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Effects of alkyl parabens on plant pathogenic fungi
Bioorganic & Medicinal Chemistry Letters 25 (2015) 1774–1777
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Effects of alkyl parabens on plant pathogenic fungi Shinsaku Ito , Satoru Yazawa , Yasutaka Nakagawa, Yasuyuki Sasaki, Shunsuke Yajima ⇑ Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan
a r t i c l e
i n f o
Article history: Received 1 December 2014 Revised 7 February 2015 Accepted 20 February 2015 Available online 7 March 2015 Keywords: Structure–activity relationship study Drug design Paraben Gallate Mitochondrial complex II Black leaf spot disease
a b s t r a c t Alkyl parabens are used as antimicrobial preservatives in cosmetics, food, and pharmaceutical products. However, the mode of action of these chemicals has not been assessed thoroughly. In this study, we determined the effects of alkyl parabens on plant pathogenic fungi. All the fungi tested, were susceptible to parabens. The effect of linear alkyl parabens on plant pathogenic fungi was related to the length of the alkyl chain. In addition, the antifungal activity was correlated with the paraben-induced inhibition of oxygen consumption. The antifungal activity of linear alkyl parabens likely originates, at least in part, from their ability to inhibit the membrane respiratory chain, especially mitochondrial complex II. Additionally, we determined that some alkyl parabens inhibit Alternaria brassicicola infection of cabbage. Ó 2015 Elsevier Ltd. All rights reserved.
Alkyl parabens (p-hydroxybenzoic acid alkyl esters) are common preservatives used in cosmetics due to their properties such as, broad antimicrobial spectrum, relatively low toxicity, good chemical stability, and non-volatility although they have been reported to have potential health risks for humans.1 Methyl and propyl parabens are mostly used as cosmetic preservatives, and as the number of carbon in the paraben alkyl chain increases, it enhances their antimicrobial activity.2 Sapra et al. recently tested the antimicrobial potential of alkyl parabens on five microorganisms.3 They reported that decyl paraben was effective against Staphylococcus aureus, Candida albicans, and Aspergillus niger, while propyl and butyl parabens showed the highest antimicrobial activity against Bacillus subtilis and Escherichia coli, respectively.3 Thus, the effect of alkyl parabens vary according to the microorganism treated with it. Short-chain alkyl parabens (ethyl and butyl) were reported to disrupt the function of mechanosensitive channels and induce potassium efflux in E. coli; however, the underlying mechanism of the antifungal activity of alkyl parabens is poorly understood.4a,b,5 In our previous study, we evaluated the antifungal properties of alkyl gallates and their mode of action.6 The length of the alkyl chain was linked to their antifungal activity, and nonyl gallate was the most potent antifungal agent. In addition, linear alkyl gallates inhibited mitochondrial complex II activity depending on their chain length, which was consistent with their antifungal
activity. Because of a structural similarity between gallates and parabens (Fig. 1), we hypothesized that alkyl parabens may also inhibit the activity of mitochondrial complex II. Therefore, we synthesized alkyl parabens and their derivatives, investigated their antifungal properties and mode of action against plant pathogenic fungi, such as, Fusarium solani, Colletotrichum acutatum, Colletotrichum dematium, and Alternaria brassicicola. These fungi are known to cause serious damage to economically important crops and fruits across the world. The chemicals were prepared as reported in earlier studies, with slight modifications.6 Substituted benzoic acids and alkanols dissolved in tetrahydrofuran were stirred at room temperature in the presence of N,N0 -dicyclohexylcarbodiimide. The reaction mixture was quenched with distilled water, and the aqueous layer was extracted with ethyl acetate. The organic layer was dried and concentrated under reduced pressure. The resulting oil was purified by a column chromatography. The structures of the alkyl parabens and their derivatives have been shown in Figure 2. Log P values were calculated by ChemBioDraw Ultra software (MA, USA). C. acutatum (CAB03) was received from Dr. Nakaune.7 A. brassicicola (MAFF No. 726527, 726705, 237450 and 305011)
⇑ Corresponding author. Tel.: +81 3 5477 2768.
E-mail address: [email protected] (S. Yajima). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.02.049 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Figure 1. Chemical structures of alkyl gallate (left) and alkyl paraben (right).
