- Email: [email protected]
Screening antifungal activities of selected medicinal plants
Journal of Ethnopharmacology 74 (2001) 89 – 96 www.elsevier.com/locate/jethpharm
Short Communication
Screening antifungal activities of selected medicinal plants Emma Nelly Quiroga, Antonio Rodolfo Sampietro 1, Marta Amelia Vattuone *,2 Ca´tedra de Fitoquı´mica, Instituto de Estudios Vegetales ‘Dr. Antonio R. Sampietro’, Facultad de Bioquı´mica, Quı´mica y Farmacia, Uni6ersidad Nacional de Tucuma´n, Ayacucho 471, 4000 San Miguel de Tucuma´n, Argentina Received 19 November 1999; received in revised form 26 August 2000; accepted 22 September 2000
Abstract Plants synthesise a vast array of secondary metabolites that are gaining importance for their biotechnological applications. The antifungal activity of the ethanolic extracts of ten Argentinean plants used in native medicine is reported. Antifungal assays included radial growth inhibition, disk and well diffusion assays and growth inhibition by broth dilution tests. The chosen test fungi were yeasts, microfungi and wood-rot causing Basidiomycetes. Extracts of Larrea di6aricata, Zuccagnia punctata and Larrea cuneifolia displayed remarkable activity in the assays against the majority of the test fungi. In addition to the former plants, Prosopanche americana also inhibited yeast growth. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fungi; Antifungal activities; Bioassays
1. Introduction Fungi occur ubiquitously and are well adapted to use a wide range of substrates as their carbon, nitrogen and energy source. The growth of many fungi is especially difficult to control because of their ability to metabolize many substances. * Corresponding author. Tel.: + 54-381-4247752, ext. 260; fax: + 54-381-4248025. E-mail address: [email protected] (M.A. Vattuone). 1 Deceased. 2 Researcher from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Buenos Aires, Argentina.
These organisms can cause serious diseases in plants, animals, and humans. Many Basidiomycetes such as Ganoderma applanatum, Lenzites elegans, Pycnoporus sanguineus and Schizophyllum commune were associated with white-rot of living trees and on logs, dead twigs and slumps (Harsh and Bisht, 1997). They can degrade wood, leading to economic losses in forestry. Therefore, it is important to find products or methods to protect living trees and timber from decay (Blanchette, 1994). Antifungal secondary metabolites isolated from the heartwood of plants (Reyes Chilpa et al., 1987) have been considered to contribute to tree
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 3 5 0 - 0
90
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
resistance against wood destroying fungi. For this reason, it is of growing interest to detect antifungal active compounds to control the development of these wood-destroying fungi. Plants produce a great deal of secondary metabolites, many of them with antifungal activity. Well-known examples of these compounds include flavonoids, phenols and phenolic glycosides, unsaturated lactones, sulphur compounds, saponins, cyanogenic glycosides and glucosinolates (Go´mez Garibay et al., 1990; Bennett and Wallsgrove, 1994; Grayer and Harborne, 1994; Osbourne, 1996). We present results from a screening for antifungal higher plant extracts obtained from selected plants of the Northwest of Argentina. The chosen plants are all used in traditional medicine.
2. Materials and methods
Penicillium notatum Westling, Trichoderma spp. and Aspergillus niger Thom and Church. These fungi were supplied by Instituto Nacional de Tecnologı´a Agraria, Tucuma´n, Argentina. The yeast strains used in this study were Saccharomyces carlsbergensis Hansen (IEV 002) and Rhodotorula spp (IEV 001). They were obtained from Laboratorio de Micologı´a, Instituto de Microbiologı´a, UNT, Argentina.
2.3. Preparation of ethanolic extracts Ten grams of ground air-dried plant material were shaken in 100 ml EtOH 96° at 40°C for 48 h (40 cycles/min). The insoluble material was filtered by filter paper (Whatman No. 4) and evaporated to dryness under reduced pressure at 40°C. The extract was weighed and dissolved in EtOH 96° at 0.100 and 0.035 g ml − 1 final concentration for antifungal tests against yeasts and mycelial fungi.
2.1. Plant material 2.4. Fungal cultures Some plants were purchased from the market and others were collected in different sites of the Northwest of Argentina. Plants were identified by Dr A.R. Sampietro at the chair in Phytochemistry of the Universidad Nacional de Tucuma´n (UNT), Argentina (Toursarkissian, 1980). Voucher specimens were deposited in the Herbarium of the Instituto de Estudios Vegetales (IEV), Facultad de Bioquı´mica, Quı´mica y Farmacia, UNT (Tucuma´n, Argentina). The used parts were leaves, stems, flowers, roots, tree barks and, in some cases, the whole plant.
2.2. Microorganisms The following wood-destroying fungi were used: L. elegans Spreng ex Fr. (IEV 012), G. applanatum (pers, ex Walls) Pat. (IEV 017), P. sanguineus (L. Ex Fr.) Murr. (IEV 006), and S. commune Fr. (IEV 009). These strains were obtained from the Botanical Institute ‘Miguel Lillo’, Tucuma´n, Argentina, and were originally isolated from local decaying wood. Other phytopatogenic fungi used were Fusarium oxysporum Schlecht,
Filamentous fungi were inoculated in 250 ml Erlenmeyer flasks with 50 ml growth medium: malt extract broth (15 g malt extract and 5 g peptone in 1 l distilled water). Then, it was incubated at 30°C until a strong mycelial growth was obtained (8–10 days). Yeasts were grown in the same medium and were incubated at 30°C for 24–48 h. Fungal cultures were maintained on malt extract broth plus 15 g Bacto agar l − 1 (1.5% malt extract, 0.5% peptone and 1.5% Bacto agar).
