Fitoterapia 74 (2003) 453–458
Antifungal activity of Brassica oleracea var. botrytis fresh aqueous juice M. Sistia,*, G. Amaglianib, G. Brandia,b a
Istituto di Scienze Tossicologiche Igienistiche e Ambientali, Universita` di Urbino, Urbino, Italy b Centro di Biotecnologie, Universita` di Urbino, Fano, Italy Received 10 March 2003; accepted 10 April 2003
Abstract The antifungal activity of fresh, aqueous Brassica oleracea var. botrytis juice against Candida albicans and other pathogenic fungi was investigated. The juice was found to be effective both in inhibiting the growth of blastoconidia and reducing the appearance of C. albicans germ tubes. Furthermore, the juice inhibited the growth of some pathogenic, filamentous fungi. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: Brassica oleracea var. botrytis; Isothyocianates; Antifungal activity
1. Introduction Glucosinolates and the products of their hydrolysis, due to an endogenous plant enzyme known as myrosinase, are found in the family of the Cruciferae and can be isolated from Brassica crops. These compounds and the products of their degradation have been proven active against various species of micro-organisms w1x. Although glucosinolates show only mild antimicrobial activity at high concentrations w2x, the products of their enzymatic hydrolysis, such as isothiocyanates (ITCs), were particularly effective in inhibiting the germination of the major postharvest fungal pathogens: Botrytis cinerea, Rhizopus stolonifer, Monilinia laxa, Mucor piriformis and Penicillium expansum w3x. Moreover, synthetic hydroxyphenyl isothiocyanate *Corresponding author. Tel.: q39-722-350585; fax: q39-722-4717. E-mail address:
[email protected] (M. Sisti). 0367-326X/03/$ - see front matter 䊚 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0367-326X(03)00108-4
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showed a pronounced antimicrobial effect towards Aspergillus niger, Aspergillus fumigatus, Staphylococcus aureus and Escherichia coli w4x. The inhibitory activity of aqueous plant Brassicaceae extracts towards Verticillium chlamidosporium w5x, Glomus mosseae spore germination has been also observed w6x. The present study reports the in vitro activity of crude aqueous juice obtained from Brassica oleracea var. botrytis leaves against Candida albicans and some pathogenic filamentous fungi. 2. Materials and methods 2.1. Preparation of juice Fresh leaves of B. oleracea var. botrytis, obtained from the Cadette, Artemis and Meridienne cultivar, were homogenized, centrifuged at 10 000 rev.ymin at 4 8C for 10 min and the liquid sterilized by filtration (0.2 mm pore size). The juice of B. oleracea var. botrytis (BJ) obtained was used immediately. 2.2. Effect of BJ on C. albicans growth The inoculum suspension was prepared by picking four–five colonies from a 24h-old culture of C. albicans on Sabouraud dextrose agar (SDA) plates followed by suspension in sterile 0.85% NaCl. The turbidity of the cell suspension was measured with a spectrophotometer at 530 nm and adjusted with sterile saline until matching the transmittance produced by a 0.5 McFarland barium sulfate standard. This procedure provided a cell suspension containing approximately 106 CFUyml, which was then confirmed by enumeration of the CFUyml on SDA. The adjusted suspension was incubated with different concentrations of cauliflower juice: 5, 10 and 20% vyv. A growth control tube without juice was included. The cultures were incubated at 37 8C with agitation for 6 h. Every 2 h serial dilutions of each culture were spread on SDA and CFUyml were determined after incubation at 37 8C for 24 h. Percentages of growth inhibition were calculated. 2.3. Evaluation of the effect of BJ on C. albicans germ tube formation The inoculum suspension was prepared as previously described. The effect of juice on tube formation was tested using 100 ml of the above yeast suspension, mixed with foetal calf serum (FCS) containing 5, 10 and 20% vyv of fresh juice from each cultivar to obtain a final volume of 1 ml (105 CFUyml). The cultures were incubated at 39 8C with agitation for 2 h and cells were microscopically analyzed to determine either the percentage of cells presenting germ tubes or the viability after staining with trypan blue dye. 2.4. Evaluation of the effect of BJ on filamentous fungi growth Filamentous fungi species were isolated from a nosocomial environment and routinely cultured in our laboratory. Inoculum preparation of filamentous fungi was
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Table 1 Variation in inoculum size for filamentous fungi using a spectrophotometric methoda Fungi
T-range (%)
Inoculum (CFUyml w106x)
Alternaria spp. Aspergillus flavipes Aspergillus fumigatus Aspergillus niger Botrytis spp. Cladosporium spp. Fusarium proliferatum Fusarium solani Microsporum canis Microsporum canis vr. Distortum Paecilomyces variotii ATCC 22319 Penicillium spp. Rizhopus spp. Trichoderma sp. Trichophyton verrucosum
85–87 83–85 76–81 79–86 86–87 80–82 84–85 78–80 85–86 83–85 82–86 78–83 84–85 80–82 78–86
0.12–2 0.17–0.7 9–2.5 0.12–0.29 0.1–0.43 0.21–0.51 0.15–2.3 0.41–0.6 0.11–0.2 0.1–0.24 0.13–0.51 0.44–0.58 1.4–5.4 0.49–2.4 1–3.9
a
Percent of transmittance (T) was measured at 530 nm.
