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Increased antifungal activity of 3- and 7-hydroxyflavone against Cladosporium herbarum and Penicillium glabrum through ester formation
Mycol. Res. 101 (8), 920–922 (1997)
920
Printed in Great Britain
Increased antifungal activity of 3- and 7-hydroxyflavone against Cladosporium herbarum and Penicillium glabrum through ester formation
H E I K E M A R T I NI1, M A R T I N W E I D E N B O> R N E R2*, S U S A N N E A D A M S1 A N D B E N N O K U N Z1 " Institut fuX r Lebensmitteltechnologie UniversitaX t Bonn, RoX merstraße 164, 53117 Bonn # Institut fuX r Angewandte Mikrobiologie UniversitaX t Giessen, Senckenbergstr. 3, 35390 Giessen, Germany
3- and 7-hydroxyflavone and their acetic acid esters were tested against Cladosporium herbarum and Penicillium glabrum for antifungal activity. The effect of the hydroxylated flavones was relatively low whereas the ester derivatives were substantially more active.
C. herbarum and P. glabrum are food-contaminating moulds capable of producing mycotoxins which are potentially dangerous to consumer health (Frisvad & Thrane, 1995). Although there are various chemical preservatives which inhibit mould growth in food (Kra$ mer, 1997) advantages are seen in finding naturally occurring substances which would be equally suitable for the preservation of foods (Weidenbo$ rner et al., 1992 ; Kunz et al., 1995). Consequently, flavonoids and isoflavonoids, which are natural components of plants with antifungal properties (O’Neill & Mansfield, 1982 ; Adesanya et al., 1986 ; Weidenbo$ rner et al., 1990 a ; Weidenbo$ rner & Jha, 1993), have been investigated. Rather high concentrations of these compounds are needed, however, to control fungal growth in food (Kra$ mer et al., 1984). Consideration has been given to increasing understanding of the mode of action of these natural fungicides and of improving their effectiveness through substitutions (Van Etten, 1976 ; Hedin & Waage, 1986 ; Weidenbo$ rner & Jha, 1994 a). There is evidence that their action is linked with lipophilicity (O’Neill & Mansfield, 1982 ; Arnoldi & Merlini, 1990 ; Weidenbo$ rner et al., 1990 b ; Lattanzio et al., 1994), suggesting it may be possible to increase fungitoxicity by replacing a hydroxy group on a flavone molecule with an acetoxy group. Although it has been reported that such substitutions do not improve the activity of certain isoflavonoids (Weidenbo$ rner & Jha, 1994 b), no comparable studies have been made with flavonoids. Therefore, the following study was conducted to examine the antifungal properties of 3- and 7-hydroxyflavone in comparison with their acetic acid esters. MATERIALS AND METHODS The fungi used were Penicillium glabrum (Wehmer) Westling and Cladosporium herbarum (Pers.) Link, which often occur on food (Weidenbo$ rner & Kunz, 1994). * Corresponding author.
