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Antifungal activity of flavonoids against storage fungi of the genus Aspergillus
Phytochemistry, Vol. 29, No. 4, pp. 1103-I 105, 1990. Printedin Great Britain.
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003 1 9422/90 $3.00 + 0.00
Pergamon Press plc
ACTIVITY OF FLAVONOIDS AGAINST STORAGE FUNGI OF THE GENUS ASPERGZLLUS
MARTIN WEIDENB~RNER, Institut fiir Pflanzenkrankheiten
HOLGER HINDORF,
der Universitat Universitlt
Key Word Index-Aspergillus structure-activity
repens;
and
PRODROMOS TSOTSONOS*
Bonn Nussallee 9, D-5300 Bonn 1, F.R.G.; *Institut Bonn Nussallee 11, D-5300 Bonn 1, F.R.G. in revised j)rm
(Receioed
activity;
HEM CHANDRA JHA*
A. amstelodami;
fiir Physiologische
Chemie der
14 July 1989)
A. chevalieri;
A. jaws;
A. petrakii;
flavonoids;
antifungal
relationship.
Abstract-Four flavones, three flavanones and one catechin were tested for fungicidal activity in malt extract broth against five storage fungi of the genus Asperyillus. Unsubstituted flavone and flavanone were highly active while the hydroxylated flavonoids possess only weak activity. Structure-activity relationships are discussed.
INTRODUCTION
RESULTS AND DISCUSSION
Flavonoids, besides other biological activities, have been shown to be active against microorganisms [l-3]. At least in some cases the presence of these substances might serve as a chemical barrier to invading microorganisms [4]. Because they are natural compounds and possess highly specific antimicrobial activity, these substances may be an alternative to conventional fungicides in the control of storage fungi which cause great losses in the storage of grains in the developing countries [S].
In our previous investigations [6-81, very encouraging results have been obtained in inhibiting the growth of fungi by the use of isoflavonoids. This work deals with the activity of flavonoids against storage fungi of the genus Aspergillus.
In this experiment the unsubstituted flavone (1) and flavanone (2) caused mycelial inhibition up to 90% in the case A. glaucus in concentration c, while A. javus and A. petrakii were inhibited only up to 70% in this concentra-
R5)@#$’ R4+R2 =
RZ =
R’ =
R4 =
Rs
=
2
1
RI
3 4 8
K’ = Kt = K’ = Ka = KS = 0” RI = K’ = K4 = KS = t.,, K3 = 0” RI= K” Ka= K4’ H,Ks= OH
”
R’
=
R2
OH OH HO
HCI
8 1103
=
R3
=
R4 =
”
6 K’ = “, K* = K3 = K* = 0” 7 K’ = K3 = K4 = O”, K’ = OMe
1104
M.
WHDENB~RNER
tion (Table 1). In contrast, the activity of the oxygenated flavonoids was rather low. Quercetin (3) was totally ineffective. Flavonol (4) inhibited mycelial growth of A. repens to 33.5% in concentration h, while this substance stimulated growth of A. checalieri in concentrations b and c to 29.0%. An inhibition of 8.7% was caused by 7hydroxyflavone (5) in the case of A. ,flavus in concentration c. The flavanones naringenin (6) and hesperetin (7) were only mildly active against fungi of the A. @mucus group. The highest inhibition was 20.7”/0 with narmgenin at concentration c using A. chevalieri as test organism (Table I). The growth of A. rheculieri was stimulated by the (+)-catechin (8) to 22.0 and 40.6% in the concentrations h and c respectively (Table 1). The high fungitoxic activity of flavone and flavanone is surprising as the isomeric counterparts of both substances, viz., isoflavone and isoflavanone, showed only low activity against Asperyillus [6]. Furthermore, Johnson et al. [9] found that the isoflavonoids were more active than other flavonoids. As regards the results of our former investigations with isoflavonoids [6--83, it seems that this conclusion is valid only in the case of substituted isoflavonoids. Flavone showed higher activity than flavanone which is corroborated by the results of O’Neill et a/. [lo], although they utilized a different method to evaluate the activity. In spite of the fact that the activity of the hydroxyflavones was low, flavonol was more effective than 7hydroxyflavone (Table 1). A hydroxyl group at position 3, however, gives no guarantee for antifungal activity, if other hydroxyl groups are present in the molecule (Table 1). O’Neill et al. [lo] found that 5,7-dihydroxyflavone (chrysin) was