Antimicrobial activity of flavonoids in medicinal plants ...

Journal of Ethnopharmacology 73 (2000) 317 – 322 www.elsevier.com/locate/jethpharm

Short communication

Antimicrobial activity of flavonoids in medicinal plants from Tafı´ del Valle (Tucumań, Argentina) Nancy E. Hernańdez *, M.L. Tereschuk, L.R. Abdala Ca´tedra de Quı´mica Orgańica y Biolo´gica, Facultad de Ciencias Naturales e Instituto Miguel Lillo, Uni6ersidad Nacional de Tucumań, Miguel Lillo 205, 4000, Tucumań, Argentina Received 15 July 1999; received in revised form 28 May 2000; accepted 6 June 2000

Abstract Preliminary studies of flavonoids have been realised in five native species from Tafı´ del Valle (Tucumań, Argentina) used in popular medicine. Most of compounds detected were flavonoids mono and dihydroxylated in B ring. Screening for antimicrobial activity against Gram positive and Gram negative microorganisms has been realised with Lippia turbinata, Satureja par6ifolia, Sambucus peru6iana, Verbena officinalis and Chenopodium gra6eolens. The total extracts of flavonoids of each plant were tested and four species studied showed antimicrobial activity. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Flavonoids; Medicinal plants; Antimicrobial activity

1. Introduction The phytochemical and biological activities of some Argentina medical plants have previously been reported (Bartotto, 1964; Ratera and Ratera, 1980; Tousarkissian, 1980; Martıńez Crovetto, 1981). A lot of species are used in popular medicine in Tafı´ del Valle, an important area of the province of Tucumań, in the north west of Argentina. Some species are widely distributed in this area (Table 1). * Corresponding author. Fax: +54-381-4212681. E-mail address: [email protected] (N.E. Hernańdez).

Not many studies of antimicrobial activity in medicinal plants from Tafı´ del Valle has been realised and only some species of genus Lippia, Satureja and Sambucus native from Guatemala have been studied against Gram positive Coccus (Caćeres et al., 1991, 1993). The flavonoids are secondary metabolites of low molecular weight, widely distributed in plant kingdom, with several biological activities. There are some reports about antimicrobial activity of flavonoids, e.g. apigenin, a monohydroxylated flavone in B ring (Palacios et al., 1983), quercetin a dihydroxylated flavonol in B ring and myricetin a trihydroxylated flavonol in B ring (Nishino et al., 1987).

0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 9 5 - 6

N.E. Hernańdez et al. / Journal of Ethnopharmacology 73 (2000) 317–322

318

On the other hand, Gnamanickam and Mansfield (1981) reported the antimicrobial activity of flavones and isoflavones against Staphylococcus epidermidis and a report of the group about the positive activity of 7-O-glycosylated flavonol and the negative activity of the 3-O-glycosylated flavonol (Tereschuk et al., 1997). Little information has been found about the content of flavonoids in the species analysed. Only anthocyanins and isoflavones have been reported in Caprifoliaceae, Labiatae, Verbenaceae and Chenopodiaceae (Harborne, 1975; Woldu and Abegaz, 1990; Wollenweber, 1994; Donnelly and Boland, 1994) and flavones have been detected in Chenopodium gra6eolens and Lippia species (Harborne, 1994a,b). In the present paper, preliminary analyses of the flavonoid content and the screening for antimicrobial activity of the flavonoid extract of five species are reported: Satureja par6ifolia (Phil) Epling (munã– munã); Lippia turbinata. Griseb (poleo); Verbena officinalis L. (verbena) and Chenopodium gra6eolens Willd (arca-yuyo); Sambucus peru6iana H.B.K. (sauco) used in popular medicine for the treatment of different diseases.

2. Materials and methods

2.1. Plant material Five species used as medicinal plants were chosen for this study. Botanical material were collected

from Tafı´ del Valle (Tucumań, Argentina) (Table 1). The plants were identified by Lic. Graciela Ponessa from the Departament of Botany, Vegetal Morphology Laboratory (F.M.L) and Lic. Nancy E. Hernańdez. Sambucus peru6iana (H.B.K.). Exsicata: ´ N. Depto. Tafı´ del Valle. Ponessa TUCUMA et,al 598591 (LIL). ´ N. Lippia turbinata Gris. Exsicata: TUCUMA Depto. Tafı´ del Valle. Ponessa. 598639 (LIL). Verbena officinalis Cham. Exsicata: ´ N. Depto. Tafı´ del Valle. TUCUMA Localidad: Rıó El Churqui. Ponessa. 597829 (LIL). ´ N. Satureja par6ifolia Phil. Exsicata: TUCUMA Depto. Tafı´ del Valle. Localidad: El Indio, Camino a Tafı´ del Valle. Ponessa. 598735 Chenopodium gra6eolens. Linne. Exsicata: ´ N. Depto. Tafı´ del Valle. TUCUMA Localidad: Rıó El Churqui. Ponessa. 597829 (LIL)

