition spectrum, presenting strong activity (MIC between0.1-0.5 mg/mL) against three ETEC and one EPEC serotypes, while C. winterianus inhibited strongly two ETEC andone EPEC serotypes. Aloysia triphylla also shows good potential to kill E. coli with moderate to strong inhibition. Otheressential oils showed antimicrobial properties, however with restrict action against serotypes ETEC 5041-1 andEPEC 0031-2. Chemical analyses performed by Gas chromatographic (GC) and mass spectrometry (GC-MS) analysesshowed the presence of compounds with known antimicrobial activity, including limonene, geranial, geraniol, trans-geranial, trans-cariophyllene and geranil acetate. The results indicate significant antibacterial activity from these oilsand suggest that they may serve as sources for compounds with therapeutic potential.
Key words: Escherichia coli, essential oil, antimicrobial activity, medicinal plants, minimal inhibitory concentration.
the use of herbal remedies has been increased inBrazil as well as in another countries. In the present Diarrhea caused by Escherichia coli infection study, essential oils from 28 native and exotic is a emergent problem in both developing and medicinal plants traditionally used in Brazil were developed world and is responsible for high rates of screened for antimicrobial activity against EPEC and mortality in newborn child and animals. Although ETEC E. coli serotypes. The essential oils from active commensal representatives founded in the intestinal plants were also characterized by gas- flora of man and animals are apathogenic, certain chromatography/ mass spectrometry analyses in strains are highly pathogenic.
order to identify their major compounds.
All diarrheagenic strains of E. coli were initially termed enteropathogenic E. coli (EPEC) but MATERIAL AND METHOD
as was learnt about their pathogenic mechanismsthey were grouped accordingly (Clarke, 2001).
According to Schmidt et al. (1997), EPEC and ETEC are the most important of these in terms of A list of the plants studied are listed in Table total diarrhea episodes on a global scale although 1. The plants were grown in the experimental field of EHEC has become more significant in developed the Research Center for Chemistry, Biology and countries in recent years due to the occurrence of Agriculture (CPQBA, State University of Campinas, numerous outbreaks. ETEC are nowadays considered São Paulo, Brazil). The plants were collected from a major cause of E. coli-associated diarrhea November/2001 to February/2003. Voucher specimens worldwide, producing one or both of two types of were deposited at the State University of Campinas enterotoxins namely heat stable enterotoxins (ST) and Herbarium (UEC) or at the herbarium at the CPQBA.
heat labile enterotoxins (LT) (Clarke, 2001).
The diarrhea is often treated with antibacterial Essential oil extraction
drugs, but this treatment is generally ineffective, duein part to the presence of drug resistant strains and The essential oils were obtained from 40 g of failure to identify drug sensitivity (Cid et al., 1996).
fresh plant parts by water-distillation using a This fact has underscored the need of a quick Clevenger-type system for 3 h. The aqueous phase development of antibacterial drugs that are more was extracted three times with 50 mL dichloromethane.
effective than those currently in use. Since medicinal The pooled organic phases were dried with sodium plants play a fundamental role in traditional medicine, sulphate, filtered and the solvent evaporated untildryness. Dried samples were stored at -25 oC in Recebido para publicação em agosto/2004 sealed glass vials.
Aceito para publicação em julho/2006 Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.139-143, 2006.
TABLE 1. Identification, voucher specimen and data on traditional use of the plants studied.
alv, leaves; rt, roots. bData from Lorenzi and Matos (2002).
Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.139-143, 2006.
) from the plants tested.-1 n.d. = not determined; * = MIC > 1.0 mg.mLb . Essential oil Yield (% w/w) and MIC results (mg.mL , leaves; rt, root; Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.139-143, 2006.
E. coli serotypes were kindly supplied by Dra.
chloramphenicol varied from 0.004 to 0.008 mg.mL-1 Tânia A. T. G. do Amaral (UNIFESP - Brazil). The for E. coli strains. There is not an agreement on the bacteria were grown overnight at 36oC in Mc Conkey acceptable inhibition level for plant materials. A Agar (Merck), and inocula for the assays were classification, based at MIC results as: strong, until prepared by diluting scraped cell mass in 0.85% NaCl 0.5 mg.mL-1; moderate, between 0.6 and 1.5 mg.mL- solution, adjusted to McFarland scale 0.5 and 1 and weak inhibitors, above 1.6 mg.mL-1 was confirmed by spectrophotometrical reading at 580 nm.
proposed (Aligianis et al., 2001). Thus in the present Minimal Inhibitory Concentration (MIC) tests work have been established 1.0 mg.mL-1 as highest were carried out using Müller-Hinton Broth on a 96 concentration and only the oils presenting MIC below wells culture testplate (Eloff,1998). culture testplate 0.5 mg.mL-1 were considered as potential (96 wells) - (Eloff,1998). The stock solutions of the oils were diluted and transferred into the first well, Among the 29 medicinal species and serial dilutions were performed so thatconcentrations in the range of 1.0-0.016 mg.mL-1 were TABLE 3. Identified compounds from the essential oils
obtained. Chloramphenicol (Merck) was used as the of A. triphyla (AT), Cymbopogum martini (CM), reference control in the range of 0.0625-0.0005 mg.mL- Cymbopogum winterianus (CW).
1. The inoculum was added to wells and the plateswere incubated at 36oC for 24 h. Antimicrobial activitywas detected by adding 20 µL of 0.5% TTC (triphenyltetrazolium chloride, Merck). MIC was defined as thelowest concentration of oil that inhibited visible growth,as indicated by the TTC staining.
