Indian J Med Res 135, February 2012, pp 252-254 Correspondence Evaluation of antibiotic & antibiotic modifying activity of pilocarpine & rutin Sir, 24 h at 37°C. Clinical isolate EC27 was resistant to neomycin and gentamicin (low level) and to amikacin and kanamycin17. S. aureus 358 (MRSA) showed resistance to methicillin. All strains were obtained from the collection of microorganisms of the Mycology Laboratory, UFPB, Paraiba, Brazil. Three standard yeast strains were utilized: Candida albicans ATCC 40227, C. krusei ATCC 6538 and C. tropicalis ATCC 13803. All these strains were maintained on HIA, and before the assays, the cells were grown in BHI for 24 h at 37°C. The antibiotics tested were the aminoglycosides amikacin, kanamycin, gentamicin and neomycin (Sigma, USA). The antifungal agent was amphotericin B (Sigma, USA). The antibiotic and antifungal solutions were prepared following the recommendations of the Clinical and Laboratory Standards Institute – CLSI18. The test compounds (pilocarpine and rutin) (obtained from Merck & Company, Germany), was dissolved (10 mg) in 1 ml dimethylsuphoxide (DMSO- Merck, Darmstadt, Germany), giving an initial concentration of 10 mg/ml. Starting with this concentration, a dilution was made to 1024 μg/ml, Secondary metabolites are small molecules1, responsible for various biological activities, acting as antibiotic, antifungal and antiviral agents to protect plants from pathogens. There are large groups of secondary metabolites, which include alkaloids and flavonoids2, described mainly by their antimicrobial3,4 and antioxidant potential5. Pilocarpine is an alkaloid present in the leaves of jaborandi (Pilocarpus microphyllus)6, which is utilized as a phytotherapeutic agent in the treatment of glaucoma and xerostomia7. Rutin is a flavonoid belonging to the subclass of flavones found in various plant sources8,9, which has shown antioxidant activity10, efficacy in the control of Aspegillus oschraceus11, anticonvulsivant effects in rats12, suppression of cellular immunity13, anticarcinogenic activity14 and antiinflammatory effect15. The irrational use of antibiotics and antifungals has resulted in the development of drug resistance. The rapid development of drug resistance and the slow-down in the development of new active drugs, have drawn attention to treatment with drug combinations16. The aim of this study was to evaluate the antibacterial and antifungal activity of rutin and pilocarpine and to determine their possible modifying effect when combined with aminoglycoside antibacterials and the antifungal amphotericin B. Table I. Evaluation of antifungal and antibacterial activities of test compounds Minimal inhibitory concentration - MIC (μg/ml) Cepas Rutin Pilocarpine Negative DMSO control 32 32 32 C. albicans (ATCC 40227) 32 32 32 C. krusei (ATCC6538) 32 32 32 C. tropicalis (ATCC 13803) 128 128 128 E. coli 27 128 128 128 E. coli (ATCC 10536) 128 128 128 S. aureus 358 128 128 128 S. aureus (ATCC 25923) The experiments were carried out in Laboratory of Microbiology and Molecular Biology, Department of Biological Chemistry, Regional University of Cariri, Crato, Brazil, with standard and multiresistant strains of Staphylococcus aureus and Escherichia coli: S. aureus ATCC 12692, S. aureus 358 (MRSA), E. coli ATCC 25922 and E. coli 27 (EC27). All strains were maintained on heart infusion agar slants (HIA; Difco, USA), and before the assays, the cells were grown in brain heart infusion broth (BHI; Difco) for 252 ARARUNA et al: ANTIBIOTIC MODIFYING ACTIVITY OF PILOCARPINE & RUTIN 253 Table II. Evaluation of antibiotic modifying activity of test compounds Concentrations (μg/ml) S. aureus 358 Rutin Pilocarpine DMSO C+ C+, only antibiotic AMI 156.25 156.25 156.25 156.25 