2.2. Preparation of extracts The plants were air-dried at room temperature. The total extracts of flavonoid content were made from the aerial parts of each plant. The material was exhaustive and successively extracted with MeOH 80% and MeOH 50%. Each methanolic extract was filtered and concentrated to dryness under vacuum. The solid residue was resuspended

Table 1 Ethnobotanical data of studied species Family

Species

Popular uses

Common name

Caprifoliaceae

Sambucus peru6iana Lippia turbinata

Buccal infections, pulmonary diseases, exciting, catartic, diaforetic (Bernal and Correa, 1990) Gastrointestinal disorders, antidiarrhoea (Fernańdez Canosa, 1953) Menstrual flux, milk secretion, antidiarrhoea (Parodi, 1881)

Sauco

Verbenaceae

Labiateae Chenopodiaceae

Verbena officinalis Satureja par6ifolia Chenopodium gra6eolens

Stomachal, diuretic, astringent, stimulant (O’Donell et al., 1943) Antialergic, sedative, inducing sleep

Poleo Verbena, yerba de Santa Ana Munã–munã Arca yuyo, yerba larca, yepasote de toro


in MeOH 80% (w/v) and used for chromatographic studies. The solid residue obtained in similar way to the previous residue, was weighed and dissolved in MeOH 80% at 200 mg/ml concentration. This extract was stored in a closed container in a refrigerator for a maximum time of 15 days.

2.3. Chromatographic techniques Separation of flavonoids was carried out by descendent two dimensional paper chromatography on sheets (30×30 cm). Methods and techniques used and colour spots interpretation are based on procedures described by Mabry et al. (1970), by Markham (1982), and by Mc Cormick (1982) which have been reported in previous papers with some modifications (Abdala de Isralilev et al., 1991).

2.4. Bacterial strains Gram negative rods: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoneae*, Salmonela enteriditis*, Shigella sp.* Gram positive spore-forming rods: Bacillus subtilis (BSGC A1) Gram positive cocci: Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (43, Instituto Microbiologıá, Facultad de Bioquı´mica, Quı´mica y Farmacia, U.N.T.), Streptococcus b-hemolı´ticus A Group ( = Streptococcus pyogenes)*. * Isolation of clinic samples from: Bacteriological section from Instituto de Microbiologıá, ‘Dr. C. Verna’, Facultad de Bioquı´mica, Quı´mica y Farmacia, U.N.T.

2.5. Bacterial cultures The tested strains were cultured twice for activation in Erlenmeyer flasks (125 ml.) containing 25 ml. of Muëller–Hinton broth at 30 – 37°C for 24 h. Optical density (OD) was determined at 600 nm. The cultures were stopped at 0.8 – 1.0 OD600

319

range (equivalent to 108 CFU), diluted until 0.1 OD600. and used for antimicrobial tesing.

2.6. Detection of antimicrobial acti6ity Three ml of 0.6% agar media containing 30 ml of an overnight culture of each microorganism were overlaid on the plates containing 2% agarized Muëller–Hinton media. The agar well diffusion method, previously described by Bennet et al. (1966), was used to detect antimicrobial activity. Wells of 3 mm were made in the agarized medium after inoculation with the test microorganism. The wells were filled with 20 ml of total extract of flavonoids. Chloramphenicol was used as a standard antibiotic in 0–250 mg ml − 1 range. The results are the average of three experiments.

