Farmar ETHNO PHARMACOLOGY ELSEVIER Journal of Ethnopharmacology 56 (1997) 227 Antimicrobial activity of flavonoids from leaves of Tagetes minuta Maria L. Tereschuk *, Marta V.Q. Riera“, Guillermo R. Castro *, Lidia R. Abdala ** * Citedra de Quimica Orginica y Biolégica, Facultad de Ciencias Naturales ¢ Instituto Miguel Lillo, Universidad Nacional de Tucwnin. Miguel Lillo 205. 4000 Tucwnn, Argentina ° Citedra de Microbiologia Superior, Facultad Bioguimica, Quimica ) Farmacia, Universidad Nacional de Tucumier - PROIMI-— Avda. Belgrano y Pe. Cares. 4000 Tucwnan, Argentina Received 25 August 1996; received in revised form 2 March 1997; accepted 11 March 1997 Abstract ‘The total extract and fractions with different solvents, obtained from leaves of Tagetes minua, showed several degrees of antimicrobial activity against Gram positive and Gram negative microorganisms. The same fractions were inactive against Lactobacillus, Zymomonas and Saccharomices species. The major component of the extract ‘quercetagetin-7-arabinosyl-galactoside, showed significant antimicrobial activity on pathogen microorganisms tested Correlation results were carried out using chloramphenicol as standard antibiotic. © 1997 Elsevier Science Iretand Lid. Keywords: Tagetes minuta; Asteraceae: Flavonoids; Antimicrobial activity 1. Introduction The folk medicine of Argentina employ plants to counteract diverse diseases. Species of the family Asteraceae are some of the most useful, and some South American members were re- ported previously to show pharmacological activi- ties. Leaves infusions from members of Tagetes (Astereceae) have been used in folk medicine to Corresponding author. Fax: +S4 81 239456; e-mail gobiaesnat unt eduar treat stomach and intestinal diseases (Zardini 1984), and several Tagetes species have been found to possess biological activity (Ross et al., 1981; Penna et al. 1994), On the other hand, antibacterial activity of flavonoids has been reported against microorgan- isms like Proteus vulgaris, Staphylococcus aureus and Staphylococeus epidermidis (Mori etal, 1987 Nishino et al., 1987). These compounds have also other important effects including enzyme inhibi- tion, antioxidant and cytotoxic effects (Middleton and Kandaswami, 1993). (0378-8741/97/S17.00 © 1997 Elsevier Science Ireland Ltd All rights reserved PULSO378- 41(97}00038-X28 ML. Teresohuk et al. /Journal of Ethnopharmacology $6 (1997) 227-232 Radius (mm) q ae ° a 5 4 oe 1 18 2 28 8 too “7 miata Fig. 1, Radius of £, col! growth inhibition (mm) versus the concentration logarithm of Tagetes mimua total extract (yg ml"). Tageres minuta is a native plant known as ‘suico’ or ‘chinchilla’ and has been used in popu- lar medicine as antimicrobial, antihelminthic, di- uretic and antispasmodic (Amat, 1983). It was selected for this work, on the basis of previously reported antimicrobial activity (Camm et al. 1975; Amat, 1983) and its flavonoid content which as been reported by Abdala de Israilev and Secligmann (1983). Although several studies on thiophenes and essential oil activity have been made in Tagetes species (Ross et al., 1981; Hethe- lyi et al, 1989; Croes et al,, 1989), flavonoid activity has not received much attention, The aim of this paper is to study the antimicro- bial activity of flavonoids from Tagetes minuta, towards some Gram positive and Gram negative microorganisms, the isolation, purification and identification of the responsible compounds, 2. Methodology 2.1, Plant material Leaves of Tagetes minuta were collected from several localities in Tucumdn, Argentina. Voucher specimens were deposited in the herbarium of Fundacién Miguel Lillo (LIL). Tagetes minuta L. ARG. Prov. TUCUMAN, Dep. Burruyacié: Agua Negra (Los Pinos), 2-V- 1937, A. Peirano s/n (LIL 58551); Piedra Buena, I8-IV-1944, L.A. Varela, (LIL 101720). Dep. Chi- cligasta: Puesto de las Pavas, 16-VI-1949, T. Meyer 15134 (LIL). Dep. Capital: 47 km next to Tafi, 24-V-1950, R. Rocha 2403 (LIL). 2.2, Preparation of extracts Extracts of Tagetes minuta were prepared by ‘maceration of 5.0 g dried leaves with 80 and 50% methanol at room temperature until drug exhaus- tion. The fractions were collected and the solvent was removed under vacuo. The solid residue was resuspended in 80% methanol at 500 ue mi~t concentration, and was designated as the total extract, Three fractions of this extract were taken up with the following solvents at room tempera- ture: chloroform, ethyl acetate, and water. 2.3, Isolation, purification and identification of biological active compounds Methods and techniques used, are based on procedures described by Mabry et al. (1970) and by Markham (1982), which have been reported inML. Tereschuk etal. / Journal of Ethnopharmacology 56 (1987) 227-232 Chloramphenicol ug/mi 250 200 100 29 160 200 260 300 350 400 460 600 660 Total extract ug/ml Fig. 2. Comparison of microbial growth inhibition of E, coli by Chloramphenicol and Tagetes minute total extract. previous papers with some modifications (Abdala de Israiley et al., 1991). 