inactive against Botrytis cinerea. This shows that even a further hydroxylation at position 5 caused no increase in antifungal activity. The isoflavone biochanin A, which possesses hydroxyl groups at 5,7 positions, showed very high activity against Rhizoctoniu solani and Sclerotium rolfsii 181. In the case of flavones, a high antifungal activity is guaranteed by at least two methoxy groups in ring A, one of which being at the Sposition [l]. Nobiletin, 5,6,7,8,3’,4’-hexamethoxyflavone is fungistatic against Deuterophoma trucheiphilu [ 11, 123 and 5,4’-dihydroxy-6,7,8,3’-tetramethoxyflavone is fungistatic too. Flavones methoxylated in ring A and in rings A and C are fungicidal to C. cucumerinum while 5hydroxy-6,7-dimethoxyand the Shydroxy-6,7,8-trimethoxyfiavone are ineffective Cl>]. In general the high activity of the Ravone sketeton can be ascribed to the absence of polar groups in the molecule. The two flavanones naringenin and hesperetin were most effective in concentration c, although the highest inhibition rate amounted to only 20% in the case of A. chedieri. The methoxy group at position 4’ of hesperetin caused no increased activity compared to naringenin (Table 1). In contrast, Biswas et al. [14] found 5,8dihydroxy-6,7-dimethoxyand the 7-hydroxy-5,6,8-trimethoxyflavanone to be active against sclerotia germination of S. rolfsii, Thanatephorus cucumerinum and Rhizoctoniu soluni. The former flavanone also inhibits spore germination of Helmithosporium oryzue, Rhizopus urtocurpi and Fusurium oxysporum ciceri. In both these substances ring A is fully substituted and contains at least two methoxy groups. The lower activity of hesperetin and naringenin might be due to the partially substituted ring A and the absence of the methoxy groups. However, 7hydroxy- and 6,7_dihydroxyflavanone, are fungicidal to
et al.
Antifungal Alternaria flavanones fungicidal
activity
solani and Curt&aria lunata [lSJ. It seems that substituted only in ring A, possess a higher activity than those substituted in ring B too
[lo]. Catechin, the only flavan-3-01 tested, had no fungicida1 effect on Aspergillus. Even in the concn c mycelial growth of A. chevalieri was stimulated upto 40% (Table 1). Similar results were obtained by O’Neill and Mansfield [lo] with 3,7,4’,5’-tetrahydroxy-, 3,4,7,4’,5’pentahydroxyand the 3,5,7,4’,5’-pentahydroxyfiavan against B. cinerea. High polarity due to several hydroxyl groups seems to reduce activity.
EXPERIMENTAL
The fungi used were all soil-borne species of the genus Aspergillus: A. repens de Bary, A. amstelodami (Thorn & Church) and A. chevalieri (Mangin) Thorn & Church of the Aspergillus gfaucus Link grpup; A.jauus Link of the Aspergillusjauus Link group and A. petrakii V6rds of the Aspergillus ochraceus Wilhelm group. They were isolated from seeds of soybean, pigeonpea, kidney-bean, peanut and cotton 1161. The quantification of mycelium growth in liquid culture was used [17, 181 to determine antifungal activities. The nutrient solution contained 30g malt,‘1 and solvent for the isoflavonoids. The solvent concentration in the solution was maintained at 1.1% level. Medium (20 ml) including the particular flavonoid in the required concentration was transferred to lOOm1 flasks and inoculated withfive small pieces of mycelium (5 mm in diameter). The flasks were incubated at 23-25” on a reciprocal shaker for 7 days. Concentrations ofjavonoids. From phytoalexin experiments [lo] it is known that concentrations in the range 10-5-10-3 mol/l exhibit fungal inhibition. Therefore the concentrations of 0.5, 2.0 and 8.0 x lo-“ mol/l denoted as a-c were used in these tests. Evaluations ofresults. Round filter papers (Schleicher & Schiill No. 595) were weighed after drying for 24 hr at 105”. After filtration of the culture the residue was dried and weighed as described above. The difference of the weighings gave the dry wt of the fungi. The mean value of eight repetitions for each concentration and fungus was used for ca!culation. The data
of flavonoids
1105
were evaluated by analysis of variance. Probability of single differences was calculated at the 5% level. Flauonoids. All substances were obtained commercially.