3- and 7-hydroxyflavone were obtained from Aldrich Chemical Co. (Steinheim, Germany). Their corresponding acetic acid esters were prepared by reaction with acetic anhydride in pyridine. The mixture was refluxed for 3 h under dry conditions, poured into ice water, and subsequently acidified with 1 N HCl. Then the ester was filtered, washed with water, and dried. All flavonoids were tested for antifungal activity against P. glabrum and C. herbarum at concentrations of 0±8, 3±5 and 8±0¬10−$ mol l−" (Table 1). The effect of the flavonoids on mycelium growth of the test fungi was investigated on solid medium (30 g malt extract, 15 g agar, 3 g peptone l−" ; in distilled water) using three different methods. The autoclaved medium (20 ml) was transferred to Petri dishes (90 mm) and allowed to solidify. Then 1 ml flavonoid solution, dissolved in either ethanol (method A) or acetone (method B), was pipetted onto the agar. After evaporation of the solvent (72 h) the dishes were inoculated with a small piece of mycelium (1 mm diam.). In method C the flavonoids were dissolved in acetone and incorporated directly into the agar at 50 °C. In this case, higher flavonoid concentrations (3±5 and 8±0¬10−$ mol l−") were chosen, because in methods A and B the flavonoids (0±8¬10−$ mol l−") were concentrated on the agar surface. Thus, comparable conditions applied within the three methods. The dishes were incubated at 25° in the dark. Mycelium diameters of C. herbarum and P. glabrum were measured after 7 d. Evaluation was carried out by measuring the diameter of the colonies three times at different sites. The mean value of five repetitions for each experiment was used for calculation. The data were evaluated by analysis of variance. Probability of single differences was calculated at the 5 % level. RESULTS AND DISCUSSION The substituted acetic acid esters were more active in reducing mycelium growth of C. herbarum and P. glabrum than were the
Heike Martini and others
921
Table 1. Growth inhibition (%) of C. herbarum and P. glabrum caused by 3- and 7-hydroxyflavone (I, III) and their acetic acid esters (II, IV) in comparison with the corresponding control
C. herbarum
P. glabrum
Method
Concn ¬10−$ mol l−"
I (%)
II (%)
Difference (%)
III (%)
IV (%)
Difference (%)
A B C C A B C C
0±8 0±8 3±5 8±0 0±8 0±8 3±5 8±0
15±6 0 10±6 14±7 35±4 2±3 0 11±9
52±3 34±4 49±0 61±8 99±5 43±0 50±9 77±7
36±7 34±4 38±4 47±1 64±1 40±7 50±9 65±8
12±8 0 3±5 5±9 22±7 2±8 5±3 5±2
16±5 8±3 35±4 32±4 42±4 22±3 40±5 30±6
3±7 8±3 31±9 26±5 19±7 19±5 35±2 25±4
hydroxylated flavones (Table 1). The rank order of activity was 3-acetoxyflavone (II) followed by 7-acetoxyflavone (IV), 3-hydroxyflavone (I), and 7-hydroxyflavone (III). The greatest differences in activity resulted from the 3-substitution and the greatest effect was seen in P. glabrum. C. herbarum was less sensitive to each group of compounds than was P. glabrum. These results confirm earlier findings (Weidenbo$ rner et al., 1990 b ; Weidenbo$ rner & Jha, 1994 a) that 3- and 7-hydroxyflavones are generally poor inhibitors of mould growth. In contrast, however, they demonstrate that the esters are the more active, irrespective of the mould or method employed (Table 1). A general increase in the antifungal activity of lipophilic flavonoids and isoflavonoids has also been observed by other authors (O’Neill & Mansfield, 1982 ; Adesanya et al., 1996 ; Weidenbo$ rner et al., 1989 ; Weidenbo$ rner & Jha, 1993). One reason for their enhanced effectiveness may be an easier penetration of fungal membranes (Harborne & Ingham, 1978). However, because neither 4«,6,7-triacetoxyisoflavone nor 3«acetoxy-6,7-dihydroxyisoflavanone exhibit hardly any fungicidal activity (Weidenbo$ rner & Jha, 1994 b), the increased antifungal potential of the acetoxy flavones tested in this study cannot be explained exclusively by higher lipophilicity. As can be seen from the data in Table 1 the position of the substitution is also critical in determining effectiveness of each substance. Since the 3-substituted acetic acid ester (II) caused higher inhibition rates than 7-acetoxyflavone (IV), it is possible that certain metal ions essential for fungal growth are blocked by chelation with members of the 3-hydroxy-4ketone group (Somaatmadja et al., 1964) and the 3-acetoxy-4ketone group, respectively. Further investigations will focus on this phenomenon. The present study demonstrates that the antifungal potential of certain flavonoids is increased significantly by a simple chemical modification. This may be a first step in the development of naturally occurring food preservatives. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG). REFERENCES Adesanya, S. A., O’Neill, T. M. & Roberts, M. F. (1986). Structure-related fungitoxicity of isoflavonoids. Physiological and Molecular Plant Pathology 29, 95–103.