3. Results and discussion The ethnobotanical approach assumes that the popular uses of plants can offer strong clues to the biological activity of those plants. The high percentage of positive results found in this and previous studies (Caćeres et al., 1991, 1993) shows that this approach is also promising for antimicrobial activity. L. turbinata (Verbenaceae) and S. par6ifolia (Labiatae), showed the major activity against the Gram positive and Gram negative microorganisms. V. officinalis (Verbenaceae) showed lower activity against both types of bacteria, while S. peru6iana (Caprifoliaceae) showed activity only against Gram positive bacteria. Ch. gra6eolens (Chenopodiaceae) showed no activity in concordance with ethnobotanical use in popular medicine but no against diseases caused by microorganisms (Table 2). On the other hand, in reference to the structure–activity relationship, preliminary assays on flavonoids and some considerations have been done. Addition of NA reagent (diphenylboryloxiethylamine) to the flavonoids, show yellow colour for a monohydroxylated system in B ring and orange colour for dihydroxylated systems. (Mc Cormick, 1982) (Table 3). The Rf values and changes of


320

Table 2 Results of the microbiological assaya Microbial species

E. coli S. epidermidis S. aureus B. subtilis P. aeruginosa Klebsiella Salmonella Shigella Streptocuccus a

Chloramphenicol equivalent concentrations (mg ml−1) Plant species L. turbinatta S. par6ifolia S. peru6iana

V. officinalis

Ch. gra6eolens

114.5 – 176.0 n.d. 176.0 – – 99.0 68.3

37.5 n.d. 37.5 68.3 – – – – –

– – – – – – – – –

129.8 – 99.0 n.d. 142.5 – – 145.2 68.3

– 37.5 60.6 52.9 – – – – –

n.d., not determinated; –, no inhibition zone.

Table 3 Chromatographic data from the species analysed Species

Compound no.

UV

UV/NH3

NA

Rf×100 (TBA/AcOH 15%)

Sambucus peru6iana

1 2 3 1 2 3 4 1 2 3 4 1 2 3 1 2 3 4

Yellow Dark Yellow–green Dark Dark Dark Dark Dark Dark Dark Dark Dark Dark Dark Dark Dark Dark Dark

Yellow Yellow–green Yellow–green Dark Yellow Yellow–green Yellow Yellow–green Yellow–green Yellow–green Yellow Yellow–green Yellow–green Yellow–green Dark green Yellow–green Yellow Yellow–green

Orange Orange Orange Yellow Yellow Yellow Yellow Yellow Yellow Orange Yellow Orange Orange Orange Yellow Yellow Yellow Yellow

60/00 70/45 75/68 27/48 27/37 51/27 46/17 43/30 40/08 65/21 65/17 17/43 34/24 60/01 26/10 41/16 53/19 59/23

Lippia turbinata

Verbena officinalis

Satureja par6ifolia

Chenopodium gra6eolens

colour from dark under UV light, to yellow – green with ammonia vapours, suggest the presence of aglycone or compounds with C3 occupied, and change to yellow indicates 7-OR derivatives (Mabry et al., 1970) (Table 3). S. peru6iana and S. par6ifolia, have 3%,4% dihydroxylated in B ring flavonoids. In both cases one of the spots identified is an aglycone, since the other two are 3-O glycosylated derivatives. However despite a similar situation was detected in

both species, the activity of S. par6ifolia is higher than S. peru6iana; this might be due to high concentration of flavonoids. L. turbinata and Ch. gra6eolens have monohydroxylated compounds in B ring, but, while L. turbinata has two 7-O glycosylated derivatives and only one 3-O-glycosylated, Ch. gra6eolens has one 7-O-glycosylated derivatives and three 3-O-glycosylated compounds. The absence of activity in Ch. gra6eolens might be due to the inhibitory effect of


the 3-O-glycosylated derivatives over 7-O-glycosylated compounds. (Tereschuk et al., 1997). V. officinalis showed a particular case, since has similar chromatographic profile of flavonoids than Ch. gra6eolens, but presents some antimicrobial activity. Identification of individual chemicals from these extracts and the confirmation of their activity are now under investigation.

4. Conclusions

Four of the five botanical species studied posses different degrees of antimicrobial activity against almost all the microorganisms used. The major compounds detected were glycosylated derivatives of mono and dihydroxylated in B ring flavonoids. The low or no activity observed when 3-O and 7-O glycosylated compounds coexist have been previously reported by Tereschuk et al. (1997). The present study tends to confirm the use in folk medicine of aerial parts of the selected species, as being safe and useful in the treatment of several diseases produced by microorganisms.

Acknowledgements To Consejo de Investigaciones de la UNT (CIUNT) for the financial support and the fellowship for students (Nancy Hernańdez). To Lic. Graciela Ponessa and Lic. Alberto Slanis, for providing plant material.

