Chemical Compounds and Antibacterial Activity of Tephrosia toxicaria Pers

Eight known compounds were isolated from the shrub Tephrosia toxicaria. Among them, 6,7-dimethoxy-chromone (1), was described by the first time from this genus, villosinol (2), which had been previously reported without its 13C-NMR data and sumatrol (3), which has its 13C-NMR data corrected. The antibacterial activity of Tephrosia toxicaria extract and obovatin (6), deguelin (7), 12a-hydroxyα-toxicarol (8), 12a-hydroxy-rotenone (10), and tephrosin (11) is also described.


INTRODUCTION
Tephrosia toxicaria Pers.(Fabaceae), also referred as T. sinapou (Buchoz), is a shrub popularly known as "timbo de caiena" in Ceara state (Northeast of Brazil) where it is used as pesticide and fishing poison [1][2] .The phytochemical studies of Tephrosia genus revealed compounds with anticinoceptive, larvicidal and antiinflammatory activities 3,4 .Previous investigations of T. toxicaria led to the identification of flavonoids, mainly rotenoids [1][2][5][6][7] . In te present work, we report the isolation of eight known compounds, including the chromone,(6,7)-dimethoxy-chromone,(1) described for the first time for this genus, the flavonoid, villosinol, (2) which has been previously reported without its 13 C-NMR data and sumatrol, (3) which has its 13 C-NMR data corrected.In addition, as a support of use of alternative source in the treatment of bacterial infections, we describe the antibacterial activity of ethanolic extract from its roots and of some its compounds.

Plant material
Pods and roots of T. toxicaria Pers.were collected in Guaraciaba do Norte (Ceara state, Brazil).A voucher specimen (#32139) is kept at the Herbarium Prisco Bezerra, Universidade Federal do Ceara -Brazil.

Antibacterial activity Microorganisms
The following strains used in this study were provided by the Oswaldo Cruz Foundation -FIOCRUZ: K. pneumoniae ATCC 10031; P. aeruginosa ATCC 15442; S. mutans ATCC 0046; S. aureus ATCC 6538.Strains isolated from clinical material of Escherichia coli 27 and Staphylococcus aureus 358 were also used.The bacteria were activated in Brain Heart Infusion (BHI, Himedia laboratories Pvt. Ltd., Mumbai, India) for 24 h at 35°C.

Antibacterial test (MIC) and resistance modulation bacterial
MIC (minimal inhibitory concentration) was determined in a microdilution assay 16 utilizing an inoculum of 100 µl of each strain, suspended in BHI broth up to a final concentration of 10 5 CFU/ml in 96-well microtiter plates, using serial dilutions (1:1).Each well received 100 µl of each (roots ethanolic extract (TTRE) and of the compounds 6, 7, 8, 10 and 11).The concentrations of the extract and organic compounds varied 512 -8 µg/ml.MICs were recorded as the lowest concentrations required to inhibit growth.
The minimum inhibitory concentration for antibiotics was determined in BHI by the microdilution test, using suspensions of 10 5 CFU/ mL and a drug concentration ranging from 2,500 to 2,4 µg/ml (1:1 serial dilutions).MIC was defined as a lower concentration at which growth was observed.For evaluation of the absence and presence of deguelin (7) and 12a-hydroxy-α-toxicarol (8) in P. aeruginosa and Staphylococcus aureus modulators of antibiotic resistance, a MIC of the antibiotics was determined in the presence or absence of subunits and as plates were incubated for 24 h at 37 o C. Each antibacterial test for MIC determination was performed in triplicate.

RESULTS AND DISCUSSION
It is worth to mention that villosinol, 2 has its 13 C-NMR data being reported for the first time, and that the 13 C-NMR assignment for sumatrol, 3 was corrected through bidimensional NMR analysis.
Table 2 shows the results concerning the modulation tests of bacterial resistance to aminoglycosides.When ( 7) and ( 8) compounds are combined with the antibiotic amikacin, tested against P. aeruginosa and S. aureus strains, the isolated and combined MIC values were the same.Synergism was observed in the combinations of (7)  with gentamicin against the two bacterial strains used in the test and in the combination of ( 7) with neomycin against P. aeruginosa, characterized by reduction of MIC by 50% compared to MIC of the antibiotics tested alone.Only the (8) combination with neomycin against S. aureus showed antagonism, increasing the MIC by 50% compared to the MIC of the neomycin tested alone.Some natural products of origin plant and phytochemicals are known to have antimicrobial properties, which may be of great importance in treatment against infections.With the increased incidence of antibiotic resistance, alternative natural plant products may be of interest 17 .Several studies have been conducted in different countries, demonstrating the efficacy of this type of treatment.Many natural products of plants were evaluated not only for direct antimicrobial activity, but also as resistance modifying agents 18,19 .
Various chemical compounds (synthetic or natural) have direct antibacterial activity against many species, expanding the activity of an antibiotic, reversing the natural resistance of bacteria to specific antibiotics, causing the inhibiting the active efflux of antibiotics through the plasma membrane and / or elimination of plasmids.Potentiation of antibiotic activity or reversion of resistance to antibiotics allows the classification of these compounds as modifiers of antibiotic activity 20,21 .
The mechanism described as "synergistic multi-effect targeting" or "Herbal shotgun" is a possible strategy that explains modulatory effects and refers to the use of plants and drugs in an approach using various-substance combinations, which may not only affect a single target, but several ones, where the different therapeutic components contribute in a synergistic-agonistic effect.This approach is not only for combinations of extracts: Combinations between complex mixtures (extracts and/or oil) and compounds chemical isolated synthetic or naturals or antibiotics are also possible 22,23 .

CONCLUSION
In conclusion, the prospective healthpromoting effects of plant secondary metabolites have encouraged the research about their chemical constitution and their potential to treat several diseases, among them bacterial infection.The plant substances and their extracts display rarely toxic side effects when compared in treatments with conventional drugs, so these results could be leads for the development of new antibacterial agent, or the compounds from T. toxicaria even may be used associates with standard antibiotics.FRLS, MVST, JNV, JQL (Master and PhD sudents), IGP (Graduate student) contributed running the laboratory work and drafted the paper; AMCA, JM, MRS, RBF, GMPS did the analysis and interpretation of data of RMN, critical revision of the manuscript.JGMC, EFFM and FFGR contributed with the microbiological tests.