performed by a modified Espinel-Ingroff et al. protocol w7x (Table 1). The suspension for each isolate was prepared growing the mould on potato dextrose agar (PDA) slants for 7 days at 35 8C. The fungal colonies were covered with approximately 1 ml of sterile 0.85% saline containing 0.05% Tween 80. The resulting mixture was withdrawn and the heavy particles were allowed to settle for 3 or 5 min. The upper homogeneous suspensions were vortexed for 15 s and the turbidity was spectrophotometrically adjusted to 68–82% transmittance (optical density ranges: 0.09–0.17) at 530 nm according to the species tested w7,8x. The adjusted suspensions were diluted 1:100 and cell viability was confirmed by plating serial dilutions onto SDA plates incubated at 35 8C. The plates were observed daily for the presence of fungal colonies which were counted as soon as possible. For the preparation of the test inoculum, conidial suspensions were diluted 1:100 with RPMI supplemented with both L-glutamine and 0.165 M MOPS (morpholinepropanesulfonic acid) buffer (34.54 gyl), without sodium bicarbonate. The pH of the medium was 7.0"0.1 at 35 8C. Ten microlitres of the final concentration inoculum prepared was added to 0.99 ml of RPMI containing 0.5, 1, 5 and 10% vyv of crude aqueous juice (corresponding to 0.054, 0.109, 0.545 and 1.09 mgyml of total ITCs, respectively, determined according to Zhang et al. w9x) of Cadette cultivar. The growth control tube contained 0.99 ml of juice-free medium and 10 ml of inoculum suspension. All tubes were incubated at 35 8C and when growth in the control tube was visible, each tube was vortexed for 10 s. The growth in each tube was compared with that of the growth control and given a numerical score as follows: 0, optically clear or the absence of growth; 1, approximately 75% reduction in growth; 2, approximately 50% reduction in growth; 3, approximately 25% reduction in growth; and 4, no reduction in growth.
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Fig. 1. Effect of fresh juice on the morphology of C. albicans germ tubes.
