Print version ISSN 0001-3714
Rev. Microbiol. vol. 29 n. 4 São Paulo Oct./Dec. 1998
Cristina M. Devia, Nora B. Pappano, Nora B. Debattista*
Laboratory of Physical-Chemistry, Department of Chemistry, San Luis National University, Chacabuco - San Luis, Argentina
Submitted: December 02, 1997; Approved: September 17, 1998.
The bacteriostatic activity of 2,4,2-trihydroxychalcone; 2,4,3-trihydroxychalcone and 2,4,4-trihydroxychalcone, prepared by condensation of 2,4-dihydroxyacetophenone and benzaldehyde substituted, against Staphylococcus aureus ATCC 25923 was assayed by agar plate method. The three compounds presented important inhibition halos. In order to elucidate structure-activity relationships, the minimal inhibitory concentrations against S. aureus were determined by the broth dilution method and the results obtained were compared to that of 2',4'-dihydroxychalcone. The sequence observed was: MIC 2,4,3-(OH)3 > MIC 2,4-(OH)2 > MIC 2,4,4-(OH)3 > > MIC 2,4,2-(OH)3. These results showed that the introduction of an electron donating group (-OH) in the aromatic B-ring causes an increase in bioactivity, and that the intensity of action depends on the position of the OH substitute.
Key Word: bacteriostatic activity, structure-activity relationship, flavonoids, trihydroxylated chalcones.
A large number of natural flavonoids with biological activity have been identified in recent decades. One group of these products, the polihydroxylated chalcones, exhibit antimicrobial (8,15), antiviral (6,9), antitumoral (7,10,12) and antiinflamatory (17) activities, and applications of therapeutic effects (12) have been reported. The increase of the bacteriostatic action due to free hydroxyl groups on the aromatic A- and B- rings has been demonstrated in a previous work, which showed that the introduction of hydroxylic groups, specially in the 4- and 4- positions, enhance the bioactivity of 2-hydroxychalcone (15).
In order to elucidate structure-activity relationships of natural and synthetic polyhydroxylated chalcones, the minimal inhibitory concentrations (MICs) of 2,4,2-trihydroxychalcone 1, 2,4,3-trihydroxychalcone 2 and 2,4,4-trihydroxychalcone 3 against Staphylococcus aureus ATCC 25923 were evaluated. These compounds were obtained by condensation of 2,4-dihydroxyacetophenone and benzaldehyde substituted under specific work conditions (2,16). The chalcone 3 (isoliquiritigenin) occurs free in barck and wood of several Leguminosae (4) as well as in Compositae (5).
MATERIALS AND METHODS
General Experimental Procedure: Chalcones were prepared by adding KOH solution (35 g in 50 ml of water) to an equimolar aldehyde and ketone solution in ethyl alcohol (50 ml). The mixture was maintained in ice. Their structures and numbering system are shown in Fig. 1.
R2=R3=H; R1= OH: 2,4,2-trihydroxychalcone
R1=R3=H; R2=OH: 2,4,3-trihydroxychalcone
R1=R2=H; R3=OH: 2,4,4-trihydroxychalcone
Figure 1. Structure of trihydroxychalcones.
