Antimicrobial Assays

3.3.1. Microbial Culture Conditions

The different strains must be cultured twice for activation in Erlenmeyer flasks (125 mL) containing 25 mL of Mueller-Hinton broth at 37°C for 12 h in a shaker, except for Lactobacillus rhamnosus, L. plantarum, Zymomonas mobilis, and Sac-charomyces cerevisiae (see Note 10). Stop the cultures at 0.8-1 OD600 (equivalent to 108 CFU ), dilute until 0.1 OD600, and use for antimicrobial testing (see Note 11).

3.3.2. Detection of Antimicrobial Activity in Solid Medium

The agar well diffusion method, previously described (41,42), with some modifications (16), is used to detect antimicrobial activity of plant extracts.

1. Inoculate 3 mL of 0.6% M-H agar media with 30 ^L of an overnight culture of each microorganism prepared as described in Subheading 3.3.1.

2. Overlay onto plates containing 2% Mueller-Hinton agar media, except for Proteus vul-garis, for which 4% agar must be used.

3. Make wells of 3 mm in the agarized medium after inoculation with the microorganism to be tested. More than five wells per plate are not recommended.

4. Fill the wells with 20 ^L of total extract of flavonoids, fractions, or pure compound at a range of concentrations from 5 to 1.000 ^g/mL. The solvent for resuspending the extract should be MeOH 80% instead of pure MeOH because the latter produces irregular inhibition zones.

5. Carry out a negative control with each solvent.

6. Maintain the culture Petri dishes at 37°C, and do not invert them until the solvent is evaporated. After that, invert the plates to avoid water vapors condensation.

7. Measure the inhibition zones from one side to the other of the circle three times and discount the well diameter (Figs. 2 and 3).

8. Use chloramphenicol, as a standard antibiotic, at a range of 0-250 ^g/mL in order to make a calibration curve. The results are the averages of three individual experiments.

Fig. 3. Inhibition zones of fractions from T. minuta against E. coli. Solvent as negative control (1); chloroform fraction (2); ethyl acetate fraction (3); and aqueous fraction (4).

Table 2

Microorganisms Suitable for Antimicrobial Screening



Gram-negative rods

Gram-positive rods

Gram-positive cocci


Escherichia coli Salmonella enteriditis Shigella sp.

Pseudomonas aeruginosa Proteus vulgaris Zymomonas mobilis Bacillus subtilis Lactobacillus rhamnosus Lactobacillus plantarum Staphylococcus aureus Staphylococcus epidermidis Micrococcus luteus Saccharomyces cerevisiae

3.3.3. Minimum Inhibitory Concentration (MIC) in Liquid Medium

MIC values against bacterial strains are performed using the Ericcson and Sherris broth dilution method (43). Inocula suspensions are prepared from 6-h broth cultures and adjusted to 0.5 McFarland turbidity equivalents. Substances and extracts must be sterilized by Millipore filtration (0.45 ^m) and add to the MH broth medium. MIC determination are performed using serial dilutions of each extract and pure substance at a range of 0-1.000 ^g/mL.

4. Notes

1. Caution: chloroform is irritating to the skin, eyes, mucous membranes, and respiratory tract. Chloroform is a carcinogen and may damage the liver and kidneys. Methanol is poisonous and can cause blindness if ingested in sufficient quantities. Adequate ventilation is necessary to limit exposure to vapors. Glacial acetic acid is volatile. Concentrated acids should be handled with great care. Wear gloves and safety glasses and work in a chemical fume hood.

2. Microorganisms suitable for antimicrobial screening are shown in Table 2.

3. The possible enzyme action occurring during this early period of isolation, leading in particular to hydrolysis of glycosides, may be avoided by plunging the plant material into boiling solvent or by rapid drying prior to extraction. The success of the extraction is directly related to the extent that chlorophyll is removed from the solvent and when the tissue debris, on repeated extraction, is completely free of green colors.

4. Depending on the room temperature, it will take the solvent approx 24 h at 30°C to approach 2 cm from the end of sheet.

5. The run is developed in about 3-4 h. It is remarkable that with 15% or 30% AcOH the run typically forms a rounded front effect.

6. Spots detected should be circled with a pencil, and Rf values are determined as follows:

Rf = Distance between origin and the centre of concentration of the flavonoid spot Distance between origin and solvent front

7. Is important to minimize contamination of a flavonoid to be used for UV spectral studies. For purification of the flavonoid used in the spectroscopic investigation, it should be extracted from the chromatographic paper with spectroscopic methanol for a few minutes only.

8. The band should be located at approx 8 cm from the top of the chromatographic paper.

9. Flavonoids isolated by PC may still contain some soluble polysaccharide impurities; it is best to purify them further by Sephadex LH-20 chromatography.