3. Results 3.1. Effects of BJ on C. albicans In a first set of experiments, we found that BJ was able to inhibit C. albicans growth in a dose-dependent manner. The inhibition resulted )95% after 4 h of incubation already with 15% of juice for all cultivar tested (data not shown). Since the germ tube represents a virulence factor in the process of Candida infection of the host cells w10x, we tested the effect of the juice on tube formation. As shown in Fig. 1, maximum activity was obtained with the Cadette cultivar (20.4%"1.7 and 70%"4.5 of inhibition of germ tube emission with 10 and 20% of juice, respectively), followed by the Artemis cultivar (11.8%"2.7 and 31%"3.2 of inhibition) and Meridienne (3.2%"2.1 and 23.4%"3.9) at the same concentrations. 3.2. Inhibitory effects on filamentous fungi growth Variability in the inoculum size for each species tested is summarized in Table 1. The data reported in Table 2 show that the most susceptible species to juice resulted M. canis var. distortum, which did not produce detectable growth for all concentrations tested even after the third reading (corresponding to 5 days after initial treatment). Alternaria spp., Cladosporium spp., M. canis and T. verrucosum did not produce detectable growth for all concentrations tested even after the second reading, whereas a very limited growth was observed for Botrytis spp., in the third reading only for the 0.5% concentration. The growth of Trichoderma spp. was inhibited
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Table 2 Effect of Brassica oleracea var. botrytis (cultivar Cadette) crude aqueous juice on filamentous fungi 2nd Readinga
1st Reading
3rd Readingb
0.50% 1% 5% 10% 0.50% 1% 5% 10% 0.50% 1% 5% 10% Alternaria spp. Aspergillus flavipes Aspergillus fumigatus Aspergillus niger Botrytis spp. Cladosporium spp. Fusarium proliferatum Fusarium solani Microsporum canis Microsporum canis vr. Dist. P. variotii ATCC 22319 Penicillium spp. Rizhopus spp. Trichoderma spp. Trichophyton verrucosum
0 2 1 2 1 0 2 3 0 0 2 2 4 1 0
0 1 1 2 0 0 0 2 0 0 2 1 4 0 0
0 1 1 2 0 0 0 2 0 0 1 1 4 0 0
0 1 1 2 0 0 0 2 0 0 1 1 4 0 0
0 4 4 4 1 0 4 4 0 0 4 4
0 4 4 4 0 0 1 4 0 0 4 4
0 1 1 4 0 0 1 3 0 0 4 4
0 1 1 4 0 0 1 3 0 0 4 4
4 0
4 0
4 0
4 0
4
4
4 4 4
4 4 4
1 1
1 1 4
1 0
1 0
1 1 2 4 1 0
1 1 2 4 1 0
1
1
1
1
0: No growth; 1: approximately 75% reduction in growth; 2: approximately 50% reduction in growth; 3: approximately 5% reduction in growth; 4: no reduction in growth. aReading after 24 h from 1st. b Reading after 48 h from 1st.
during the first 24 h of treatment. However, in the following days, the development of the mycelium was observed in culture. Rizhopus spp. was found the most resistant fungi to juice activity, producing a growth equal to that of the control. The other strains showed variable sensitivity to juice. 4. Discussion Myrosinase-induced hydrolysis of glucosinolates yields a wide variety of products. Most of them contain aliphatic groups which form ITCs. Most ITCs have antimicrobial and pesticide activities and act in biodefencive mechanisms. Thus, naturally occurring and synthetic ITCs have been studied extensively w1x in medicine. This study indicates that the addition of the crude aqueous juice of B. oleracea var. botrytis leaves to the culture medium inhibits the growth of C. albicans and of various filamentous fungi. Furthermore, some cultivar were able to reduce the appearance of germ tubes after 2 h of exposure without affecting cell growth. This inhibition of germ tube formation is probably due to the different ITCs content of the cultivars. In fact, our findings (data not shown) demonstrate that the Cadette cultivar, that is, the most active in germ tube inhibition, has the highest amount (10.9 mgyml) of total ITCs in its aqueous juice. The filamentous fungi tested showed variable sensitivity: the species most inhibited were two dermatophytes (M. canis, T. verrucosum), Botrytis spp. and Cladosporium spp. Although the mechanism is not yet clear, we can speculate that one possible mechanism for fresh juice inhibition of fungal growth and germ tube
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formation in C. albicans is due to an interaction with cellular sulfhydryl compounds. In fact, it has been shown that ITC compounds exert their antimicrobial effects by blocking enzyme sulfhydryl groups or other proteins involved in ATP synthesis w4x. Other findings suggest that ITCs could be used for their antimicrobial properties in various food products w3,11–14x and soil-borne plant pests w15x. The results of the present study indicate the antifungal properties of the crude aqueous juice of B. oleracea var. botrytis leaves may be effective in reducing the proliferation of pathogens associated with foods. References w1 x w2 x w3 x w4 x w5 x w6 x w7 x w8 x w9 x
w10x w11x w12x w13x w14x w15x
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