REFERENCES 1. Tomas-Barberan, F. A., Maillard, M. and Hostettmann, K. (1988) in PIant FIavonoids in Biology and Medicine, Vol. 2. (Cody, V., Middleton, Jr. E., Harborne, J. B. and Beretz, A., eds), p. 61. Alan, R. Liss, New York. 2. Harborne, J. B. (1983) in Plant and Fungal Toxins (Keeler, R. F. and Tu, A. T., eds), p. 743. Marcel Dekker, New York. 3. Piatelli, M. and Impellizzeri, G. (1971) Phytochemistry 10, 2657. 4. Harborne, J. B. (1977) Biochem. Syst. Ecol. 5, 7. 5. Neergaard, P. (1977) in Seed Pathology. MacMillan Press, London. 6. Weidenbiirner, M., Jha, H. C., Hindorf, H., Weltzien, H. C. and Zilliken, F. (1987) Med. Fat. Landhouww. Rijksuniv. Gent. 52, 933. 7. Weidenbiirner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. and Egge, H. (1989) Phytochemistry 28, 3317. 8. Weidenbtirner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. and Egge, H. (1989) Phytochemistry 28 (in press). 9. Johnson, G., Maag, D. D., Johnson, D. K. and Thomas, R. D. (1976) Physiol. Plant Pathol. 8, 225. 10. O’Neill, T. M. and Mansfield, J. W. (1982) Trans. B. Mycol. Sot. 19, 229. 11. Pinkas, J., Lavie, D. and Chorin, M. (1968) Phytochemistry 7, 169. 12. Ben Aziz, A. (1967) Science 155, 1026. 13. Tomas-Barberan, F. A., Msonthi, 3. D. and Hostettmann, K. (1988) Phytochemistry 27, 753. 14. Biswas, P. Bhattacharyya, A., Bose, P. C., Mukherjee, N. and Adityachaudhury, N. (1981) Experientia 37, 397. 15. Chowdhury, A., Mukherjee, N. and Adityachaudhury, N. (1974) Experientia 30, 1022. 16. Weidenbiirner. M. and Hindorf, H. (1989) Seed Sci. Technol. 17, 383. 17. Smith, D. A. (1976) Physiol. Plant Pathol. 9, 45. 18. Van Etten, H. D. and Bateman, D. F. (1971) Phytopatholoyy 61, 1363.
ANTIFUNGAL
003 1 9422/90 $3.00 + 0.00
Pergamon Press plc
ACTIVITY OF FLAVONOIDS AGAINST STORAGE FUNGI OF THE GENUS ASPERGZLLUS
MARTIN WEIDENB~RNER, Institut fiir Pflanzenkrankheiten
HOLGER HINDORF,
der Universitat Universitlt
Key Word Index-Aspergillus structure-activity
repens;
and
PRODROMOS TSOTSONOS*
Bonn Nussallee 9, D-5300 Bonn 1, F.R.G.; *Institut Bonn Nussallee 11, D-5300 Bonn 1, F.R.G. in revised j)rm
(Receioed
activity;
HEM CHANDRA JHA*
A. amstelodami;
fiir Physiologische
Chemie der
14 July 1989)
A. chevalieri;
A. jaws;
A. petrakii;
flavonoids;
antifungal
relationship.
Abstract-Four flavones, three flavanones and one catechin were tested for fungicidal activity in malt extract broth against five storage fungi of the genus Asperyillus. Unsubstituted flavone and flavanone were highly active while the hydroxylated flavonoids possess only weak activity. Structure-activity relationships are discussed.
INTRODUCTION
RESULTS AND DISCUSSION
Flavonoids, besides other biological activities, have been shown to be active against microorganisms [l-3]. At least in some cases the presence of these substances might serve as a chemical barrier to invading microorganisms [4]. Because they are natural compounds and possess highly specific antimicrobial activity, these substances may be an alternative to conventional fungicides in the control of storage fungi which cause great losses in the storage of grains in the developing countries [S].
In our previous investigations [6-81, very encouraging results have been obtained in inhibiting the growth of fungi by the use of isoflavonoids. This work deals with the activity of flavonoids against storage fungi of the genus Aspergillus.