S. Ito et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1774–1777
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Figure 2. Chemical structures and log P values of parabens.
was purchased from NIAS Genebank (Tsukuba city, Japan). The other fungi were received from Prof. Natsuaki (Tokyo Univ. of Agric.). We estimated the role of alkyl parabens against plant pathogenic fungi (Tables 1 and 2). Antifungal activities were determined by measuring the minimum inhibitory concentration (MIC).6 Methyl, ethyl, propyl, butyl, and pentyl parabens were not effective against all the fungal strains tested in this study. As for gallates,6 an increase in the chain length of linear alkyl parabens led to enhanced antifungal activity. Among the linear alkyl parabens tested (1–7), nonyl paraben (7), which was less active than nonyl gallate (11), had the highest antifungal activity. Consistent with the antifungal activity of alkyl gallates,6 branched alkyl parabens showed stronger activity than linear alkyl parabens with similar
Table 1 MIC values (lM) of alkyl parabens and derivatives against plant pathogenic fungi
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Benomyl Boscalid
F. solani
C. dematium
C. acutatum
C. acutatum (CAB03)
>50 >50 >50 >50 >50 50 50 25 >50 25 6.25 12.5 >50 25 50 3.13* 3.13
>50 >50 >50 >50 >50 50 50 50 >50 25 12.5 25 >50 6.25 50 25* nd
>50 >50 >50 >50 >50 50 25 25 50 25 6.25 12.5 >50 25 >50 12.5* nd
>50 >50 >50 >50 >50 >50 50 25 50 25 12.5 12.5 >50 25 >50 50 nd
nd: not determined. * The values of the previously described.6
log P values. Compounds 10 and 12, which have 4-hydroxyl group, showed the increased antifungal activities in comparison with the compounds 13 and 15, which do not have 4-hydroxyl group, respectively. This result suggests that the introduction of 4-hydroxyl group is important for these derivatives to show the antifungal activities. In addition, all A. brassicicola strains were more sensitive to branched alkyl parabens than the other fungal strains tested (Table 2). Notably, 3,3-dimethylbutyl paraben (10) had the highest antifungal activity against A. brassicicola.8 Interestingly, only monohydroxy benzoates (10 and 14) showed higher antifungal activity against A. brassicicola than against the other fungi. In addition, some alkyl parabens and gallates (8, 10, 11, 12 and 14) were effective against benomyl-resistant C. acutatum (CAB03). Though benomyl is a fungicide used worldwide, the rapid development of resistant strains has limited its use. These results suggest the possibility of using alkyl parabens as antifungal agents. It has been proposed that the mode of antimicrobial action of alkyl parabens depends on their ability to disrupt the native membrane due to their hydrophobicity.9 Though mechanosensitive channels are one of the targets of short-chain alkyl parabens (methyl to butyl), the target of longer-chain alkyl parabens has not been investigated. As previously described, the structurally similar linear alkyl gallates inhibit the mitochondrial complex II activity. In addition, such inhibitory activity against complex II was correlated with the antifungal property of linear alkyl gallates.6 These findings helped to determine the effects of alkyl parabens on oxygen consumption and activity of complex II in fungi.10,11 The inhibitory action on oxygen consumption was enhanced by increasing the chain length of linear alkyl parabens in A. brassicicola and F. solani (Fig. 3A and B). Especially, nonyl paraben (7) showed the highest activity consistent with the antifungal activities of linear alkyl parabens. Branched alkyl paraben (10) also inhibited the oxygen consumption, although its inhibition was slightly weaker than that of the nonyl parabens (7). In addition, nonyl and 3,3-dimethylbutyl parabens (7 and 10, respectively)
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S. Ito et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1774–1777 Table 2 MIC values (lM) of alkyl parabens and derivatives against A. brassicicola
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Benomyl
A. brassicicola
A. brassicicola (726527)
A. brassicicola (726705)
A. brassicicola (237450)
A. brassicicola (305011)
>50 >50 >50 >50 >50 25 25 12.5 25 6.25 12.5 12.5 >50 6.25 50 6.25*
>50 >50 >50 >50 >50 50 25 12.5 50 12.5 12.5 25 >50 12.5 25 nd
>50 >50 >50 >50 50 50 25 25 12.5 6.25 6.25 12.5 >50 12.5 50 nd
>50 >50 >50 >50 50 25 25 50 50 12.5 6.25 25 >50 25 25 nd
>50 >50 >50 >50 50 50 50 25 12.5 12.5 12.5 25 >50 12.5 50 nd
nd: not determined. * The values of the previously described.6
Figure 4. Inhibition of mitochondrial complex II by alkyl parabens. Values in parentheses indicate the concentration of the chemicals (lM). The data are means ± SD of three samples. ⁄⁄ indicates significant differences from control (DMSO treatment) (t-test, P <0.01).