2.5. Test for antifungal acti6ity The in vitro biological activity of alcoholic plant extracts was assessed on basis of hyphal radial growth rate of filamentous fungi, and growth rate of yeasts in the presence and absence of the plant extract. Different volumes of each alcoholic extract (0.035 g ml − 1) from 0.2 to 0.4 ml were poured into assay flasks containing 16 ml hot sterilised growth medium (malt agar 1.5%) and the final volume was adjusted to 17 ml with EtOH. After it was thoroughly mixed with a vortex, aliquots of 5 ml treated medium were
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
poured into Petri dishes (60× 15 mm). Control plates were treated with EtOH only. The plates were left to stand overnight after pouring in a sterile hood to remove the solvent completely. The assay was performed by placing a 3-mm diameter plug of growing mycelia onto the centre of a Petri dish containing the alcoholic extract in the medium. This plug was taken from an 8- to 10-day culture. The fungal strains P. notatum, Trichoderma spp. and A. niger, cultured at 30°C for 7 – 10 days, easily produced spores. A spore suspension was prepared in 0.9% NaCl solution at a known inoculum density (2.5× 104 ml − 1 fungal spores). Ten microliter aliquots of this suspension were placed onto the centre of the Petri dish as already described. These suspensions were prepared immediately before the test was carried out. Plates were incubated aerobically at 30°C in a humidity environment to prevent the growth medium from drying out. Three replicates were run simultaneously. The radial growth of mycelia (colony diameter (mm)) in all plates was measured 5 days after inoculation. Each datum point is the mean of at least four measurements of a growing colony. The percentage of growth inhibition was calculated from mean values as (Reyes Chilpa et al., 1997):
91
plus 2% Bacto agar cooled at 45°C. This was then poured into 9.1 cm diameter Petri dishes, and maintained for 1 h at room temperature. Small wells were cut in the agar plate using a cork borer (5 mm). A fixed volume of different alcoholic extract concentration and a carrier solvent control were loaded in the wells. The dishes were then inverted and preincubated at 4°C for 2 h to allow uniform diffusion into the agar. After preincubation, the plates were inverted and incubated aerobically at 30°C for 48 h. The diameter of the inhibition zone around each well was recorded in four different directions.
2.6.2. Paper disk diffusion assays Agar plates were poured in the same mode as already described. Sterile paper disks (Whatman No. 4 paper, 5 mm diameter) were impregnated with 10 ml different vegetal alcoholic extract dilutions of known concentrations. The disks were allowed to dry and then six disks were spaced on the agar surface of each Petri dish. A disk negative control (10 ml ethanol) was included. The diameter of the inhibition zone around the disks was measured after 48 h at 30°C. The values were the average (mm) of four measurements per disk, taken at four different directions in order to mistake minimisation. The experiment involved the use of at least three plates for each test.
% Inhibition= mycelial growth in control− mycelial growth in extract plant × 100 mycelial growth in control
2.6. Test for biological acti6ity against yeasts In these assays, two agar diffusion techniques ( Camm et al., 1975; Cole, 1994; Torres et al., 1998) and broth dilution tests were employed. The yeast cultures were prepared by inoculating the microorganism to be tested in a 250 ml Erlenmeyer flask containing 50 ml malt-broth medium, followed by incubation at 30°C for 24 – 48 h or until the turbidity matched the Mac Farland tube No. 1.
2.6.1. Agar well diffusion assays An aliquot of 50 ml inoculum preparation was added to 20 ml melted extract malt-broth medium
2.6.3. Dilution test This test was used for minimal inhibitory concentration (MIC) determination. A 96-well plastic microplate was used. Each well contained malt extract broth (15 g malt extract and 5 g peptone in 1 l distilled water), plus a fixed volume of serially diluted plant extracts. Each well was inoculated with a 10 ml aliquot of the yeast inoculum prepared according to Section 2 in a final volume of 0.15 ml. The controls contained malt extract broth plus plant extract. Incubations were performed at 30°C for 24 h. Yeast growth was quantified by A550 nm using an automatic microplate reader Model 550 (Bio-Rad Laboratories, Richmond, CA). MIC was defined as the lowest concentration of plant extract to which no yeast growth was observed after incubation at 30°C for 24 h.
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
92
3. Results and discussion The antifungal activity of crude ethanolic extracts of ten medicinal plants used in the traditional medicine of the Northwest of Argentina was tested. Ethnobotanical data are shown in Table 1. As this table shows, the antifungal screening was performed in the whole plant when the selected plant was herbaceous or a woody shrub. When it was a tree, only defined parts were chosen. The plant antifungal activity was assayed against eight filamentous fungi and two yeasts. The growth inhibitory activities of the alcoholic extracts of the different Argentine plants against filamentous fungi are summarized in Table 2. The data indicate that alcoholic extracts of Zuccagnia punctata and Larrea di6aricata have considerable in vitro activity against all filamentous fungi tested. Extracts of Larrea cuneifolia have no activity only against F. oxysporum. The Schkuhria pinnata extract did not inhibit the growth of all the filamentous fungi assayed.
Some members of the Aspergillus and Fusarium genera are well known to produce aflatoxins. These secondary metabolites are potent carcinogens, hepatotoxins, teratogens and inmunosuppresive compounds (Ciegler, 1975). F. oxysporum produces phytotoxic fusaric acid and lycomarasmin (Ueno et al., 1977). A. niger produces potent mycotoxins on foodstuffs and is the most prevalent fungus affecting corn (Marassas, 1991). These fungi represent threats not only to the health of crops, but also to animals and humans ingesting contaminated feeds and foods. The alcoholic extract from L. di6aricata effectively reduced the growth of P. notatum in maltagar medium. Samples of 2, 3 and 4 mg dry extract/plate (0.4, 0.6 and 0.8 mg ml − 1 dry matter in culture medium) produce 47, 68 and 89% of inhibition, respectively (Fig. 1). Extract from Z. punctata inhibited mycelial growth of G. applanatum and L. elegans (Fig. 2). The aqueous and alcoholic extracts from Phytolacca dioica and Zinnia peru6iana showed antimicrobial activity against Gram-positive and
Table 1 Ethnobotanical data of the studied plants Family
Botanical name
Common name
Used parts
Popular uses
Zygophyllaceae
Larrea di6aricata Cav
Jarilla, jarilla hembra
Whole plant
Zygohyllaceae
Larrea cuneifolia Cav
Jarilla, jarilla macho
Whole plant
Equisetaceae Compositae
Cola de caballo Canchalahua
Whole plant Whole plant
Compositae Leguminosae
Equisetum giganteum L. Schkuhria pinnata Lam O. Kuntzi Zinnia peru6iana L Zuccagnia punctata Cav
Whole plant Whole plant
Leguminosae
Acacia ca6en (Mol) Molina
Chinita del campo Jarilla pispito, jarilla de la Puna, pus-pus Churqui, tusca, espinillo
Vulnerary, antirheumatic, emmenagoge, antiinflamatory Vulnerary, antrheumatic, emmenagoge, antiinflamatory Diuretic, astringent Antibiotic, vulnerary, phototoxic Vulnerary, phototoxic Vulnerary
Phytolaccaceae
Phytolacca dioica L o Pircunia dioica (L) Moq. Prosopanche americana (R. Br) Baillon o P.burmeisteri De Bary Schinus molle L.