Arnoldi, A. & Merlini, L. (1990). Lipophilicity-antifungal relationships for some isoflavonoid phytoalexins. Journal of Agricultural and Food Chemistry 38, 834–838. Frisvad, J. C. & Thrane, U. (1995). Mycotoxin production by food-borne fungi. In Introduction to Food-borne Fungi, 4th ed. (ed. R. A. Samson, E. S. Hoekstra, J. C. Frisvad & O. Filtenborg), pp. 251–260. Centraalbureau voor Schimmelcultures : Baarn and Delft. Harborne, J. B. & Ingham, J. L. (1978). Biochemical aspects of the coevolution of higher plants with their fungal parasites. In Biochemical Aspects of Plant and Animal Coevolution (ed. J. B. Harborne), pp. 343–405. Academic Press : New York. Hedin, P. A. & Waage, S. K. (1986). Roles of flavonoids in plant resistance to insects. In Plant Flavonoids in Biology and Medicine (ed. V. Cody, E. Middleton Jr, J. B. Harborne & A. Beretz), pp. 87–100. Alan R. Liss : New York. Kra$ mer, J. (1997). Lebensmittel-Mikrobiologie, 3rd ed. Eugen Ulmer Verlag : Stuttgart. Kra$ mer, R. P., Hindorf, H., Jha, H. C. & Zilliken, F. (1984). Antifungal activity of soybean and chickpea isoflavones and their reduced derivatives. Phytochemistry 23, 2203–2205. Kunz, K., Weidenbo$ rner, M. & Kunz, B. (1995). Die Nutzung von Gewu$ rzen in Weizenbrot zur Kontrolle der lebensmittelrelevanten Schimmelpilze Cladosporium herbarum, Eurotium repens, Penicillium expansum und Rhizopus stolonifer. Chemie, Mikrobiologie und Technologie der Lebensmittel 17, 1–5. Lattanzio, V., De Cicco, V., Di Venere, D., Lima, G. & Salerno, M. (1994). Antifungal activity of phenolics against fungi commonly encountered during storage. Italian Journal of Food Science 6, 23–30. O’Neill, T. M. & Mansfield, J. W. (1982). Antifungal activity of hydroxyflavans and other flavonoids. Transactions of the British Mycological Society 79, 229–237. Somaatmadja, D., Powers, J. J. & Hamdy, M. K. (1964). Anthocyanins. VI. Chelation studies on anthocyanins and other related compounds. Journal of Food Science, 29, 655–660. Van Etten, H. (1976). Antifungal activity of pterocarpans and other selected isoflavonoids. Phytochemistry 15, 655–659. Weidenbo$ rner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. & Egge, H. (1989). Antifungal activity of isoflavonoids against storage fungi of the genus Aspergillus. Phytochemistry 28, 3317–3319. Weidenbo$ rner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. & Egge, H. (1990 a). Antifungal activity of isoflavonoids in different reduced stages on Rhizoctonia solani and Sclerotium rolfsii. Phytochemistry 29, 801–803. Weidenbo$ rner, M., Hindorf, H., Jha, H. C. & Tsotsonos, P. (1990 b). Antifungal activity of flavonoids against storage fungi of the genus Aspergillus. Phytochemistry 29, 1103–1105. Weidenbo$ rner, M., Hindorf, H., Weltzien, H. C. & Jha, H. C. (1992). An effective treatment of legume seeds with flavonoids and isoflavonoids against storage fungi of the genus Aspergillus. Seed Science & Technology 20, 447–463. Weidenbo$ rner, M. & Jha, H. C. (1993). Antifungal activity of flavonoids and their mixtures against different fungi occurring on grain. Pesticide Science 38, 347–351.
Antifungal activity in hydroxyflavones Weidenbo$ rner, M. & Jha, H. C. (1994 a). Antifungal activity of flavonoids in relation to degree of hydroxylation, methoxylation, and glycosidation. Acta Horticulturae 381, 702–709. Weidenbo$ rner, M. & Jha, H. C. (1994 b). Structure–activity relationships (Accepted 17 January 1997)
922 among isoflavonoids with regard to their antifungal properties. Mycological Research 98, 1376–1378. Weidenbo$ rner, M. & Kunz, B. (1994). Contamination of different muesli components by fungi. Mycological Research 98, 583–586.