Figure 3. Inhibition of oxygen consumption by alkyl parabens. Oxygen consumption in (A) A. brassicicola and (B) F. solani was determined using an oxygen electrode. Alkyl parabens were added at a concentration of 200 lM. The data are means ± SD of three samples. ⁄ and ⁄⁄ indicate significant differences from control (DMSO treatment) (t-test, P <0.05 and P <0.01, respectively).
inhibited the activity of complex II in F. solani (Fig. 4). Furthermore, boscalid, which is a mitochondrial complex II inhibitor, showed about 10-fold higher in the antifungal activity and complex II inhibition than compound 7 (Table 1, Fig. 4). These results strongly indicate that the antifungal activity of linear alkyl parabens against plant pathogenic fungi is due to the inhibition of complex II.
However, there may be other mechanisms of antifungal action for branched alkyl paraben, because the inhibitory activity of 3,3-dimethylbutyl paraben (10) against oxygen consumption was less effective than its antifungal activity. A. brassicicola is a necrotrophic fungal pathogen that causes black leaf spot disease in many cruciferous crops including cabbage (Brassica oleracea L.). Branched alkyl parabens were more effective against A. brassicicola than against other plant pathogenic fungi. Thus, we estimated the effect of alkyl parabens on A. brassicicola infection of detached cabbage leaves. The infection test was performed according to a previously described method, with slight modifications.12,13 The results showed that paraben-treated plants were more resistant to A. brassicicola than untreated plants (Fig. 5A). Quantification of infection levels of A. brassicicola in the plants was carried out by measuring the infective gene copy numbers in the fungal genome by real-time PCR.14,15 Alkyl paraben treatment reduced fungal genomic DNA levels in a dose-dependent manner (Fig. 5B). These results indicate that alkyl parabens have the potential to reduce crop losses due to A. brassicicola infection. In this Letter, we demonstrated that alkyl parabens showed the antifungal activity against plant fungal pathogens. In particular, branched alkyl parabens (8–10) were more potent in inhibiting fungal growth than the linear alkyl parabens with the similar log P values. In addition, linear alkyl parabens inhibited oxygen consumption, which was linked to the increase in the chain length
S. Ito et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1774–1777
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although the specific mode of action was poorly understood. Our findings provide a new perspective for the antifungal properties of alkyl parabens against plant fungal pathogens, via the inhibition of oxygen consumption, especially in mitochondrial complex II. Acknowledgment We thank Dr. Ryoji Nakaune (National Agricultural and Food Research Organization, Japan) for kindly providing C. acutatum (CAB03). References and notes
Figure 5. Effect of alkyl parabens on infection of cabbage with A. brassicicola. (A) The appearance of cabbage leaf after three days of fungal inoculation. (B) Fungal DNA was quantified using AbSCD1 gene specific primer.12 AbSCD1 forward: 50 -GCA GAC AGC TAC GAT AGC AA-30 , AbSCD1 reverse: 50 -GAT GCA TTT GCG GAG AC-30 . Genomic DNA was quantified using Beckman DU530 UV/Vis Spectrophotometer (CA, USA). ⁄⁄ indicates significant differences from control (DMSO treatment) (t-test, P <0.01). Values in parentheses indicate the concentration of the chemicals (lM).