In this experiment the unsubstituted flavone (1) and flavanone (2) caused mycelial inhibition up to 90% in the case A. glaucus in concentration c, while A. javus and A. petrakii were inhibited only up to 70% in this concentra-
R5)@#$’ R4+R2 =
RZ =
R’ =
R4 =
Rs
=
2
1
RI
3 4 8
K’ = Kt = K’ = Ka = KS = 0” RI = K’ = K4 = KS = t.,, K3 = 0” RI= K” Ka= K4’ H,Ks= OH
”
R’
=
R2
OH OH HO
HCI
8 1103
=
R3
=
R4 =
”
6 K’ = “, K* = K3 = K* = 0” 7 K’ = K3 = K4 = O”, K’ = OMe
1104
M.
WHDENB~RNER
tion (Table 1). In contrast, the activity of the oxygenated flavonoids was rather low. Quercetin (3) was totally ineffective. Flavonol (4) inhibited mycelial growth of A. repens to 33.5% in concentration h, while this substance stimulated growth of A. checalieri in concentrations b and c to 29.0%. An inhibition of 8.7% was caused by 7hydroxyflavone (5) in the case of A. ,flavus in concentration c. The flavanones naringenin (6) and hesperetin (7) were only mildly active against fungi of the A. @mucus group. The highest inhibition was 20.7”/0 with narmgenin at concentration c using A. chevalieri as test organism (Table I). The growth of A. rheculieri was stimulated by the (+)-catechin (8) to 22.0 and 40.6% in the concentrations h and c respectively (Table 1). The high fungitoxic activity of flavone and flavanone is surprising as the isomeric counterparts of both substances, viz., isoflavone and isoflavanone, showed only low activity against Asperyillus [6]. Furthermore, Johnson et al. [9] found that the isoflavonoids were more active than other flavonoids. As regards the results of our former investigations with isoflavonoids [6--83, it seems that this conclusion is valid only in the case of substituted isoflavonoids. Flavone showed higher activity than flavanone which is corroborated by the results of O’Neill et a/. [lo], although they utilized a different method to evaluate the activity. In spite of the fact that the activity of the hydroxyflavones was low, flavonol was more effective than 7hydroxyflavone (Table 1). A hydroxyl group at position 3, however, gives no guarantee for antifungal activity, if other hydroxyl groups are present in the molecule (Table 1). O’Neill et al. [lo] found that 5,7-dihydroxyflavone (chrysin) was inactive against Botrytis cinerea. This shows that even a further hydroxylation at position 5 caused no increase in antifungal activity. The isoflavone biochanin A, which possesses hydroxyl groups at 5,7 positions, showed very high activity against Rhizoctoniu solani and Sclerotium rolfsii 181. In the case of flavones, a high antifungal activity is guaranteed by at least two methoxy groups in ring A, one of which being at the Sposition [l]. Nobiletin, 5,6,7,8,3’,4’-hexamethoxyflavone is fungistatic against Deuterophoma trucheiphilu [ 11, 123 and 5,4’-dihydroxy-6,7,8,3’-tetramethoxyflavone is fungistatic too. Flavones methoxylated in ring A and in rings A and C are fungicidal to C. cucumerinum while 5hydroxy-6,7-dimethoxyand the Shydroxy-6,7,8-trimethoxyfiavone are ineffective Cl>]. In general the high activity of the Ravone sketeton can be ascribed to the absence of polar groups in the molecule. The two flavanones naringenin and hesperetin were most effective in concentration c, although the highest inhibition rate amounted to only 20% in the case of A. chedieri. The methoxy group at position 4’ of hesperetin caused no increased activity compared to naringenin (Table 1). In contrast, Biswas et al. [14] found 5,8dihydroxy-6,7-dimethoxyand the 7-hydroxy-5,6,8-trimethoxyflavanone to be active against sclerotia germination of S. rolfsii, Thanatephorus cucumerinum and Rhizoctoniu soluni. The former flavanone also inhibits spore germination of Helmithosporium oryzue, Rhizopus urtocurpi and Fusurium oxysporum ciceri. In both these substances ring A is fully substituted and contains at least two methoxy groups. The lower activity of hesperetin and naringenin might be due to the partially substituted ring A and the absence of the methoxy groups. However, 7hydroxy- and 6,7_dihydroxyflavanone, are fungicidal to
et al.
Antifungal Alternaria flavanones fungicidal
activity
solani and Curt&aria lunata [lSJ. It seems that substituted only in ring A, possess a higher activity than those substituted in ring B too
[lo]. Catechin, the only flavan-3-01 tested, had no fungicida1 effect on Aspergillus. Even in the concn c mycelial growth of A. chevalieri was stimulated upto 40% (Table 1). Similar results were obtained by O’Neill and Mansfield [lo] with 3,7,4’,5’-tetrahydroxy-, 3,4,7,4’,5’pentahydroxyand the 3,5,7,4’,5’-pentahydroxyfiavan against B. cinerea. High polarity due to several hydroxyl groups seems to reduce activity.