2,4,2-Trihydroxychalcone 1: The reaction mixture was kept in the refrigerator at 4ºC for 30 days. It was diluted with water and acidified with concentrated HCl. The KCl formed was separated by filtration. The total solution was treated with ethyl acetate for to obtain the desired product and dried with Na2 SO4. This extract was concentrated and purified by silica gel and Sephadex LH 20 columns chromatography using benzene and methanol as eluents, respectively. All eluates collected were analyzed by thin layer chromatography (TLC) (polyamide 11 F254, methanol-acetic acid-water, 90:5:5), and spots on the plate were visualized under UV light. After removal of methanol under reduced pressure at 50ºC, an orange crystalline solid was obtained. The structure of 1 was determined by the chromatographic and spectroscopic data: Rf (TLC): 0.163; UVlmax (MeOH) nm: 370; 307; 251; 220. These data are in agreement with those reported by Dhar and Shing (3). 1H NMR (200.13 Mhz, CD3OD): d 6.28 (d, J = 2.3 Hz, H3); 6.4 (dd, J = 8.9, 2.3 Hz, H5); 7.91 (d, J = 8.9 Hz, H6); 7.85 (d, J = 15.6 Hz, H a); 8.14 (d, J = 15.6 Hz, Hß); 6.85 (dd, J = 7.6, 1.7 Hz, H3); 7.24 (td, J = 7.6, 7.6, 1.7 Hz, H4); 6.85 (td, J = 7.6, 7.6, 1.7 Hz, H5); 7.66 (dd, J = 7.6, 1.7 Hz, H6). 13 C NMR (50.33 Mhz, CD3OD): d 114.7, 162.5, 103.8, 166.4, 109.1, 133.3 (C1-C6); 193.9 (C=O, ß); 121.2, 141.3 (Ca, Cß); 123.1, 158.7, 117.0, 132.9, 120.8, 130.0 (C1-C6).
2,4,3-Trihydroxychalcone 2: The reaction mixture, kept in the refrigerator at 4ºC for 7 days was diluted with water and acidified with HCl, resulting in a yellow product. The precipitate was suction filtered, water washed, dried and recrystallized from ethanol-water to give 2,4,3-trihydroxychalcone, that exhibit the expected spectroscopic properties (3). Rf (TLC): 0.185; UVlmax (MeOH) nm: 358; 314; 253; 210. 1H NMR (200.13 Mhz, CD3OD): d 6.28 (d, J = 2.3 Hz, H3); 6.4 (dd, J = 9.0, 2.3 Hz, H5); 7.95 (d, J = 9.0 Hz, H6); 7.66 (d, J = 15.4 Hz, H a); 7.75 (d, J = 15.4 Hz, Hß); 7.12 (br.t, J = 2.1 Hz, H2); 6.84 (dt, J = 7.6, 2.1, 2.1 Hz, H4); 7.21 (dt, J = 7.6, 2.1, 2.1 Hz, H5); 7.25 (dd, J= 7.6, 2.1 Hz, H6). 13 C NMR (50.33Mhz, CD3OD): d 114.6, 167.6, 103.8, 166.7, 109.3, 133.5 (C1-C6); 193.3 (C=O, ß); 121.6, 145.3 (Ca, Cß); 137.6, 115.8, 159.0, 118.8, 122.3, 131.0 (C1-C6).
2,4,4-Trihydroxychalcone 3: The compound was prepared as described for 2,4,2-trihydroxychalcone 1, resulting in a yellow-orange crystalline solid, with Rf (TLC): 0.174; UVlmax (MeOH) nm: 368; 242; 205. 1 H NMR (200.13 Mhz, CD3OD): d 6.26 (d, J = 2.3 Hz, H3); 6.39 (dd, J = 8.9, 2.3 Hz, H5) 7.96 (d, J = 8.9 Hz, H6); 7.6 (d, J = 15.4 Hz, Ha); 7.78 (d, J = 15.6 Hz, Hb); 7.61 (d, J = 8.6 Hz, H2); 6.83 (d, J = 8.6 Hz, H3); 6.83 (d, J = 8.6 Hz, H5); 7.61 (d, J =8.6 Hz, H6). 13 C NMR (50.33 Mhz, CD3OD): d 114.6, 167.5, 103.8, 166.4, 109.2, 133.3 (C1 - C6); 193.5 (C=O, ß); 118.3, 145.6 (Ca, Cß); 122.8, 131.8, 116.9, 161.5, 116.9, 131.8 (C1-C6).
For the assignment of 1H NMR and 13C NMR spectra, resonance double experiences, DD COSY, XHCORR and COLOC spectra were used.