Hydnoraceae
Anacardiaceae
Ombu´ Guaycuru´ santiaguen˜o, flor de tierra Molle, pimienta del diablo, aguaribay
Leaf, stem, bark Antiseptic, vulnerary, of a tree cicatrizant Whole plant Vulnerary, antifeverish, vermifugy Flowers, roots Vulnerary, hemostatic, expectorant Whole plant
Antiinflamatory, emmenagoge, vulnerary
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
93
Table 2 Radial growth inhibition of alcoholic extracts against filamentous fungia Fungib
Alcoholic extractsc 1
2
3
4
5
6
7
8
9
A
++
++
−
++
−
+++
−
+
+
B
++
++
−
−
−
+++
−
−
−
C
++
+
−
+
−
++
+
−
+
D
+
−
−
−
−
++
−
−
−
E
++
++
−
++
+
++
+
−
+
F
+++
++
−
+
+
++
−
−
9
G
++
++
−
−
−
++
−
−
−
H
++
+
−
+
9
+++
−
−
−
10
− − − − + + + + a The extract concentration (dry matter) in the culture medium was 0.8 mg ml−1 in all cases. The inhibition was reported as (−) B10% growth inhibition, ( 9 ) between 10 and 20%, (+) between 20 and 40%, (++) between 40 and 80%, and (+++) 80%. (n= 12.) b A, Lenzites elegans; B, Schizophyllum commune; C, Pycnoporus sanguineus; D, Ganoderma applanatum; E, Fusarium oxysporum; F, Penicillium notatum; G, Aspergillus niger; H, Trichoderma spp. c 1, Larrea di6aricata Cav.; 2, Larrea cuneifolia Cav.; 3, Schkuhria pinnata (Lam) O. Kuntzi; 4, Zinnia peru6iana (L), L.; 5, Equisetum giganteum L.; 6, Zuccagnia punctata Cav.; 7, Acacia ca6en (Mol) Molina; 8, Phytolacca dioica L.; 9, Prosopanche americana (R.Br) Baillon; 10, Schinus molle L.
gram-negative microorganisms (Amani et al., 1998). Otherwise, alcoholic extracts of P. dioica and Z. peru6iana did not show significant antifungal activity against the eight filamentous fungi tested at the highest dose used. L. elegans, P. sanguineus and F. oxysporum were average sensitive in vitro to alcoholic extracts of flowers and roots of Prosopanche americana. A peptide designed Pa-AMP was isolated and purified from seeds of P. americana. This peptide showed inhibitory activity for pathogenic fungi and Gram-positive and negative bacteria (Minami et al., 1998). It was found that the extract from Schinus molle inhibits the growth of F. oxysporum, P. notatum, A. niger and Trichoderma spp. Triterpe-
nes and sesquiterpenolactones isolated from leaves and fruits of S. molle showed antifungal activity when they were tested against Candida albicans, Trichophyton mentagraphytes and A. niger. Also Aspergillus alternata, Aspergillus fla6us, Mycrosporum griseum, P. cyclopium and P. italicum showed growth inhibition in vitro by their leaf essential oils (Ross et al., 1980; Dishit et al., 1986). The responses of the two assayed yeasts (S. carlsbergensis and Rhodotorula spp.) are shown in Table 3. It was found that extracts of L. di6aricata, L. cuneifolia, Z. punctata and P. americana reduced the growth of both yeasts. Fig. 3 shows growth inhibition of S. carlsbergensis by various amounts of L. di6aricata extract. Similar results
94
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96 Table 3 Paper disk diffusion of plant alcoholic extracts on yeast growtha Plant alcoholic extracts Yeast strains
Fig. 1. Growth inhibition of Penicillium notatum by alcoholic extract of Larrea di6aricata. C, Control; 2, 3 and 4 mg dry matter/plate.
were obtained with the agar well diffusion assays (not shown). Table 4 shows the MICs observed for the effect of different plant extracts on yeasts. L. cuneifolia exhibited a better inhibition activity against S. carlsbergensis than L. di6aricata and P. americana.
Fig. 2. Antifungal activity of Zuccagnia punctata extract against the wood destroying fungi Ganoderma applanatum and Lenzites elegans. C, Control; 2; 3 and 4 mg dry matter/plate.
L. di6aricata Cav. L. cuneifolia Cav. S. pinnata Lam O. Kuntzi Z. peru6iana (L), L. E. giganteum L. Z. punctata Cav. A. ca6en (Mol.) Molina P. dioica L. P. americana (R.Br) Baillon S. molle L.
S. carlsbergensis
Rhodotorula spp.
++ ++ −
+++ ++ −
− − ++ −
− − ++ −
− ++
− ++
+
−
a The growth inhibition of the two yeasts was determined by paper disk diffusion assays, using 0.2 mg dry matter/disk. The inhibition is reported as: (−) without inhibition, (+) dr =1.0 mm, (++) dr =1.0–1.5 mm, and (+++) dr =1.5–2.0 mm, where dr is diameter of the inhibition zone. (n =12.)
Otherwise, L. di6aricata and L. cuneifolia showed the highest inhibition of Rhodotorula spp. growth. The MICs of L. cuneifolia against S. carlsbergensis and Rhodotorula spp. were 100 and 133 mg ml − 1, respectively, while the MIC of an accepted antifungal compound isolated and purified from
Fig. 3. Disk diffusion assay. Effect of growing amounts of Larrea di6aricata extract on Saccharomyces carlsbergensis growth. Control and 25, 33, 50, 100 and 200 mg dry matter/ disk in the clockwise sense.
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96 Table 4 MICs of different plant extracts against yeastsa Plant extracts
Yeasts S. carlsbergensis (mg ml−1)
Larrea di6aricata Cav. Larrea cuneifolia Cav. Zuccagnia punctata Cav. Prosopanche americana (R.Br) Baillon
Rhodotorula spp. (mg ml−1)
200 100 400
100 133 200
200
266
a
MICs were determined by dilution tests as indicated in Section 2. Data were taken from the inhibition produced by plant extracts (20–500 mg dry extract ml−1 were used). (n= 8; S.D. =0.01–0.09).