of alkyl paraben. Moreover, these parabens inhibited A. brassicicola infection of plants. Parabens had been used as preservatives in food and cosmetics because of their broad antimicrobial spectrum,
1. Darbre, P. D.; Harvey, P. W. J. Appl. Toxicol. 2008, 28, 561. 2. Soni, M. G.; Taylor, S. L.; Greenberg, N. A.; Burdock, G. A. Food Chem. Toxicol. 2002, 40, 1335. 3. Sapra, A.; Kumar, P.; Kakkar, S.; Narasimhan, B. Drug Res. (Stuttg) 2014, 64, 17. 4. (a) Kamaraju, K.; Sukharev, S. Biochemistry 2008, 47, 10540; (b) Nguyen, T.; Clare, B.; Guo, W.; Martinac, B. Eur. Biophys. J. 2005, 34, 389. 5. Bredin, J.; Davin-Régli, A.; Pagès, J. M. J. Antimicrob. Chemother. 2005, 55, 1013. 6. Ito, S.; Nakagawa, Y.; Yazawa, S.; Sasaki, Y.; Yajima, S. Bioorg. Med. Chem. Lett. 2014, 24, 1812. 7. Nakaune, R.; Nakano, M. Fungal Genet. Biol. 2007, 44, 1324. 8. 1H NMR spectra were recorded with a JEOL superconducting magnet 400 MHz. 1 H NMR (CD3OD): 0.97 (9H, s), 1.65 (2H, t J = 7.16 Hz), 4.30 (2H, t J = 7.16 Hz), 6.79 (2H, s), 7.83 (2H, s). 9. Fukahori, M.; Akatsu, S.; Sato, H.; Yotsuyanagi, T. Chem. Pharm. Bull. 1996, 44, 1567; Ma, Y.; Marquis, R. E. Lett. Appl. Microbiol. 1996, 23, 329. 10. Oxygen consumption was measured at 37 °C in 2 mL air-saturated glycerol peptone medium (0.12% KH2PO4, 3% glycerol, 0.2% polypeptone, 0.02% MgSO4, 0.00012% CuSO4, 0.00021% FeCl3, 0.000102% NaMoO4, 0.000021% FeSO4, 0.000006% CoCl2, and 0.000006% CaCl2) supplemented with 200 lM parabens. The assay was started by addition of fungi (25 mg), and the decrease in oxygen was monitored. After 5 min, the inhibition rate of oxygen consumption was calculated by comparing the difference in oxygen consumption between the control and the paraben-treated fungi. 11. Takaya, N.; Kuwazaki, S.; Adachi, Y.; Suzuki, S.; Kikuchi, T.; Nakamura, H.; Shiro, Y.; Shoun, H. J. Biochem. 2003, 133, 461. 12. Lin, T. C.; Fan, M. C.; Wang, S. Y.; Huang, J. W. J. Agric. Food Chem. 2011, 59, 1667. 13. A. brassicicola was grown on PDA agar for 8 days. Sterile water was added to prepare spore suspensions (4 105 spores/mL). The surfaces of two-week-old cabbage leaves were sterilized with 70% (v/v) ethanol and then placed on sterile and moist filter papers in 10 cm dishes. Aliquots (10 lL) of spore suspensions of A. brassicicola were dropped onto the detached cabbage leaves, and 10 lL of alkyl parabens (1% DMSO) were added to it. Equal volumes of sterile distilled water (1% DMSO) and spore suspension were used as control. After three days, lesions were examined and genomic DNA was extracted. 14. Su’udi, M.; Park, J. M.; Park, S. R.; Hwang, D. J.; Bae, S. C.; Kim, S.; Ahn, I. P. Microbiology 1946, 2013, 159. 15. Genomic DNA was isolated using ISOPLANT (Wako Pure Chemical Industries Ltd, Japan). qRT-PCR was performed on a Takara Thermal Cycler Dice Real Time System using a SYBR premix and Ex Taq (Takara, Japan).