2.4, Bacterial strains Bacillus subtilis (BSGC. Al), Escherichia coli (ATCC 25922), Lactobacillus rhamnosus (ATCC 7469), Lactobacillus plantarum (21, Instituto Mi- crobiologia, Facultad de Bioquimica, Quimica y Farmacia, U.N.T.), Pseudomonas aeruginosa (ATCC 27853), Saccharomyces cerevisiae PAI (PROIMI, Planta Piloto de Procesos Industriales Microbidlogicos), Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (43, Instituto Microbiologia, Facultad de Bioquimica, Quimica y Farmacia, U.N.T.), Zymomonas mobilis FloB3 (PROIMD). 2.8. Bacterial cultures The tested strains were cultured twice for acti- vation in 125 ml Erlenmeyer containing 25 ml of Mueller-Hinton or nutrient broths at 30-37°C for 24 hb. Optical density was determined at 600 am (Metrolab 325 BD, Argentina). The cultures were stopped at 0.8-1.0 optical density ranges (equiva- lent to 10* CFU), diluted until 0.1 O.D. and used for antimicrobial tests. 2.6. Detection of antimicrobial activity Three ml of 6 g 1! agar media containing 30 1 of overnight culture of each microorganism were overlaid on the plates, containing 20 g 1~' agarized Mueller Hinton. media. The agar-well diffusion method was used to detect antimicrobial, activity (Bennet et al., 1966). Wells of 3 mm were made in the agarized medium after inoculation with the test microorganism. Then, the wells were filled with 20 j11 of total extract, chloroform frac- tion, ethyl acetate fraction, aqueous fraction and pure flavonoids. Chloramphenicol was used as a standard antibiotic in 0-250 zg ml! range. The results are recorded as the means of triplicate experiments230 MLL. Tereschuk et a. /Sourmal of Etlnopharmacology $6 (1987) 227-232 Table | Activity of diferent Tageres minura extracts on some microorganisms tested expressed as chloramphenicol concentrations (1g mt") Microorganisms Fractions Total extract Bacillus subi 0 Escherichia col i Peeudmonas aeruginosa It Staphylococeus aureus bo ‘Staphylococcus epidermidis 12 « Quercetagetin 7arabinosy! galactoside, 3. Results Serial dilutions of total extracts from leaves of Tagetes minuta were first tested against Es- cherichia coli. Growth inhibition was detected in the 4,00-500.0 jeg ml" range of extracts (Fig. 1) Correlation zones of inhibition between chlo- ramphenicol used as standard antibiotic, and Tagetes minuta total extract against E.coli showed an hyperbolic response (Fig. 2). At higher concentrations (200-500 zg mi '), total extracts of Tagetes minuta showed the same inhibition of growth as 200-220 ug ml~' of chloramphenicol In this range, the sensitivity of chloramphenicol ‘was approximately twice that of the total plant extract. However, when low concentrations of Tagetes minuta total extract were assayed (below 504g ml~"), approximately three times higher concentrations of chloramphenicol had to be used in order to obtain the same inhibition of growth Total extract, ethyl acetate and aqueous frac- tions, showed different inhibitory properties against B. subtilis, E. coli, S. aureus, S. epider- ‘midis, and P. aeruginosa (Table 1). However. L. rhamnosus, L. plantarum, S. cerevisae, and Z. mobilis were not inhibited by any fraction. The chloroform fraction did not show any antimicro- bial activity against all microorganisms tested in ‘our experimental conditions. The chromato- graphic profile of chloroform fraction did not show any flavonoids or related compounds. The analysis of chromatographic maps, from the total extract, showed seven spots; quere- Chloramphenicol equivalent concentrations (ag mt") AGEL Aqueous Isa ° 6 0 130 130 38 99 0 192 ctagetin, quercetagetin-7-0-arabinosyl-galactoside, quercetagetin-3-0-arabinosyl-galactoside, _quere- etagetin-T-glucoside, patuletin-7-glucoside, pat- uletin and isorhamnetin (Table 2), and. these results were in accordance with previous work (Abdala de Istailev and Seeligmann, 1983). The ethyl acetate fraction showed six spots. The com- pound not observed in ethyl acetate fractions was patuletin-7-glycoside, and only five spots were found in the water fraction, most of them from the total extract, except quercetageti binosyl-galactoside and patuletin-7-glycoside. The major components of the total extract were: quercetagetin-7-arabinosyl-galactoside and quer etagetin-3-arabinosyl-galactoside. These _flavo- noids were selected for testing antimicrobial activity because quercetagetin and its derivatives are characteristic of Tagetes species. The flavonoid quercetagetin-3-arabinosyl-galae toside did not show antimicrobial activity, while quercetagetin-7-arabinosyl-galactoside showed a very important activity against all of the microor- ganisms tested (Table 1). 