Anemarrhena asphodeloides against Saccaromyces cere6isiae was 200 mg ml − 1 (Iida et al., 1999). Although studies on growth inhibition of fungi by isolated plant compounds suggest a role in plant defense, such in vitro tests may not always give an accurate indication of the significance of these compounds in restricting fungal growth in the plant (Mansfield, 1983). As plants are sessile organisms, they are confined to the place where they grow. They are capable of sensing the presence of potential phytopatogens (fungi, bacteria and viruses) and mounting defense mechanisms. Plants can produce antifungal compounds to protect themselves from biotic attack that could be essential for fungi infection resistance (Wojtaszek, 1997). The understanding of the inhibitory mechanism would provide better directions toward the development of efficient production and application of technologies associated with biofungicide plant materials. This is the first report showing the antifungal activities of different Argentine plants. Further studies, including the isolation and structure – function relationships of the antifungal natural products are now in progress. Acknowledgements The authors are grateful to the Ca´tedra de Micologı´a, Instituto de Microbiologı´a, Facultad de
95
Bioquı´mica, Quı´mica y Farmacia de la Universidad Nacional de Tucuma´n (UNT) and to the Instituto Nacional de Tecnologı´a Agropecuaria (INTA), Tucuma´n, Argentina for the generous gift of fungus cultures. This work was supported by grants from the Secretarı´a de Ciencia y Te´cnica from the UNT, Tucuma´n, Argentina.
References Amani, S.M., Isla, M.I., Vattuone, M.A., Poch, M.J., Cudmani, N.G., Sampietro, A.R., 1998. Antimicrobial activities in some argentine medicinal plants. In: V. Martino et al. (Eds.), Proceedings of the WOCMAP-2: Pharmacognosy, Pharmacology, Phyto-medicines, Toxicology, Acta Horticulturae, ISHS, vol. 501, pp. 115 – 118. Bennett, R.N., Wallsgrove, R.M., 1994. Secondary metabolites in plant defence mechanisms. New Phytology 127, 617 – 633. Blanchette, R.A., 1994. Degradation of the lignocellulose complex in wood. Canadian Journal of Botany 73, S999 – S1010. Camm, E.L., Towers, G.H.N., Mitchell, J.C., 1975. UV-mediated antibiotic activity of some Compositae species. Phytochemistry 14, 2007 – 2011. Ciegler, A., 1975. Mycotoxins, occurrence, chemistry, biological activity. Lloydia 38, 481 – 483. Cole, M.D., 1994. Key antifungal, antibacterial and anti-insect assays. A critical review. Biochemical Systems and Ecology 22, 837 – 856. Dishit, A., Naqvi, A., Husain, A., 1986. A new source of natural fungitoxicant. Applied and Environmental Microbiology 51, 1085 – 1088. Go´mez Garibay, F., Reyes Chilpa, R., Quijano, L., Caldero´n Pardo, J.S., Rı´os Castillo, T., 1990. Methoxifurans auranols with fungostatic activity from Lonchocarpus castilloi. Phytochemistry 29, 459 – 463. Grayer, R.J., Harborne, J.J., 1994. A survey of antifungal compounds from higher plants, 1982 – 1993. Phytochemistry 37, 19 – 42. Harsh, N.S.K., Bisht, N.S., 1997. Wood decaying fungi of Kumaun Himalaya. In: Sati, S.C., Saxena, J., Dubey, R.C. (Eds.), Recent Researches in Ecology, Environment and Pollution, vol. X. Today and Tomorrow, New Delhi-5, pp. 69 – 93. Iida, Y., Oh, K.B., Saito, M., Matsuoka, H., Kurata, H., Natsume, M., Abe, H., 1999. Detection of antifungal activity in Anemarrhena asphodeloides by sensitive BCT method and isolation of its active compound. Journal of Agricultural and Food Chem 47, 584 – 587. Mansfield, J.W., 1983. In: Callow, J.A. (Ed.), Antimicrobial Compounds in Biochemical Plant Pathology. Wiley, Chichester, UK, pp. 237 – 265.
96
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
Marassas, W.F.O., 1991. Toxigenic Fusaria. In: Smith, J.E., Henderson, R.E. (Eds.), Mycotoxins and Animal Foods. CRC Press, Boca Rato´n, FL, pp. 119–139. Minami, Y., Higuchi, S., Yagi, F., Tadera, K., 1998. Isolation and some properties of the antimicrobial peptide (PaAMP) from the seeds of pokeweed (Phytolacca americana). Bioscience and Biotechnology of Biochemistry 62, 2076– 2078. Osbourne, A.E., 1996. Preformed antimicrobial compounds and plant defense against fungal attack. The Plant Cell 8, 1821 – 1831. Reyes Chilpa, R., Pe´rez Morales, V., Del Angel Blanco, S., 1987. Influencia de los extractivos en la resistencia natural de seis maderas tropicales al hongo de pudricio´n morena Lenzites trabea. Bio´tica (Me´xico) 12, 7–13. Reyes Chilpa, R., Quiroz Va´zquez, R.I., Jime´nez Estrada, M., Navarro-Ocan˜a, A., Cassani Herna´ndez, J., 1997. Antifungal activity of selected plant secondary metabolites against
.
Coriolus 6ersicolor. Journal of Tropical Forest Products 3, 110 – 113. Ross, S., El-Keltawi, N., Megalla, S., 1980. Antimicrobial activity of some Egyptian aromatic plants. Fitoterapia LI, 201 – 205. Torres, M., Bacells, M., Sala, N., Sanchis, V., Canela, R., 1998. Bactericidal and fungicidal activity of Aspergillus ochraceus metabolites and some derivatives. Pesticide Science 53, 9 – 14. Toursarkissian, M., 1980. Plantas Medicinales de la Argentina. Editorial Hemisferio Sur, S.A., Bs. As., Argentina, pp. 5, 39, 42, 51, 60, 67, 75 and 98. Ueno, Y., Ishhii, K., Sawano, M., Ohtsubo, K., Mutsuda, Y., Tanaka, T., Kurata, H., Ichinoe, M., 1977. Toxicological approaches to the metabolites of Fusaria. Japanese Journal of Experimental Medicine 47, 177 – 184. Wojtaszek, P., 1997. Oxidative burst an early plant response to pathogen infection. Biochemistry Journal 322, 681 – 692.