KAN 2.500 2.500 2.500 2.500 GEN 19.53 ≤1.22 19.53 19.53 E. coli 27 NEO 312.5 39.06 312.5 312.5 AMI 156.25 156.25 156.25 156.25 KAN 312.5 312.5 312.5 312.5 GEN 19.53 19.53 19.53 19.53 NEO 156.25 156.25 156.25 156.25 and further dilutions were made serially 1:2 in culture medium, obtaining concentrations of 512 to 8 μg/ml. The minimal inhibitory concentration (MIC, µg/ml) was determined in 10 per cent BHI by the broth microdilution method, using a suspension of 105 cfu/ml and a drug concentration of 1024-1 μg/ml19. To evaluate the test compounds for drug modifying activity when combined with antibiotics and antifungals, a subinhibitory concentration was determined as the MIC/8 values of 16 μg/ml for EC27 and MRSA, and 10 μg/ml for C. albicans, C. krusei and C. tropicalis. The plates were incubated for 24 h at 37 °C, utilizing resarzurin to read bacterial growth and no stain for fungi. does not inhibit the growth of Staphylococcus aureus. The use of drugs in combination has been extensively studied, mainly because of the emergence of resistant strains24. The MIC values for rutin and pilocarpine showed no antibacterial or antifungal activity against the strains tested (Table I). Pilocarpine, however, increased bacterial sensitivity to the aminoglycosides gentamicin and neomycin, when added concomitantly (Table II). Pilocarpine combined with these aminoglycosides altered synergistically the MIC values against S. aureus but not E. coli. Neither of the compounds tested showed an antifungal modifying effect (> 1024 μg/ ml) with amphotericin B against the Candida strains tested. Mariana K.A. Araruna*+, Samara A. Brito** , Maria F.B. Morais-Braga†, Karla K.A. Santos† , Teogenes M. Souza†, Tiago R. Leite†† Jose G.M. Costa** & Henrique D.M. Coutinho† * Laboratorio de Farmacologia e Quimica Molecular (LFQM), **Laboratorio de Pesquisa de Produtos Naturais (LPPN), †Laboratorio de Microbiologia e Biologia Molecular (LMBM), †† Laboratorio de Botanica Aplicada (LBA), Universidade Regional do Cariri – URCA, Crato, Ceara, Brazil + For correspondence: Universidade Regional do Cariri, Departamento de Química Biológica, Crato, CE, Brazil. Av. Cel. Antônio Luiz Nº 1161, Cep:63105-000 marianakessia@yahoo.com.br The MIC values determined in the antifungal and antibacterial assays were equal to that of the negative DMSO control, suggesting that this activity was due to DMSO which is considered toxic only at higher concentration20. Rutin has been extensively studied for its various pharmacological properties, such as its anti-candida activity9,21. Missau et al 22 used the direct bioautography method and showed anti-fungal activity against three strains of Candida, with a significant effect against only C. krusei. Bolle et al23 found that this method could result in decomposition of the test substances during the assay. Our findings corroborated with that of Pereira et al11 who showed that rutin, isolated from the plant species Solanum palinacanthum The synergistic effect of pilocarpine indicates a new therapeutic possibility for the treatment of diseases associated with S. aureus infection and an alternative for the resistance shown by this microorganism against certain aminoglycosides. Further studies are required to evaluate the toxicity and antibacterial activity of this compound in vivo. Competing interests: The authors declare that they have no competing interests. References 1. Salisbury FB, Ross CW. Plant physiology, 4th ed. Belmont: Wadsworth Publishing Company; 1992. p. 681. 2. Simões CMO, Schenkel EP, Gosmann G, de Mello JCP, Mentz LA, Petrovick PR. Farmacognosia: da planta ao medicamento, 6th ed. Editora da UFRGS; Florianópolis: Editora da UFSC; 2007. p. 1104. 3. 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