920
Printed in Great Britain
Increased antifungal activity of 3- and 7-hydroxyflavone against Cladosporium herbarum and Penicillium glabrum through ester formation
H E I K E M A R T I NI1, M A R T I N W E I D E N B O> R N E R2*, S U S A N N E A D A M S1 A N D B E N N O K U N Z1 " Institut fuX r Lebensmitteltechnologie UniversitaX t Bonn, RoX merstraße 164, 53117 Bonn # Institut fuX r Angewandte Mikrobiologie UniversitaX t Giessen, Senckenbergstr. 3, 35390 Giessen, Germany
3- and 7-hydroxyflavone and their acetic acid esters were tested against Cladosporium herbarum and Penicillium glabrum for antifungal activity. The effect of the hydroxylated flavones was relatively low whereas the ester derivatives were substantially more active.
C. herbarum and P. glabrum are food-contaminating moulds capable of producing mycotoxins which are potentially dangerous to consumer health (Frisvad & Thrane, 1995). Although there are various chemical preservatives which inhibit mould growth in food (Kra$ mer, 1997) advantages are seen in finding naturally occurring substances which would be equally suitable for the preservation of foods (Weidenbo$ rner et al., 1992 ; Kunz et al., 1995). Consequently, flavonoids and isoflavonoids, which are natural components of plants with antifungal properties (O’Neill & Mansfield, 1982 ; Adesanya et al., 1986 ; Weidenbo$ rner et al., 1990 a ; Weidenbo$ rner & Jha, 1993), have been investigated. Rather high concentrations of these compounds are needed, however, to control fungal growth in food (Kra$ mer et al., 1984). Consideration has been given to increasing understanding of the mode of action of these natural fungicides and of improving their effectiveness through substitutions (Van Etten, 1976 ; Hedin & Waage, 1986 ; Weidenbo$ rner & Jha, 1994 a). There is evidence that their action is linked with lipophilicity (O’Neill & Mansfield, 1982 ; Arnoldi & Merlini, 1990 ; Weidenbo$ rner et al., 1990 b ; Lattanzio et al., 1994), suggesting it may be possible to increase fungitoxicity by replacing a hydroxy group on a flavone molecule with an acetoxy group. Although it has been reported that such substitutions do not improve the activity of certain isoflavonoids (Weidenbo$ rner & Jha, 1994 b), no comparable studies have been made with flavonoids. Therefore, the following study was conducted to examine the antifungal properties of 3- and 7-hydroxyflavone in comparison with their acetic acid esters. MATERIALS AND METHODS The fungi used were Penicillium glabrum (Wehmer) Westling and Cladosporium herbarum (Pers.) Link, which often occur on food (Weidenbo$ rner & Kunz, 1994). * Corresponding author.
3- and 7-hydroxyflavone were obtained from Aldrich Chemical Co. (Steinheim, Germany). Their corresponding acetic acid esters were prepared by reaction with acetic anhydride in pyridine. The mixture was refluxed for 3 h under dry conditions, poured into ice water, and subsequently acidified with 1 N HCl. Then the ester was filtered, washed with water, and dried. All flavonoids were tested for antifungal activity against P. glabrum and C. herbarum at concentrations of 0±8, 3±5 and 8±0¬10−$ mol l−" (Table 1). The effect of the flavonoids on mycelium growth of the test fungi was investigated on solid medium (30 g malt extract, 15 g agar, 3 g peptone l−" ; in distilled water) using three different methods. The autoclaved medium (20 ml) was transferred to Petri dishes (90 mm) and allowed to solidify. Then 1 ml flavonoid solution, dissolved in either ethanol (method A) or acetone (method B), was pipetted onto the agar. After evaporation of the solvent (72 h) the dishes were inoculated with a small piece of mycelium (1 mm diam.). In method C the flavonoids were dissolved in acetone and incorporated