Short Communication
Screening antifungal activities of selected medicinal plants Emma Nelly Quiroga, Antonio Rodolfo Sampietro 1, Marta Amelia Vattuone *,2 Ca´tedra de Fitoquı´mica, Instituto de Estudios Vegetales ‘Dr. Antonio R. Sampietro’, Facultad de Bioquı´mica, Quı´mica y Farmacia, Uni6ersidad Nacional de Tucuma´n, Ayacucho 471, 4000 San Miguel de Tucuma´n, Argentina Received 19 November 1999; received in revised form 26 August 2000; accepted 22 September 2000
Abstract Plants synthesise a vast array of secondary metabolites that are gaining importance for their biotechnological applications. The antifungal activity of the ethanolic extracts of ten Argentinean plants used in native medicine is reported. Antifungal assays included radial growth inhibition, disk and well diffusion assays and growth inhibition by broth dilution tests. The chosen test fungi were yeasts, microfungi and wood-rot causing Basidiomycetes. Extracts of Larrea di6aricata, Zuccagnia punctata and Larrea cuneifolia displayed remarkable activity in the assays against the majority of the test fungi. In addition to the former plants, Prosopanche americana also inhibited yeast growth. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fungi; Antifungal activities; Bioassays
1. Introduction Fungi occur ubiquitously and are well adapted to use a wide range of substrates as their carbon, nitrogen and energy source. The growth of many fungi is especially difficult to control because of their ability to metabolize many substances. * Corresponding author. Tel.: + 54-381-4247752, ext. 260; fax: + 54-381-4248025. E-mail address: [email protected] (M.A. Vattuone). 1 Deceased. 2 Researcher from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Buenos Aires, Argentina.
These organisms can cause serious diseases in plants, animals, and humans. Many Basidiomycetes such as Ganoderma applanatum, Lenzites elegans, Pycnoporus sanguineus and Schizophyllum commune were associated with white-rot of living trees and on logs, dead twigs and slumps (Harsh and Bisht, 1997). They can degrade wood, leading to economic losses in forestry. Therefore, it is important to find products or methods to protect living trees and timber from decay (Blanchette, 1994). Antifungal secondary metabolites isolated from the heartwood of plants (Reyes Chilpa et al., 1987) have been considered to contribute to tree
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 3 5 0 - 0
90
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
resistance against wood destroying fungi. For this reason, it is of growing interest to detect antifungal active compounds to control the development of these wood-destroying fungi. Plants produce a great deal of secondary metabolites, many of them with antifungal activity. Well-known examples of these compounds include flavonoids, phenols and phenolic glycosides, unsaturated lactones, sulphur compounds, saponins, cyanogenic glycosides and glucosinolates (Go´mez Garibay et al., 1990; Bennett and Wallsgrove, 1994; Grayer and Harborne, 1994; Osbourne, 1996). We present results from a screening for antifungal higher plant extracts obtained from selected plants of the Northwest of Argentina. The chosen plants are all used in traditional medicine.
2. Materials and methods
Penicillium notatum Westling, Trichoderma spp. and Aspergillus niger Thom and Church. These fungi were supplied by Instituto Nacional de Tecnologı´a Agraria, Tucuma´n, Argentina. The yeast strains used in this study were Saccharomyces carlsbergensis Hansen (IEV 002) and Rhodotorula spp (IEV 001). They were obtained from Laboratorio de Micologı´a, Instituto de Microbiologı´a, UNT, Argentina.
2.3. Preparation of ethanolic extracts Ten grams of ground air-dried plant material were shaken in 100 ml EtOH 96° at 40°C for 48 h (40 cycles/min). The insoluble material was filtered by filter paper (Whatman No. 4) and evaporated to dryness under reduced pressure at 40°C. The extract was weighed and dissolved in EtOH 96° at 0.100 and 0.035 g ml − 1 final concentration for antifungal tests against yeasts and mycelial fungi.
2.1. Plant material 2.4. Fungal cultures Some plants were purchased from the market and others were collected in different sites of the Northwest of Argentina. Plants were identified by Dr A.R. Sampietro at the chair in Phytochemistry of the Universidad Nacional de Tucuma´n (UNT), Argentina (Toursarkissian, 1980). Voucher specimens were deposited in the Herbarium of the Instituto de Estudios Vegetales (IEV), Facultad de Bioquı´mica, Quı´mica y Farmacia, UNT (Tucuma´n, Argentina). The used parts were leaves, stems, flowers, roots, tree barks and, in some cases, the whole plant.
2.2. Microorganisms The following wood-destroying fungi were used: L. elegans Spreng ex Fr. (IEV 012), G. applanatum (pers, ex Walls) Pat. (IEV 017), P. sanguineus (L. Ex Fr.) Murr. (IEV 006), and S. commune Fr. (IEV 009). These strains were obtained from the Botanical Institute ‘Miguel Lillo’, Tucuma´n, Argentina, and were originally isolated from local decaying wood. Other phytopatogenic fungi used were Fusarium oxysporum Schlecht,
Filamentous fungi were inoculated in 250 ml Erlenmeyer flasks with 50 ml growth medium: malt extract broth (15 g malt extract and 5 g peptone in 1 l distilled water). Then, it was incubated at 30°C until a strong mycelial growth was obtained (8–10 days). Yeasts were grown in the same medium and were incubated at 30°C for 24–48 h. Fungal cultures were maintained on malt extract broth plus 15 g Bacto agar l − 1 (1.5% malt extract, 0.5% peptone and 1.5% Bacto agar).