10. Lactobacillus strains are cultured aerobically in MRS broth at 30°C for 12 h at 250 rpm. Zymomonas mobilis should be grown in Zym broth at 30°C for 12 h without agitation. Saccharomyces cerevisiae should be cultured at 30°C for 12 h at 200 rpm.

11. Prior to carrying out the antimicrobial screening, let the microbial dilution sit for 10 min at 37°C without agitation. This culture could be maintained at 4°C for a week for further antimicrobial analyses.


1. Middleton, E. and Kandwasami, C. (1994) The Impact of plant flavonoids on mammalian biology: implications for immunity, inflammation and cancer. In: The Flavonoids: Advances in Research Since 1986 (Harborne, J. B. ed.). Chapman and Hall, London, pp. 619-621.

2. Croft, K. (1998) The chemistry and biological effects of flavonoids and phenolic acids. Ann. NY Acad. Sci. 20, 435-442.

3 Hertog, M. G. L., Fesens, E. J. M, Hollman, P. C. H., Katan, M. B., and Kromhout, D. (1993) Dietary antioxidants: flavonoids and the risk of coronary heart disease: the Zutphen Elderly Study. Lancet 342, 1007-1011.

4. Szent-Gyorgyi, A. (1938) Physiol. Chem. 225, 126.

5. Pathak, D, Pathak, K., and Singla, A. K. (1991) Flavonoids as medicinal agents. Recent advances. Review. Fitoterapia LXII, 371.

6. Harborne, J. B. (1994) The Flavonoids. Advances in research since 1986. Chapman and Hall, London.

7. Djordjevic, M. A., Redmond, J. W., Batley, M., and Rolfe, B. G. (1987) Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhyzobium trifolii. EMBO J. 6, 1173.

8. Firmin, J. L., Wilson, K. E., Rossen, L., and Johnston, A. W. B. (1986) Flavonoid activation of nodulation genes in Rhyzobium reversed by other compounds present in plants. Nature 324, 90.

9 Welton, A. F., Hurley, J., and Will, P. (1988) Flavonoids and arachidonic acid metabolism. Prog Clin Biol Res 280, 301-312.

10 Hertog, M. G. L, Kromhout, D., and Aravanis, C (1995) Flavonoid intake in long-term risk of coronary heart disease and cancer in seven countries study. Arch. Intern. Med. 155, 381-386.

11 Soejarto, D. D. and Farnsworth, N. R. (1989). Tropical rain forest: potential source of new drugs? Perspect Biol. Med. 32, 244-256.

12 Farnsworth, N. R. (1994) Ethnopharmacology and drug development. In: Ethnobotany and the Search for New Drugs (Prance, G. T., ed.). Wiley, Chichester (Ciba Foundation Symposium 185), pp. 42-59.

13. Farnsworth, N. R. (1984) The role of medicine plants in drug development. In: Natural Products and Drug Development (Krogsgaard-Larsen, P., Christensen, S. B., and Kofod, H., eds.). Balliere, Tindall and Cox, London, pp. 8-98.

14. Farnsworth, N. R and Soejarto, D. D. (1991) Global importance of medicinal plants. In: Conservation of Medicinal Plants (Akerele, O., Heywood, V., and Synge, H., eds.). Cambridge University Press, Cambridge, pp. 25-51.

15 Eloff, J. N. (1998) Which extractant should be used for the screening and isolation of antimicrobial components from plants? J. Ethnopharmacol. 60, 1-8.

16 Tereschuk, M. L., Quarenghi de Riera, M, Castro, G. R. and Abdala, L. R. (1997) Antimicrobial activity of flavonoids from leaves of Tagetes minuta. J. Ethnopharmacol. 56, 227-232.

17. González, A., Vázquez, A., Ferreira, F., and Moyna, P. (1993) Biological screening of Uruguayan medicinal plants. J. Ethnopharmacol. 39, 217-220.

18 Quarenghi, M. V., Tereschuk, M. L., Baigorí, M. D., and Abdala, L. R. (2000) Antimicrobial activity of flowers from Anthemis cotula. Fitoterapia 71, 710-712.

19. Penna, C. A., Radice, M., Gutkind, G. O., et al. (1994) Antibacterial and antifungal activities of some Argentinean plants. Fitoterapia 65, 172-174.

20 Zardini, E. M. (1984). Etnobotánica de las compuestas argentinas con especial referencia a su uso farmacológico. Acta Farm. Bonaerense 3, 169-194.