directly into the agar at 50 °C. In this case, higher flavonoid concentrations (3±5 and 8±0¬10−$ mol l−") were chosen, because in methods A and B the flavonoids (0±8¬10−$ mol l−") were concentrated on the agar surface. Thus, comparable conditions applied within the three methods. The dishes were incubated at 25° in the dark. Mycelium diameters of C. herbarum and P. glabrum were measured after 7 d. Evaluation was carried out by measuring the diameter of the colonies three times at different sites. The mean value of five repetitions for each experiment was used for calculation. The data were evaluated by analysis of variance. Probability of single differences was calculated at the 5 % level. RESULTS AND DISCUSSION The substituted acetic acid esters were more active in reducing mycelium growth of C. herbarum and P. glabrum than were the
Heike Martini and others
921
Table 1. Growth inhibition (%) of C. herbarum and P. glabrum caused by 3- and 7-hydroxyflavone (I, III) and their acetic acid esters (II, IV) in comparison with the corresponding control
C. herbarum
P. glabrum
Method
Concn ¬10−$ mol l−"
I (%)
II (%)
Difference (%)
III (%)
IV (%)
Difference (%)
A B C C A B C C
0±8 0±8 3±5 8±0 0±8 0±8 3±5 8±0
15±6 0 10±6 14±7 35±4 2±3 0 11±9
52±3 34±4 49±0 61±8 99±5 43±0 50±9 77±7
36±7 34±4 38±4 47±1 64±1 40±7 50±9 65±8
12±8 0 3±5 5±9 22±7 2±8 5±3 5±2
16±5 8±3 35±4 32±4 42±4 22±3 40±5 30±6
3±7 8±3 31±9 26±5 19±7 19±5 35±2 25±4
hydroxylated flavones (Table 1). The rank order of activity was 3-acetoxyflavone (II) followed by 7-acetoxyflavone (IV), 3-hydroxyflavone (I), and 7-hydroxyflavone (III). The greatest differences in activity resulted from the 3-substitution and the greatest effect was seen in P. glabrum. C. herbarum was less sensitive to each group of compounds than was P. glabrum. These results confirm earlier findings (Weidenbo$ rner et al., 1990 b ; Weidenbo$ rner & Jha, 1994 a) that 3- and 7-hydroxyflavones are generally poor inhibitors of mould growth. In contrast, however, they demonstrate that the esters are the more active, irrespective of the mould or method employed (Table 1). A general increase in the antifungal activity of lipophilic flavonoids and isoflavonoids has also been observed by other authors (O’Neill & Mansfield, 1982 ; Adesanya et al., 1996 ; Weidenbo$ rner et al., 1989 ; Weidenbo$ rner & Jha, 1993). One reason for their enhanced effectiveness may be an easier penetration of fungal membranes (Harborne & Ingham, 1978). However, because neither 4«,6,7-triacetoxyisoflavone nor 3«acetoxy-6,7-dihydroxyisoflavanone exhibit hardly any fungicidal activity (Weidenbo$ rner & Jha, 1994 b), the increased antifungal potential of the acetoxy flavones tested in this study cannot be explained exclusively by higher lipophilicity. As can be seen from the data in Table 1 the position of the substitution is also critical in determining effectiveness of each substance. Since the 3-substituted acetic acid ester (II) caused higher inhibition rates than 7-acetoxyflavone (IV), it is possible that certain metal ions essential for fungal growth are blocked by chelation with members of the 3-hydroxy-4ketone group (Somaatmadja et al., 1964) and the 3-acetoxy-4ketone group, respectively. Further investigations will focus on this phenomenon. The present study demonstrates that the antifungal potential of certain flavonoids is increased significantly by a simple chemical modification. This may be a first step in the development of naturally occurring food preservatives. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG). REFERENCES Adesanya, S. A., O’Neill, T. M. & Roberts, M. F. (1986). Structure-related fungitoxicity of isoflavonoids. Physiological and Molecular Plant Pathology 29, 95–103.