2.5. Test for antifungal acti6ity The in vitro biological activity of alcoholic plant extracts was assessed on basis of hyphal radial growth rate of filamentous fungi, and growth rate of yeasts in the presence and absence of the plant extract. Different volumes of each alcoholic extract (0.035 g ml − 1) from 0.2 to 0.4 ml were poured into assay flasks containing 16 ml hot sterilised growth medium (malt agar 1.5%) and the final volume was adjusted to 17 ml with EtOH. After it was thoroughly mixed with a vortex, aliquots of 5 ml treated medium were
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
poured into Petri dishes (60× 15 mm). Control plates were treated with EtOH only. The plates were left to stand overnight after pouring in a sterile hood to remove the solvent completely. The assay was performed by placing a 3-mm diameter plug of growing mycelia onto the centre of a Petri dish containing the alcoholic extract in the medium. This plug was taken from an 8- to 10-day culture. The fungal strains P. notatum, Trichoderma spp. and A. niger, cultured at 30°C for 7 – 10 days, easily produced spores. A spore suspension was prepared in 0.9% NaCl solution at a known inoculum density (2.5× 104 ml − 1 fungal spores). Ten microliter aliquots of this suspension were placed onto the centre of the Petri dish as already described. These suspensions were prepared immediately before the test was carried out. Plates were incubated aerobically at 30°C in a humidity environment to prevent the growth medium from drying out. Three replicates were run simultaneously. The radial growth of mycelia (colony diameter (mm)) in all plates was measured 5 days after inoculation. Each datum point is the mean of at least four measurements of a growing colony. The percentage of growth inhibition was calculated from mean values as (Reyes Chilpa et al., 1997):
91
plus 2% Bacto agar cooled at 45°C. This was then poured into 9.1 cm diameter Petri dishes, and maintained for 1 h at room temperature. Small wells were cut in the agar plate using a cork borer (5 mm). A fixed volume of different alcoholic extract concentration and a carrier solvent control were loaded in the wells. The dishes were then inverted and preincubated at 4°C for 2 h to allow uniform diffusion into the agar. After preincubation, the plates were inverted and incubated aerobically at 30°C for 48 h. The diameter of the inhibition zone around each well was recorded in four different directions.
2.6.2. Paper disk diffusion assays Agar plates were poured in the same mode as already described. Sterile paper disks (Whatman No. 4 paper, 5 mm diameter) were impregnated with 10 ml different vegetal alcoholic extract dilutions of known concentrations. The disks were allowed to dry and then six disks were spaced on the agar surface of each Petri dish. A disk negative control (10 ml ethanol) was included. The diameter of the inhibition zone around the disks was measured after 48 h at 30°C. The values were the average (mm) of four measurements per disk, taken at four different directions in order to mistake minimisation. The experiment involved the use of at least three plates for each test.
% Inhibition= mycelial growth in control− mycelial growth in extract plant × 100 mycelial growth in control
2.6. Test for biological acti6ity against yeasts In these assays, two agar diffusion techniques ( Camm et al., 1975; Cole, 1994; Torres et al., 1998) and broth dilution tests were employed. The yeast cultures were prepared by inoculating the microorganism to be tested in a 250 ml Erlenmeyer flask containing 50 ml malt-broth medium, followed by incubation at 30°C for 24 – 48 h or until the turbidity matched the Mac Farland tube No. 1.
2.6.1. Agar well diffusion assays An aliquot of 50 ml inoculum preparation was added to 20 ml melted extract malt-broth medium
2.6.3. Dilution test This test was used for minimal inhibitory concentration (MIC) determination. A 96-well plastic microplate was used. Each well contained malt extract broth (15 g malt extract and 5 g peptone in 1 l distilled water), plus a fixed volume of serially diluted plant extracts. Each well was inoculated with a 10 ml aliquot of the yeast inoculum prepared according to Section 2 in a final volume of 0.15 ml. The controls contained malt extract broth plus plant extract. Incubations were performed at 30°C for 24 h. Yeast growth was quantified by A550 nm using an automatic microplate reader Model 550 (Bio-Rad Laboratories, Richmond, CA). MIC was defined as the lowest concentration of plant extract to which no yeast growth was observed after incubation at 30°C for 24 h.
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
92
3. Results and discussion The antifungal activity of crude ethanolic extracts of ten medicinal plants used in the traditional medicine of the Northwest of Argentina was tested. Ethnobotanical data are shown in Table 1. As this table shows, the antifungal screening was performed in the whole plant when the selected plant was herbaceous or a woody shrub. When it was a tree, only defined parts were chosen. The plant antifungal activity was assayed against eight filamentous fungi and two yeasts. The growth inhibitory activities of the alcoholic extracts of the different Argentine plants against filamentous fungi are summarized in Table 2. The data indicate that alcoholic extracts of Zuccagnia punctata and Larrea di6aricata have considerable in vitro activity against all filamentous fungi tested. Extracts of Larrea cuneifolia have no activity only against F. oxysporum. The Schkuhria pinnata extract did not inhibit the growth of all the filamentous fungi assayed.
Some members of the Aspergillus and Fusarium genera are well known to produce aflatoxins. These secondary metabolites are potent carcinogens, hepatotoxins, teratogens and inmunosuppresive compounds (Ciegler, 1975). F. oxysporum produces phytotoxic fusaric acid and lycomarasmin (Ueno et al., 1977). A. niger produces potent mycotoxins on foodstuffs and is the most prevalent fungus affecting corn (Marassas, 1991). These fungi represent threats not only to the health of crops, but also to animals and humans ingesting contaminated feeds and foods. The alcoholic extract from L. di6aricata effectively reduced the growth of P. notatum in maltagar medium. Samples of 2, 3 and 4 mg dry extract/plate (0.4, 0.6 and 0.8 mg ml − 1 dry matter in culture medium) produce 47, 68 and 89% of inhibition, respectively (Fig. 1). Extract from Z. punctata inhibited mycelial growth of G. applanatum and L. elegans (Fig. 2). The aqueous and alcoholic extracts from Phytolacca dioica and Zinnia peru6iana showed antimicrobial activity against Gram-positive and
Table 1 Ethnobotanical data of the studied plants Family
Botanical name
Common name
Used parts
Popular uses
Zygophyllaceae
Larrea di6aricata Cav
Jarilla, jarilla hembra
Whole plant
Zygohyllaceae
Larrea cuneifolia Cav
Jarilla, jarilla macho
Whole plant
Equisetaceae Compositae
Cola de caballo Canchalahua
Whole plant Whole plant
Compositae Leguminosae
Equisetum giganteum L. Schkuhria pinnata Lam O. Kuntzi Zinnia peru6iana L Zuccagnia punctata Cav
Whole plant Whole plant
Leguminosae
Acacia ca6en (Mol) Molina
Chinita del campo Jarilla pispito, jarilla de la Puna, pus-pus Churqui, tusca, espinillo
Vulnerary, antirheumatic, emmenagoge, antiinflamatory Vulnerary, antrheumatic, emmenagoge, antiinflamatory Diuretic, astringent Antibiotic, vulnerary, phototoxic Vulnerary, phototoxic Vulnerary
Phytolaccaceae
Phytolacca dioica L o Pircunia dioica (L) Moq. Prosopanche americana (R. Br) Baillon o P.burmeisteri De Bary Schinus molle L.