Antibacterial assays: Antibacterial activity was examined by the method of agar plate and broth dilution:
1. Agar plate method: 0.2 ml of a 1:4 dilution of a 18 hours broth culture of S.aureus ATCC 25923 was plated on agar surface, where wells of 10 mm diameter were made. In each well was filled with 0.1 ml of the test substance containing 30 - 130 µg/0.1 ml. After incubation at 37ºC, for 18h, the inhibition zones were measured with a vernier. Chalcones derivatives were diluted in ethanol-water mixture. No inhibitory effect was observed for ethanol in the concentration used in the tests.
2. Broth dilution method: 4 ml of 1/400 diluted inocula, prepared with a fresh culture of S. aureus ATCC 25923, were added to 100 ml of Mueller-Hinton broth (MHB). Aliquots of 7 ml were distributed in test tubes, followed by addition of chalcones at concentrations of 13-55 µg/ml. After incubation for 24 hours at 35ºC, the minimal inhibitory concentration (MIC, µg/ml) was determined by turbidimetry at 720 nm (13).
RESULTS AND DISCUSSION The structures of the prepared compounds were confirmed by chromatographic and spectroscopic data.
Although agar plate method does not yield an absolute measurement of antimicrobial activity, it provides a rapid and preliminary screening determination of the relative activity of the tested compounds. Every chalcone derivatives analysed showed significant bacteriostatic activity (Table 1), with the best activity achieved by compound 1.
Broth dilution method (14) was able to determine more accurate MIC values: 23.0µg/ml; 55.0 µg/ml and 33.5 µg/ml, for compounds 1, 2 and 3 respectively.
Results obtained in a previous work led us to propose inhibition mechanisms in which the molecular region responsible for the biological properties involves the carbonyl group (1).
The inhibition sequence presented by trihydroxylated chalcones when compared to 2',4'-dihydroxychalcone which presented a MIC of 36.5 µg/ml, was:
MIC 2 > MIC 2,4-(OH)2-CHALCONE > MIC 3 > MIC 1
This sequence clearly shows that the introduction of an electron donating group (-OH) in the aromatic B-ring increased bioactivity, the intensity depending on the position of the substituent OH. The closes the active region (C=O) is to the hydroxyl group, the strongest the inhibition effect.
The lower efficacy of 2,4,3-trihydroxychalcone can be attributed to the occurrence of torsion at the C (carbonylic) Ca axis, which forms an angle between the plane that contains the B-ring and the double bond Ca = Cb and the plane that includes the rest of the molecule in chalcone 3-R- substituted (16).
These results suggest that chalcone synthesis should be directed to select compounds with greater bioactivity, like the introduction of hydroxyl groups in the B-ring of the molecule.
ACKNOWLEDGEMENTS The present work was supported by the San Luis National University.
Relação estrutura atividade biológica de chalconas trihidroxiladas sintéticas
A atividade bacteriostática de 2,4,2-trihidroxichalcona, 2,4,3-trihidroxichalcona e 2,4,4-trihidroxichalcona, preparadas por condensação de 2,4-dihidroxiacetofenona e benzaldeido convenientemente substituído, contra Staphylococcus aureus ATCC 25923 foi avaliada pela técnica de difusão em placas. Importantes halos de inibição foram observados para os três compostos. Com o propósito de esclarecer a relação estrutura-atividade biológica, as concentrações inibitórias mínimas (CIM) frente a S. aureus foram determinadas, empregando o método da diluição em caldo. Os resultados obtidos foram semelhantes à 2,4-dihidroxichalcona, observando-se a seguinte ordem: CIM 2,4,3-(OH)3 > CIM 2,4-(OH)2 > CIM 2,4,4-(OH)3 > CIM 2,4,2-(OH). A seqüência obtida mostra que a introdução de um grupo doador de eletrons (OH) no anel aromático B provoca um aumento da bioatividade, sendo a intensidade dependente da posição do substituinte -OH.
Palavras-chave: atividade bacteriostática, relação estrutura-atividade, flavonóides, chalconas trihidroxiladas.
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* Corresponding author. Mailing address: Laboratory of Physical-Chemistry. Department of Chemistry, San Luis National University, Chacabuco 917, 5700 - San Luis, Argentina. FAX: (+54-0652-30224), E-mail: email@example.com