Gas chromatographic (GC) and mass
spectrometry (GC-MS) analyses

The identification of volatile constituents was performed using a Hewlett-Packard 5890 Series II gaschromatograph, equipped with a HP-5971 massselective detector and capillary column HP-5 (25 m x0.2 mm x 0.33 mm diam.). GC and GC-MS were doneusing split/splitless injection, with injector set at220oC, column set at 60oC, with heating ramp of 3oC.min-1and final temperature 240oC for 7 min, andthe FID detector set at 250oC. Helium was used ascarrier gas at 1 mL.min-1. The GC-MS electronionization system was set at 70 eV. A sample of theessential oil was solubilized in ethyl acetate for theanalyses. Retention indices (RI) were determined byco-injection of hydrocarbon standards. The oilcomponents were identified by comparison with datafrom literature (Adams, 2001), the profiles from the Wiley 138 and Nist 98 libraries, and by co-injection of MW = molecular weight. bRI = retention index. cResults expressed authentic standards, when available.
as % of area.
RESULT AND DISCUSSION
Antimicrobial activity tests
Oil yields of the plant expressed in relation investigated, C. martinii exhibited a more to dry weight plant material, are presented in Table 2.
reach inhibition spectrum, presenting strong activity Most plants had oil yield below 1% (w/w), though (MIC between 0.1-0.5 mg.mL-1) against three ETEC higher amount was obtained from A. triphylla (1.60% and one EPEC serotypes. The other Cymbopogon w/w), C. martinii (1.77% w/w), Piper mollicomum specie studied, C. winterianus also inhibited strongly (2.98% w/w) and P. regnelli (2.7% w/w).
two ETEC and one EPEC. Aloysia triphylla shows MIC results of the oils obtained from the plants good potential to kill E. coli with moderate to strong tested are shown in Table 2. Considering the standard inhibition (MIC between 0.5-0.8 mg.mL-1). The most antibiotic used as control, the MIC from of other essential oils presented antimicrobial Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.139-143, 2006.
properties, however with restrict action against serotypes ETEC 5041-1 and mainly against EPEC0031-2, which demonstrate a more susceptibility from Adams, R.P., 2001. Identification of essential oils these strains to essential oils.
components by Gas Chromatography/Quadrupole The negative results obtained against the Mass Spectroscopy. Allured Publishing Corporation,Illinois, USA.
gram-negative bacteria by the rest of the plants were Aligiannis, N., Kalpotzakis, E., Mitaku, S., Chinou, I.B., not surprising as, in general, these bacteria are more 2001. Composition and antimicrobial activity of the resistant than Gram-positive bacteria (Kudi et al., essential oils of two Origanum species. Journal of Agricultural and Food Chemistry 40, 4168-4170.
Oils with good antimicrobial potential were Clarke, S.C., 2001. Diarrhoeagenic Escherichia coli – an subjected to GC and GC-MS analyses (Table 3). The emerging problem? Diagnostic Microbiology and majority constituents were identified using the data Infections Disease 41, 93-98.
sources available. Among the identified compounds, Cid, D., Blanco, M., Blanco, J.E., Quiteira, J.A.R.S., Fuente, some were previously reported to have antimicrobial R., Blanco, J. 1996. Serogroups, toxins and antibioticresistance of Escherichia coli strains isolated from activity, including 1,8-cineole, limonene and linalool diarrhoeic goat kids in Spain. Veterinary Microbiology (Mazzanti et al., 1998) and germacrene-D (Ngassapa 53, 349-354.
et al., 2003).
Ellof, J.N.,1998. A sensitive and quick microplate method The results of the present study indicate that to determine the minimal inhibitory concentration of the essential oils obtained from 3 out of 28 plants plant extracts for bacteria. Planta Medica 64, 711-713.
commonly used in Brazilian folk medicine had good Kudi, A.C., Umoh, J.U., Eduvie, L.O., Gefu, J., 1999.
anti-E. coli activity. The essential oils from A. triphylla, Screening of some Nigerian medicinal plants for C. martini and C. winterianus presented strong activity antibacterial activity. Jounal of Ethnopharmacology against E. coli serotypes, with good oil yield. This 67, 225-228.
Lorenzi, H., Matos, F. J.A., 2002. Plantas medicinais do study corroborates the importance of Brasil: nativas e exóticas cultivadas. Instituto ethnopharmacology survey data in the selection of Plantarum, 512 pp.
plants for bioactivity screening representing an Mazzanti, G., Battinelli, L., Salvatore, G., 1998. Antimicrobial expressive contribution to the characterization of the properties of the linalool-rich essential oil of Hyssopus anti-E. coli activity of traditional Brazilian medicinal officinalis L. var decumbens (Lamiaceae). Flavour plants, as previous studies have not focused into this and Fragrance Journal 13, 289-294.
type of assays. Subsequently, bioguided fractionation Ngassapa, O., Runyoro, D.K.B., Harvala, E., Chinou, I.B., will be conducted to the potential plants for 2003. Composition and antimicrobial activity of identification of the active compounds.
essential oils of two populations of Tanzanian Lippiajavanica (Burm.f.) Spreng. (Verbenaceae). Flavour andFragrance Journal 18, 221-224.
Schmidt, H., Henkel, B., & Karch, H., 1997. A gene cluster Research was supported by a grant from closely related to type II secretion pathway operons FAPESP (SP, Brazil).
of gram-negative bacteria is located on the largeplasmid of enterohemorrhagic Escherichia coli O157strains. FEMS Microbiology Letters 148, 265-272.
Rev. Bras. Pl. Med., Botucatu, v.8, n.esp., p.139-143, 2006.

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