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Medicinal plants have been presented as a valuable source of preservation of human health. In special, Stryphnodendron barbatiman has been employed due to its antimicrobial activity. This plant is rich in tannins and has been used in popular medicine for the treatment of gastrointestinal disorder, treatment of lesions, and also as anti-inflammatory microbicide. Citrobacter freundii is a member of the family Enterobacteriaceae and is one of the major causes of opportunistic infections. This microorganism is a bacterium (bacillus) aerobic gram-negative with a length in the range of 1 to 5 mm. C. freundii is commonly found in water, soil, food and occasionally in the gastrointestinal tract of animals and humans. In this paper, we have demonstrated the antibacterial activity of S. barbatiman by observing cellular death by using inhibition halo approach. Atomic force microscopy and FT-IR spectroscopy results suggested that interaction between the main active components of S. barbatiman with cellular wall of C. freundii gives rise to cellular wall damage, and then leads this microorganism to death.