4, Discussion E. coli was selected as a first bacterial target for T. minusa extract due 10 its importance as a possible enteropathogen bacteria, The growth was considerably inhibited by the total flavonoids ex- tract at low concentrations compared with chlo- ramphenicol.MLL, Tereschak etal Table 2 Idenification of flavonoids produced by leaves of Tageses minuta ournal of Eshnopharmacology 56 (1997) 227-232 21 Compounds Rr uv NH, NA Querectagetin 22,00 4 4 ° Quercetagetin-7arabinosyl-galactoside 05:06 4 a 0 Quercetagetin--arabinossb-gaactoside 18/12 4 ay pm Quereetagetin--glucoside 05:03 a ab po Patuletin 804 dy ay © Patuletn-7-gucoside asi ay y ° Isorhamnetin so02 dy Wy y Abbreviations: d, dark; db, dark brown; dy, dark yellow: 0, orange; ro, red orange; y, yellow The different antimicrobial activities of 7. minuta total extract, ethyl acetate, and aqueous fractions against B. subvilis, E. coli, S. epidermidis, S. aureus and P. aeruginosa could be explained by the effect of flavonoid concentrations in each solvent andjor the different flavonoid compo: tions of the fractions. These results are in agree- ment with # previous work, where 100 eg ml~' of quercetagetin were an active agent against P. cul- garis, but not against S. aureus (Mori et al., 1987) E.coli and P. aeruginosa showed similar antibiotic profiles with ethyl acetate and aqueous fractions (Table 1). These results might suggest that quere- clagetin-3-arabinosyl-galactoside, absent in the aqueous extract, and patuletin-7-glycoside, not present in any of both fractions, were not respon- sible of antimicrobial activity. This was sup- ported, in part, by the negative results obtained with pure querecetagetin-3-arabinosyl-galactoside at the same concentration. The activity of pure quercetagetin-7-arabinosyl-galactoside, showed similar activity profiles than those observed with all extracts, against these two Gram negative bac- teria. These results might be explained by the high concentration of this flavonoid in the total extract and in the fractions. (Table 1) On the contrary, in two Gram positive Staph) locoecus species, aqueous fraction showed higher activity than ethyl acetate, This could be ex- plained by the flavonoid concentrations, and the possible interfering activities of quercetagetin-3- arabinosyl-galactoside, compound not detected in aqueous extracts, Similar values were observed between the pure quercetagetin-7-arabinosyl- galactoside and the ethyl acetate extract, but lower than those observed in the aqueous extract These results might be explained by the synergic action of the other flavonoids present in each fraction. A similar situation with total extract was detected. but the values were higher. This might be due to the high concentration of flavonoids in this extract or to the presence of some other active compound. Bacillus. subtilis seems to have different. be- haviour against the total extract and the fractions. The activity of the ethyl acetate fraction is higher than the total extract. The aqueous fraction and quercetagetin-7-arabinosyl-galactoside have the same values, but lower than both extracts The absence of antimicrobial activity against non-pathogenic human bacteria as Z. mobilis, S. cerevisiae and all Lactobacillus species could be beneficial for intestinal disease treatments, in which the intestinal flora must be preserved. Further investigations are necessary to deter- mine the antimicrobial qualities of minor flavonoids produced by leaves of 7. minuta, and to establish the possible mechanism of action of the most active compound, in microorganisms Acknowledgements The financial support from the Consejo de In- vestigaciones de la Universidad Nacional de Tu- cumin (CIUNT), Consejo Nacional de Investigaciones Cientificas_y Técnicas. (CON ICET) of Argentina, and Third World Academy232 MAL, Tereschuk etal. Journal of Ethnopharmacology 56 (1997) 227-232 of Sciences (TWAS, Italy) are gratefully acknow!- edged, We would like to thank Biog. Silvia Valz Gianinnet for critical reading of this manuscript, ‘The technical assistance of Mr Salvador Borchia is also gratefully acknowledged. References Ablala de Isriley, L.R.A.. Seligmann, P. 1983. Distribution of flavonoids from leaves of three species of Tagetes Com- positae and their chemosystematics significance (In Spanish). Lilloa 36, 5-14 Abdala de lrilev, LR, Del Pero de Martinez, M.A. Seelig- ‘mann, P1991. Mysiesin in Tagetes) (Asteraceae) chemosystematic significance. Phytochemistry 30, 4037. ‘Amat, A.G.« 1983. Pharmacological research for major taxons ‘of Bonacrenses Compositae (in Spanish). Acta Farm Bonaerense 2, 23-36 Bennet, JV., Brodie JL. Benner,J.L.. Kirby. WM.M., 1966 Simplified acurate method for antibiotic assay of clnicel specimens. Appl. Microbiol. 14, 170-177, Camm, B.L., Towers, G.H.N.. Mitchell J.C, 1975. UV-medi- ‘ated antibiotic activity of some Composite species, Phyto- ‘chemistry 14, 2007-201 Crocs, ALF. 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