Antimicrobial and Antioxidant Secondary Metabolites from Trifolium baccarinii Chiov. (Fabaceae) and Their Mechanisms of Antibacterial Action

Abstract

The treatment of infectious diseases with antimicrobial agents continues to present problems in modern-day medicine with many studies showing significant increase in the incidence of bacterial resistance to several antibiotics. The screening of antimicrobial activity of plant extracts and natural products has shown that medicinal plants are made up of a potential source of new anti-infective agents. The aim of this study was to evaluate the antimicrobial and antioxidant activities of extracts and compounds from the whole plant Trifolium baccarinii Chiov. and to determine their modes of antibacterial action. The plant extracts were prepared by maceration in organic solvents. The antimicrobial activities were evaluated using the broth microdilution method. The antioxidant activity was evaluated using the 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) assays. The mechanisms of antibacterial action were determined by lysis, salt tolerance assays, and antioxidant enzyme activities. The cytotoxic effect on the erythrocytes was determined by a spectrophotometric method. Biochanin A, formononetin, luteolin, luteolin-4'-O-β-D-glucopyranoside, 4,7,2'-trihydroxy-4'-methoxyisoflavanol, sissotrin, 1-methyl-β-D-glucopyranoside, ononin, D-mannitol, and 3-O-β-D-glucuronopyranosylsoyasapogenol B were isolated from Trifolium baccarinii. The MeOH, EtOAc, and n-BuOH extracts as well as biochanin A, formononetin, luteolin, luteolin-4'-O-β-D-glucopyranoside, 4,7,2'-trihydroxy-4'-methoxyisoflavanol, and sissotrin from Trifolium baccarinii displayed the highest antimicrobial and antioxidant activities. The MeOH extract and 4,7,2'-trihydroxy-4'-methoxyisoflavanol exhibited antibacterial activity through the bacteriolytic effect and reduction of the antioxidant defenses in the bacterial cells. The present study portrays Trifolium baccarinii as a potential natural source of antibacterial, antifungal, and antioxidant agents.

Figure 1

Chemical structures of compounds isolated from Trifolium baccarinii (1–10). 1: biochanin A; 2: formononetin; 3: luteolin; 4: luteolin-4′-O-β-D-glucopyranoside; 5: 4,7,2′-trihydroxy-4′-methoxyisoflavanol; 6: sissotrin; 7: 1-methyl-β-D-glucopyranoside; 8: ononin; 9: D-mannitol; and 10: 3-O-β-D-glucuronopyranosylsoyasapogenol B.

Figure 2

Bacteriolytic effect of the MeOH extract and compound 5 against Pseudomonas aeruginosa and Staphylococcus aureus. Results represent the mean ± standard deviation of the triplicate OD at each incubation time.

Figure 3

Effect of the Trifolium baccarinii methanol extract (a) and compound 5 (b) on the reduction of salt tolerance of Staphylococcus aureus. MIC: minimum inhibitory concentration; bars represent the mean ± standard deviation of the triplicate CFU. For the same figure, values with different letters are significantly different at p < 0.05 according to the Waller-Duncan test.

Figure 4

Antioxidant catalase (a) and superoxide dismutase (b) activities in Staphylococcus aureus ATCC25923 and methicillin-resistant Staphylococcus aureus 03 (MRSA03) treated with the MeOH extract and compound 5. Bars represent the mean ± SD of three independent experiments carried out in triplicate. For the same microorganism and enzyme, values earmarked by different superscript letters (a–g) are significantly different according to one-way ANOVA and the Waller-Duncan test (p < 0.05).