EXPERIMENTAL
The fungi used were all soil-borne species of the genus Aspergillus: A. repens de Bary, A. amstelodami (Thorn & Church) and A. chevalieri (Mangin) Thorn & Church of the Aspergillus gfaucus Link grpup; A.jauus Link of the Aspergillusjauus Link group and A. petrakii V6rds of the Aspergillus ochraceus Wilhelm group. They were isolated from seeds of soybean, pigeonpea, kidney-bean, peanut and cotton 1161. The quantification of mycelium growth in liquid culture was used [17, 181 to determine antifungal activities. The nutrient solution contained 30g malt,‘1 and solvent for the isoflavonoids. The solvent concentration in the solution was maintained at 1.1% level. Medium (20 ml) including the particular flavonoid in the required concentration was transferred to lOOm1 flasks and inoculated withfive small pieces of mycelium (5 mm in diameter). The flasks were incubated at 23-25” on a reciprocal shaker for 7 days. Concentrations ofjavonoids. From phytoalexin experiments [lo] it is known that concentrations in the range 10-5-10-3 mol/l exhibit fungal inhibition. Therefore the concentrations of 0.5, 2.0 and 8.0 x lo-“ mol/l denoted as a-c were used in these tests. Evaluations ofresults. Round filter papers (Schleicher & Schiill No. 595) were weighed after drying for 24 hr at 105”. After filtration of the culture the residue was dried and weighed as described above. The difference of the weighings gave the dry wt of the fungi. The mean value of eight repetitions for each concentration and fungus was used for ca!culation. The data
of flavonoids
1105
were evaluated by analysis of variance. Probability of single differences was calculated at the 5% level. Flauonoids. All substances were obtained commercially.
REFERENCES 1. Tomas-Barberan, F. A., Maillard, M. and Hostettmann, K. (1988) in PIant FIavonoids in Biology and Medicine, Vol. 2. (Cody, V., Middleton, Jr. E., Harborne, J. B. and Beretz, A., eds), p. 61. Alan, R. Liss, New York. 2. Harborne, J. B. (1983) in Plant and Fungal Toxins (Keeler, R. F. and Tu, A. T., eds), p. 743. Marcel Dekker, New York. 3. Piatelli, M. and Impellizzeri, G. (1971) Phytochemistry 10, 2657. 4. Harborne, J. B. (1977) Biochem. Syst. Ecol. 5, 7. 5. Neergaard, P. (1977) in Seed Pathology. MacMillan Press, London. 6. Weidenbiirner, M., Jha, H. C., Hindorf, H., Weltzien, H. C. and Zilliken, F. (1987) Med. Fat. Landhouww. Rijksuniv. Gent. 52, 933. 7. Weidenbiirner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. and Egge, H. (1989) Phytochemistry 28, 3317. 8. Weidenbtirner, M., Hindorf, H., Jha, H. C., Tsotsonos, P. and Egge, H. (1989) Phytochemistry 28 (in press). 9. Johnson, G., Maag, D. D., Johnson, D. K. and Thomas, R. D. (1976) Physiol. Plant Pathol. 8, 225. 10. O’Neill, T. M. and Mansfield, J. W. (1982) Trans. B. Mycol. Sot. 19, 229. 11. Pinkas, J., Lavie, D. and Chorin, M. (1968) Phytochemistry 7, 169. 12. Ben Aziz, A. (1967) Science 155, 1026. 13. Tomas-Barberan, F. A., Msonthi, 3. D. and Hostettmann, K. (1988) Phytochemistry 27, 753. 14. Biswas, P. Bhattacharyya, A., Bose, P. C., Mukherjee, N. and Adityachaudhury, N. (1981) Experientia 37, 397. 15. Chowdhury, A., Mukherjee, N. and Adityachaudhury, N. (1974) Experientia 30, 1022. 16. Weidenbiirner. M. and Hindorf, H. (1989) Seed Sci. Technol. 17, 383. 17. Smith, D. A. (1976) Physiol. Plant Pathol. 9, 45. 18. Van Etten, H. D. and Bateman, D. F. (1971) Phytopatholoyy 61, 1363.