References Abdala de Isralilev, L.R., del, M.A., de Martıńez, P., Seeligmann, P., 1991. Myricetin in Tagetes (Asteraceae): chemosystematic significance. Phytochemistry (UK) 30, 4037 – 4038. Bartotto, J.C., 1964. Flora Medicinal. Bs. As, Arenaria, pp. 9–301. Bennet, J.V., Brodie, J.L., Benner, J.L., Kirby, W.M.M., 1966. Simplified accurate method for antibiotic assay

321

of clinical specimens. Applied Microbiology 14, 2170 – 2177. Bernal, H., Correa, J.E., 1990. Especies vegetales promisorias (de los paı´ses del convenio Andre´s Bello). Bogota´, Colombia. Caćeres, A., Alvarez, A.V., Ovando, A.E., Samayoa, B.E., 1991. Plants used in Guatemala for the treatment of respiratory diseases I. Screening of 68 plants against Grampositive bacteria. Journal of Ethnopharmacology 31, 193 – 208. Caćeres, A., Figueroa, L., Taracena, A.M., Samayoa, B.E., 1993. Plants used in Guatemala for the treatment of respiratory diseases II. Evaluation of the sensitivity of 16 plants against Gram-positive bacteria. Journal of Ethnopharmacology 39, 77 – 82. Donnelly, D.M.X., Boland, G., 1994. Neoflavonoids. In: Harborne, J.B. (Ed.), The Flavonoids Advances in Research since 1986. Chapman and Hall, London, pp. 261 – 265. Fernańdez Canosa, J., 1953. Lippia integrifolia (Griseb) Huron. Anatomohistologıá y microscopıá cuantitativa de la hoja. Revista Asociacioń Bioquı´mica Argentina XVII 88, 225 – 230. Gnamanickam, S.S., Mansfield, J. (1981). Phytochemistry 20, 997. Harborne, J.B., 1975. The Flavonoids. Chapman & Hall, London, p. 635. Harborne, J.B., 1994a. The Flavonoids. Advances in Research Since 1986. Chapman & Hall, London, p. 261. Harborne, J.B., 1994b. The Flavonoids. Advances in Research Since 1986. Chapman & Hall, London, p. 265. Mabry, T.J., Markhan, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer Verlag, New York, p. 11. Markham, K.R., 1982. Techniques of flavonoids identification. Academic Press, New York, p. 3. Martıńez Crovetto, R., 1981. Plantas utilizadas en Medicina del noroeste de Corrientes. Ministerio de Cultura de la Nacioń, Tucumań, pp. 13 – 125. Mc Cormick S.P., 1982. Flavonoid chemistry of Passiflora subgenus Plectostemma. Ph.D. Thesis. The University of Texas at Austin. Nishino, C., Enoki, N., Tawata, S., Mori, A., Kobayashi, K., Fukushima, M., 1987. Antibacterial activity of flavonoids against S. epidermidis, a skin bacterium. Agricultural Biological Chemistry 51, 139 – 143. O’Donell, C.A., Rodriguez, J.M., 1943. Contribucioń al conocimiento histolo´gico de la materia me´dica indı´gena. Archives Farmaceutico Bioquı´mica Tucumań 1 (1), 17 – 34. Palacios, P., Gutkind, G., Rondina, R., De Torres, R., Coussio, J., 1983. Acta Farmaceutico Bonaerense 2, 5 – 10. Parodi, D., 1881. Ensayo de Botańica Me´dica Argentina PhD Thesis. Bs. As. Imprenta P.E. Com.: Fac. Nac. Cs. Me´dicas, p. 103.

322


Ratera, E.L., Ratera, M.O., 1980. Plantas de la Flora Argentina Empleadas en Medicina Popular. Hemisferio Sur, Bs. As, pp. 227 – 232. Tereschuk, M.L., Quarenghi de Riera, M., Castro, G.R., Abdala, L.R., 1997. Antimicrobial activity of flavonoids from leaves of Tagetes minuta. Journal of Ethnopharmacology (UK) 56 (3), 227–232.

Tousarkissian, M., 1980. Plantas Medicinales de la Argentina: sus nombres Botańicos, Vulgares, usos y Distribucioń Geogra´fica. Hemisferio Sur, Bs. As, pp. 1 – 139. Woldu, Y., Abegaz, B., 1990. Phytochemistry 29, 2013. Wollenweber, E., 1994. Flavones and Flavonols. In: Harborne, J.B. (Ed.), The Flavonoids Advances in Research Since 1986. Chapman & Hall, London, p. 261.

.