Bacteriostatic action of synthetic polyhydroxylated chalcones against Escherichia coli

In previous work the bacteriostatic action of trihydroxylated chalcones against Staphylococcus aureus ATCC 25 923 was investigated. In this work the action of 2 ́,4 ́,2-(OH) 3 -chalcone, 2 ́,4 ́,3-(OH) 3 chalcone and 2 ́,4 ́,4-(OH) 3 –chalcone against Escherichia coli ATCC 25 922 was evaluated. Growth kinetic curves of E.coli were made in nutritive broth added with increasing drug concentrations. The specific growth rates of the microorganisms were calculated by a kinetic turbidimetric method, which was previously probed and the minimal inhibitory concentrations (MIC ́s) were evaluated by a mechanism of action proposed. The MICs of 2 ́,4 ́,3-(OH) 3 -chalcone and 2 ́,4 ́,2-(OH) 3 -chalcone were 46 μg/ml and 122 μg/ml, respectively. The 2 ́,4 ́,4-(OH) 3 –chalcone was inactive. The MIC value of 2 ́, 4 ́, 3-(OH) 3 -chalcone (46 μg/ml), more active than 2 ́, 3-(OH) 2 -chalcone (72.2 μg/ml) may be due to the introduction of an electron donating group (-OH) at position 4 ́ in the aromatic Aring, which activates the region that includes the 2 ́-hydroxyl neighbur group and the α,βunsaturated carbonyl group. BIOCELL 2004, 28(1): 31-34 ISSN 0327 9545 PRINTED IN ARGENTINA


Materials and Methods
Microbial strain: E. coli ATCC 25 922 (acquired in American Type Culture Collection) maintained by successive subcultures in trypticase soy agar (BBL) at 4ºC and by liofilization.

Results
Compounds assayed were efficient against E. coli ATCC 25 922.The number of cfu/ml at different times was obtained by the expression of the turbidimetric kinetic method.
Considering the microbial growth law where t: time in min; N o : cfu/ml at t = 0; N t : cfu/ml at time t; µ: specific growth rate in min -1 , values for E. coli specific growth rates in media with increasing drug concentrations were obtained from the ln N t vs. t plot in the exponential growth phase.Results from the E. coli growth tests in presence of 2´,4´,3 -trihydroxychalcone are shown in Figure 2.

Turbidimetric kinetic method:
A 24 h culture of E.coli ATCC 25 922 in slant agar were transferred to 30 ml of Müller-Hinton broth (Oxoid) and incubated for 18 h at 35ºC with permanent stirring, in order to be used as inoculum.Erlenmeyers containing 100 ml of culture medium with increasing drug concentrations to be tested were inoculated with 2 ml of inoculum and stirred in a Rosi 1000 culture chamber at 35ºC and 180 rpm, including one without drug as control.Aliquots were extracted at 20 min intervals during 5 h and transmittance (T) was registered in a UV-Visible recording spectrophotometer Shimadzu 160 A. T values were related to the number cfu/ml (colony In Table 1 values of T, ln N t (evaluated from equation 1) and t are informed.
Table 2 exhibits values for the microbial specific growth rates and the drug concentrations added to the culture media.

Discussion
The results were interpreted satisfactorily by means of the bacteriostatic inhibition mechanism previously proposed for dihydroxylated chalcones (Pappano et al., 1994).Thus, the variation of the specific growth rate (µ) with the drug concentration follows the relation where µ: specific growth rate (min -1 ); µ T : specific growth rate in medium without drug (min -1 ) (control); k: specific inhibition rate (ml /(µg.min)) and C: drug concentration (µg/ml).
The graphical representation of equation ( 3) is shown in Figure 3 for the assayed compound and minimal inhibitory concentrations were evaluated by extrapolation at µ = 0.
The minimal inhibitory concentration (MIC) values obtained for the compounds tested in our study were as follows: 2´,4´,3-trihydroxychalcone 46 µg/ml These values allowed us to conclude that the presence of a new OH group at 4´ position in the structure of 2´, 3-dihydroxychalcone (MIC = 72.2µg/ml) increases the bacteriostatic activity of the base compound.The increase of eff icacy on the part of 2´,4´,3trihydroxychalcone could be attributed to the presence of a OH group at 4´ position in connection with the active region that comprises the -OH at 2´ position and the α,β unsaturated carbonyl group.On the other side, it was found that the presence of a OH group in 4 position of 2´,4´,4-trihydroxychalcone, in contrast to the results of the same chalcones against Staphylococcus aureus (Devia et al., 1998), annuls the action of the same one.The similar behavior was observed for 2´,4´,2trihydroxychalcone regarding 2´,4´-dihydroxychalcone. Different wall structure of Gram positive and Gram negative bacteria could be the reason of those results.