Hydnoraceae
Anacardiaceae
Ombu´ Guaycuru´ santiaguen˜o, flor de tierra Molle, pimienta del diablo, aguaribay
Leaf, stem, bark Antiseptic, vulnerary, of a tree cicatrizant Whole plant Vulnerary, antifeverish, vermifugy Flowers, roots Vulnerary, hemostatic, expectorant Whole plant
Antiinflamatory, emmenagoge, vulnerary
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
93
Table 2 Radial growth inhibition of alcoholic extracts against filamentous fungia Fungib
Alcoholic extractsc 1
2
3
4
5
6
7
8
9
A
++
++
−
++
−
+++
−
+
+
B
++
++
−
−
−
+++
−
−
−
C
++
+
−
+
−
++
+
−
+
D
+
−
−
−
−
++
−
−
−
E
++
++
−
++
+
++
+
−
+
F
+++
++
−
+
+
++
−
−
9
G
++
++
−
−
−
++
−
−
−
H
++
+
−
+
9
+++
−
−
−
10
− − − − + + + + a The extract concentration (dry matter) in the culture medium was 0.8 mg ml−1 in all cases. The inhibition was reported as (−) B10% growth inhibition, ( 9 ) between 10 and 20%, (+) between 20 and 40%, (++) between 40 and 80%, and (+++) 80%. (n= 12.) b A, Lenzites elegans; B, Schizophyllum commune; C, Pycnoporus sanguineus; D, Ganoderma applanatum; E, Fusarium oxysporum; F, Penicillium notatum; G, Aspergillus niger; H, Trichoderma spp. c 1, Larrea di6aricata Cav.; 2, Larrea cuneifolia Cav.; 3, Schkuhria pinnata (Lam) O. Kuntzi; 4, Zinnia peru6iana (L), L.; 5, Equisetum giganteum L.; 6, Zuccagnia punctata Cav.; 7, Acacia ca6en (Mol) Molina; 8, Phytolacca dioica L.; 9, Prosopanche americana (R.Br) Baillon; 10, Schinus molle L.
gram-negative microorganisms (Amani et al., 1998). Otherwise, alcoholic extracts of P. dioica and Z. peru6iana did not show significant antifungal activity against the eight filamentous fungi tested at the highest dose used. L. elegans, P. sanguineus and F. oxysporum were average sensitive in vitro to alcoholic extracts of flowers and roots of Prosopanche americana. A peptide designed Pa-AMP was isolated and purified from seeds of P. americana. This peptide showed inhibitory activity for pathogenic fungi and Gram-positive and negative bacteria (Minami et al., 1998). It was found that the extract from Schinus molle inhibits the growth of F. oxysporum, P. notatum, A. niger and Trichoderma spp. Triterpe-
nes and sesquiterpenolactones isolated from leaves and fruits of S. molle showed antifungal activity when they were tested against Candida albicans, Trichophyton mentagraphytes and A. niger. Also Aspergillus alternata, Aspergillus fla6us, Mycrosporum griseum, P. cyclopium and P. italicum showed growth inhibition in vitro by their leaf essential oils (Ross et al., 1980; Dishit et al., 1986). The responses of the two assayed yeasts (S. carlsbergensis and Rhodotorula spp.) are shown in Table 3. It was found that extracts of L. di6aricata, L. cuneifolia, Z. punctata and P. americana reduced the growth of both yeasts. Fig. 3 shows growth inhibition of S. carlsbergensis by various amounts of L. di6aricata extract. Similar results
94
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96 Table 3 Paper disk diffusion of plant alcoholic extracts on yeast growtha Plant alcoholic extracts Yeast strains
Fig. 1. Growth inhibition of Penicillium notatum by alcoholic extract of Larrea di6aricata. C, Control; 2, 3 and 4 mg dry matter/plate.
were obtained with the agar well diffusion assays (not shown). Table 4 shows the MICs observed for the effect of different plant extracts on yeasts. L. cuneifolia exhibited a better inhibition activity against S. carlsbergensis than L. di6aricata and P. americana.
Fig. 2. Antifungal activity of Zuccagnia punctata extract against the wood destroying fungi Ganoderma applanatum and Lenzites elegans. C, Control; 2; 3 and 4 mg dry matter/plate.
L. di6aricata Cav. L. cuneifolia Cav. S. pinnata Lam O. Kuntzi Z. peru6iana (L), L. E. giganteum L. Z. punctata Cav. A. ca6en (Mol.) Molina P. dioica L. P. americana (R.Br) Baillon S. molle L.
S. carlsbergensis
Rhodotorula spp.
++ ++ −
+++ ++ −
− − ++ −
− − ++ −
− ++
− ++
+
−
a The growth inhibition of the two yeasts was determined by paper disk diffusion assays, using 0.2 mg dry matter/disk. The inhibition is reported as: (−) without inhibition, (+) dr =1.0 mm, (++) dr =1.0–1.5 mm, and (+++) dr =1.5–2.0 mm, where dr is diameter of the inhibition zone. (n =12.)
Otherwise, L. di6aricata and L. cuneifolia showed the highest inhibition of Rhodotorula spp. growth. The MICs of L. cuneifolia against S. carlsbergensis and Rhodotorula spp. were 100 and 133 mg ml − 1, respectively, while the MIC of an accepted antifungal compound isolated and purified from
Fig. 3. Disk diffusion assay. Effect of growing amounts of Larrea di6aricata extract on Saccharomyces carlsbergensis growth. Control and 25, 33, 50, 100 and 200 mg dry matter/ disk in the clockwise sense.
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96 Table 4 MICs of different plant extracts against yeastsa Plant extracts
Yeasts S. carlsbergensis (mg ml−1)
Larrea di6aricata Cav. Larrea cuneifolia Cav. Zuccagnia punctata Cav. Prosopanche americana (R.Br) Baillon
Rhodotorula spp. (mg ml−1)
200 100 400
100 133 200
200
266
a
MICs were determined by dilution tests as indicated in Section 2. Data were taken from the inhibition produced by plant extracts (20–500 mg dry extract ml−1 were used). (n= 8; S.D. =0.01–0.09).