Arnoldi, A. & Merlini, L. (1990). Lipophilicity-antifungal relationships for some isoflavonoid phytoalexins. Journal of Agricultural and Food Chemistry 38, 834–838. Frisvad, J. C. & Thrane, U. (1995). Mycotoxin production by food-borne fungi. In Introduction to Food-borne Fungi, 4th ed. (ed. R. A. Samson, E. S. Hoekstra, J. C. Frisvad & O. Filtenborg), pp. 251–260. Centraalbureau voor Schimmelcultures : Baarn and Delft. Harborne, J. B. & Ingham, J. L. (1978). Biochemical aspects of the coevolution of higher plants with their fungal parasites. In Biochemical Aspects of Plant and Animal Coevolution (ed. J. B. Harborne), pp. 343–405. Academic Press : New York. Hedin, P. A. & Waage, S. K. (1986). Roles of flavonoids in plant resistance to insects. In Plant Flavonoids in Biology and Medicine (ed. V. Cody, E. Middleton Jr, J. B. Harborne & A. Beretz), pp. 87–100. Alan R. Liss : New York. Kra$ mer, J. (1997). Lebensmittel-Mikrobiologie, 3rd ed. Eugen Ulmer Verlag : Stuttgart. Kra$ mer, R. P., Hindorf, H., Jha, H. C. & Zilliken, F. (1984). Antifungal activity of soybean and chickpea isoflavones and their reduced derivatives. Phytochemistry 23, 2203–2205. Kunz, K., Weidenbo$ rner, M. & Kunz, B. (1995). Die Nutzung von Gewu$ rzen in Weizenbrot zur Kontrolle der lebensmittelrelevanten Schimmelpilze Cladosporium herbarum, Eurotium repens, Penicillium expansum und Rhizopus stolonifer. Chemie, Mikrobiologie und Technologie der Lebensmittel 17, 1–5. Lattanzio, V., De Cicco, V., Di Venere, D., Lima, G. & Salerno, M. (1994). Antifungal activity of phenolics against fungi commonly encountered during storage. Italian Journal of Food Science 6, 23–30. O’Neill, T. M. & Mansfield, J. W. (1982). Antifungal activity of hydroxyflavans and other flavonoids. Transactions of the British Mycological Society 79, 229–237. Somaatmadja, D., Powers, J. J. & Hamdy, M. K. (1964). Anthocyanins. VI. Chelation studies on anthocyanins and other related compounds. Journal of Food Science, 29, 655–660. Van Etten, H. (1976). Antifungal activity of pterocarpans and other selected isoflavonoids. Phytochemistry 15, 655–659. Weidenbo$ rner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. & Egge, H. (1989). Antifungal activity of isoflavonoids against storage fungi of the genus Aspergillus. Phytochemistry 28, 3317–3319. Weidenbo$ rner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. & Egge, H. (1990 a). Antifungal activity of isoflavonoids in different reduced stages on Rhizoctonia solani and Sclerotium rolfsii. Phytochemistry 29, 801–803. Weidenbo$ rner, M., Hindorf, H., Jha, H. C. & Tsotsonos, P. (1990 b). Antifungal activity of flavonoids against storage fungi of the genus Aspergillus. Phytochemistry 29, 1103–1105. Weidenbo$ rner, M., Hindorf, H., Weltzien, H. C. & Jha, H. C. (1992). An effective treatment of legume seeds with flavonoids and isoflavonoids against storage fungi of the genus Aspergillus. Seed Science & Technology 20, 447–463. Weidenbo$ rner, M. & Jha, H. C. (1993). Antifungal activity of flavonoids and their mixtures against different fungi occurring on grain. Pesticide Science 38, 347–351.
Antifungal activity in hydroxyflavones Weidenbo$ rner, M. & Jha, H. C. (1994 a). Antifungal activity of flavonoids in relation to degree of hydroxylation, methoxylation, and glycosidation. Acta Horticulturae 381, 702–709. Weidenbo$ rner, M. & Jha, H. C. (1994 b). Structure–activity relationships (Accepted 17 January 1997)
922 among isoflavonoids with regard to their antifungal properties. Mycological Research 98, 1376–1378. Weidenbo$ rner, M. & Kunz, B. (1994). Contamination of different muesli components by fungi. Mycological Research 98, 583–586.