21. Cáceres, A., Figueroa, L., Taracena, A., and Samayoa, B. (1993) Plants used in Guatemala for treatment of respiratory diseases. 2: Evaluation of activity of 16 plants against grampositive bacteria. J. Ethnopharmacol. 39, 77-82.

22. Cáceres, A., Torres, M., Ortiz, S., Cano, F., and Jauregui, E. (1993) Plants used in Guatemala for the treatment of gastrointestinal disorders. IV. Vibriocidal activity of five American plants used to treat infections. J. Ethnopharmacol. 39, 73-75.

23. Tereschuk , M. L., Gallegos Serruto, S., Quarenghi, M. V., and Abdala, L. R. (1999) Comparative assays of the antimicrobial activity between two species of Tagetes (Astaeraceae). Acta Horticulturae 501, 223-225.

24. Strother, J. L. (1977) Tageteae—systematic review. In: The Biology and Chemistry of the Compositae (Heywood, V., Harborne, J. B., and Turner, B. L., eds.). Academic, London, pp. 769-796.

25 Soule, J. A. (1996) Novel annual and perennial Tagetes. In: Progress in New Crops (Janick, J., ed.). ASHS Press, Arlington, VA, pp. 546-551.

26 Amat, A. G. (1983) Investigación farmacológica de los taxones mayoritarios de compuestas bonaerenses. Acta Farm. Bonaerense 2, 23-36.

27 Neher, R. T. (1966) Monograph of the genus Tagetes (Compositae). PhD Thesis, Indiana University, Bloomington, IN.

28 Macedo, M. E., Consoli, R. A., Grandi, T. S., et al. (1997) Screening of Asteraceae (Compositae) plant extracts for larvicidal activity against Aedes fluviatilis (Diptera:Culicidae). Mem. Inst. Oswaldo Cruz 92, 565-570.

29 Abad, M. J., Bermejo, P., Sánchez Palomino, S., Chiriboga, X., and Carrasco, L. (1999) Antiviral activity of some South American medicinal plants. Phytother. Res. 13,142-146.

30. Abdala, L. R. and Seeligmann, P. (1983) Distribución de los flavonoids de hojas de tres especies de Tagetes (Compositae) y su significado quimiosistemático. Lilloa 36, 5-14.

31. Tereschuk, M. L., Vázquez, A., Pezaroglo, H., Baigorí, M., and Abdala, L. R. (2002) Determinación estructural de compuestos con actividad antibacteriana en especies de Tagetes del noroeste argentino. I Congreso Latinoamericano de Fitoquímica-IV Meeting de la Sociedad Latinoamericana de Fitoquímica, Buenos Aires, Argentina.

32 Hollman, P. Bijsman, M., Gameren, Y, Cnossen, E., de Vries, J., and Katan, M. (1999) The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man. Free Rad. Res. 31, 569-573.

33. Wagner, H. and Bladt, S. (1996) Plant Drug Analysis: A Thin Layer Chromatography Atlas, 2nd ed. Springer Verlag, Berlin, Heidelberg, New York, pp. 169-224.

34. Mabry T. J., Markhan K. R., and Thomas M. B. (1970) The systematic Identification of Flavonoids. Springer-Verlag, New York, p.11.

35. Markham K. R. (1982) Techniques of Flavonoids Identification. Academic, New York.

36. Imperato, F. (1998) Two new flavonol glycosides from the fern Pteridum aquilinum. Ilnd International Electronic Conference on Synthetic Organic Chemistry (ECSOC-2), no. 074.

37. Harborne, J. B. (1998) Phytochemical Methods, 3rd ed. Chapman and Hall, London, pp. 63-82 and pp. 235-255.

38. Abdala, L. R., Del Pero de Martinez, M. A., and Seeligmann,P. (1991) Myricetin in Tagetes (Asteraceae). Chemosystematic significance. Phytochemistry 30, 4037-4038.

39 Crozier, A., Jensen, E., Lean, M. E. L., and McDonald, M. S. (1997) Quantitative analysis of flavonoids by reversed-phase high-performance liquid chromatography. J. Chromatogr. A. 761, 315-321.

40 Merken, H. and Beecher, G. (2000) Measurement of food flavonoids by high-performance liquid chromatography: a Review. J. Agric. Food Chem. 48, 577-599.

41. Bennet, J. V., Brodie, J. L., Benner, J. L., and Kirby, W. M. M. (1966) Simplified accurate method for antibiotic assay of clinical specimens. Appl. Microbiol. 14, 170-177.

42. Vanden Berghe D.A and Vlietink, A.J. (1991) Methods in Plant Biochemistry, vol. 6. Academic Press, London, p. 47.

43. Ericcson, H. M. and Sherris, J. C. (1971) Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol. Microbiol. Scand. 217(suppl).

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