Anemarrhena asphodeloides against Saccaromyces cere6isiae was 200 mg ml − 1 (Iida et al., 1999). Although studies on growth inhibition of fungi by isolated plant compounds suggest a role in plant defense, such in vitro tests may not always give an accurate indication of the significance of these compounds in restricting fungal growth in the plant (Mansfield, 1983). As plants are sessile organisms, they are confined to the place where they grow. They are capable of sensing the presence of potential phytopatogens (fungi, bacteria and viruses) and mounting defense mechanisms. Plants can produce antifungal compounds to protect themselves from biotic attack that could be essential for fungi infection resistance (Wojtaszek, 1997). The understanding of the inhibitory mechanism would provide better directions toward the development of efficient production and application of technologies associated with biofungicide plant materials. This is the first report showing the antifungal activities of different Argentine plants. Further studies, including the isolation and structure – function relationships of the antifungal natural products are now in progress. Acknowledgements The authors are grateful to the Ca´tedra de Micologı´a, Instituto de Microbiologı´a, Facultad de
95
Bioquı´mica, Quı´mica y Farmacia de la Universidad Nacional de Tucuma´n (UNT) and to the Instituto Nacional de Tecnologı´a Agropecuaria (INTA), Tucuma´n, Argentina for the generous gift of fungus cultures. This work was supported by grants from the Secretarı´a de Ciencia y Te´cnica from the UNT, Tucuma´n, Argentina.
References Amani, S.M., Isla, M.I., Vattuone, M.A., Poch, M.J., Cudmani, N.G., Sampietro, A.R., 1998. Antimicrobial activities in some argentine medicinal plants. In: V. Martino et al. (Eds.), Proceedings of the WOCMAP-2: Pharmacognosy, Pharmacology, Phyto-medicines, Toxicology, Acta Horticulturae, ISHS, vol. 501, pp. 115 – 118. Bennett, R.N., Wallsgrove, R.M., 1994. Secondary metabolites in plant defence mechanisms. New Phytology 127, 617 – 633. Blanchette, R.A., 1994. Degradation of the lignocellulose complex in wood. Canadian Journal of Botany 73, S999 – S1010. Camm, E.L., Towers, G.H.N., Mitchell, J.C., 1975. UV-mediated antibiotic activity of some Compositae species. Phytochemistry 14, 2007 – 2011. Ciegler, A., 1975. Mycotoxins, occurrence, chemistry, biological activity. Lloydia 38, 481 – 483. Cole, M.D., 1994. Key antifungal, antibacterial and anti-insect assays. A critical review. Biochemical Systems and Ecology 22, 837 – 856. Dishit, A., Naqvi, A., Husain, A., 1986. A new source of natural fungitoxicant. Applied and Environmental Microbiology 51, 1085 – 1088. Go´mez Garibay, F., Reyes Chilpa, R., Quijano, L., Caldero´n Pardo, J.S., Rı´os Castillo, T., 1990. Methoxifurans auranols with fungostatic activity from Lonchocarpus castilloi. Phytochemistry 29, 459 – 463. Grayer, R.J., Harborne, J.J., 1994. A survey of antifungal compounds from higher plants, 1982 – 1993. Phytochemistry 37, 19 – 42. Harsh, N.S.K., Bisht, N.S., 1997. Wood decaying fungi of Kumaun Himalaya. In: Sati, S.C., Saxena, J., Dubey, R.C. (Eds.), Recent Researches in Ecology, Environment and Pollution, vol. X. Today and Tomorrow, New Delhi-5, pp. 69 – 93. Iida, Y., Oh, K.B., Saito, M., Matsuoka, H., Kurata, H., Natsume, M., Abe, H., 1999. Detection of antifungal activity in Anemarrhena asphodeloides by sensitive BCT method and isolation of its active compound. Journal of Agricultural and Food Chem 47, 584 – 587. Mansfield, J.W., 1983. In: Callow, J.A. (Ed.), Antimicrobial Compounds in Biochemical Plant Pathology. Wiley, Chichester, UK, pp. 237 – 265.
96
E.N. Quiroga et al. / Journal of Ethnopharmacology 74 (2001) 89–96
Marassas, W.F.O., 1991. Toxigenic Fusaria. In: Smith, J.E., Henderson, R.E. (Eds.), Mycotoxins and Animal Foods. CRC Press, Boca Rato´n, FL, pp. 119–139. Minami, Y., Higuchi, S., Yagi, F., Tadera, K., 1998. Isolation and some properties of the antimicrobial peptide (PaAMP) from the seeds of pokeweed (Phytolacca americana). Bioscience and Biotechnology of Biochemistry 62, 2076– 2078. Osbourne, A.E., 1996. Preformed antimicrobial compounds and plant defense against fungal attack. The Plant Cell 8, 1821 – 1831. Reyes Chilpa, R., Pe´rez Morales, V., Del Angel Blanco, S., 1987. Influencia de los extractivos en la resistencia natural de seis maderas tropicales al hongo de pudricio´n morena Lenzites trabea. Bio´tica (Me´xico) 12, 7–13. Reyes Chilpa, R., Quiroz Va´zquez, R.I., Jime´nez Estrada, M., Navarro-Ocan˜a, A., Cassani Herna´ndez, J., 1997. Antifungal activity of selected plant secondary metabolites against
.
Coriolus 6ersicolor. Journal of Tropical Forest Products 3, 110 – 113. Ross, S., El-Keltawi, N., Megalla, S., 1980. Antimicrobial activity of some Egyptian aromatic plants. Fitoterapia LI, 201 – 205. Torres, M., Bacells, M., Sala, N., Sanchis, V., Canela, R., 1998. Bactericidal and fungicidal activity of Aspergillus ochraceus metabolites and some derivatives. Pesticide Science 53, 9 – 14. Toursarkissian, M., 1980. Plantas Medicinales de la Argentina. Editorial Hemisferio Sur, S.A., Bs. As., Argentina, pp. 5, 39, 42, 51, 60, 67, 75 and 98. Ueno, Y., Ishhii, K., Sawano, M., Ohtsubo, K., Mutsuda, Y., Tanaka, T., Kurata, H., Ichinoe, M., 1977. Toxicological approaches to the metabolites of Fusaria. Japanese Journal of Experimental Medicine 47, 177 – 184. Wojtaszek, P., 1997. Oxidative burst an early plant response to pathogen infection. Biochemistry Journal 322, 681 – 692.