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Effects of alkyl parabens on plant pathogenic fungi Shinsaku Ito , Satoru Yazawa , Yasutaka Nakagawa, Yasuyuki Sasaki, Shunsuke Yajima ⇑ Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan
a r t i c l e
i n f o
Article history: Received 1 December 2014 Revised 7 February 2015 Accepted 20 February 2015 Available online 7 March 2015 Keywords: Structure–activity relationship study Drug design Paraben Gallate Mitochondrial complex II Black leaf spot disease
a b s t r a c t Alkyl parabens are used as antimicrobial preservatives in cosmetics, food, and pharmaceutical products. However, the mode of action of these chemicals has not been assessed thoroughly. In this study, we determined the effects of alkyl parabens on plant pathogenic fungi. All the fungi tested, were susceptible to parabens. The effect of linear alkyl parabens on plant pathogenic fungi was related to the length of the alkyl chain. In addition, the antifungal activity was correlated with the paraben-induced inhibition of oxygen consumption. The antifungal activity of linear alkyl parabens likely originates, at least in part, from their ability to inhibit the membrane respiratory chain, especially mitochondrial complex II. Additionally, we determined that some alkyl parabens inhibit Alternaria brassicicola infection of cabbage. Ó 2015 Elsevier Ltd. All rights reserved.
Alkyl parabens (p-hydroxybenzoic acid alkyl esters) are common preservatives used in cosmetics due to their properties such as, broad antimicrobial spectrum, relatively low toxicity, good chemical stability, and non-volatility although they have been reported to have potential health risks for humans.1 Methyl and propyl parabens are mostly used as cosmetic preservatives, and as the number of carbon in the paraben alkyl chain increases, it enhances their antimicrobial activity.2 Sapra et al. recently tested the antimicrobial potential of alkyl parabens on five microorganisms.3 They reported that decyl paraben was effective against Staphylococcus aureus, Candida albicans, and Aspergillus niger, while propyl and butyl parabens showed the highest antimicrobial activity against Bacillus subtilis and Escherichia coli, respectively.3 Thus, the effect of alkyl parabens vary according to the microorganism treated with it. Short-chain alkyl parabens (ethyl and butyl) were reported to disrupt the function of mechanosensitive channels and induce potassium efflux in E. coli; however, the underlying mechanism of the antifungal activity of alkyl parabens is poorly understood.4a,b,5 In our previous study, we evaluated the antifungal properties of alkyl gallates and their mode of action.6 The length of the alkyl chain was linked to their antifungal activity, and nonyl gallate was the most potent antifungal agent. In addition, linear alkyl gallates inhibited mitochondrial complex II activity depending on their chain length, which was consistent with their antifungal
activity. Because of a structural similarity between gallates and parabens (Fig. 1), we hypothesized that alkyl parabens may also inhibit the activity of mitochondrial complex II. Therefore, we synthesized alkyl parabens and their derivatives, investigated their antifungal properties and mode of action against plant pathogenic fungi, such as, Fusarium solani, Colletotrichum acutatum, Colletotrichum dematium, and Alternaria brassicicola. These fungi are known to cause serious damage to economically important crops and fruits across the world. The chemicals were prepared as reported in earlier studies, with slight modifications.6 Substituted benzoic acids and alkanols dissolved in tetrahydrofuran were stirred at room temperature in the presence of N,N0 -dicyclohexylcarbodiimide. The reaction mixture was quenched with distilled water, and the aqueous layer was extracted with ethyl acetate. The organic layer was dried and concentrated under reduced pressure. The resulting oil was purified by a column chromatography. The structures of the alkyl parabens and their derivatives have been shown in Figure 2. Log P values were calculated by ChemBioDraw Ultra software (MA, USA). C. acutatum (CAB03) was received from Dr. Nakaune.7 A. brassicicola (MAFF No. 726527, 726705, 237450 and 305011)
⇑ Corresponding author. Tel.: +81 3 5477 2768.
E-mail address: [email protected] (S. Yajima). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.02.049 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Figure 1. Chemical structures of alkyl gallate (left) and alkyl paraben (right).
S. Ito et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1774–1777
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Figure 2. Chemical structures and log P values of parabens.
was purchased from NIAS Genebank (Tsukuba city, Japan). The other fungi were received from Prof. Natsuaki (Tokyo Univ. of Agric.). We estimated the role of alkyl parabens against plant pathogenic fungi (Tables 1 and 2). Antifungal activities were determined by measuring the minimum inhibitory concentration (MIC).6 Methyl, ethyl, propyl, butyl, and pentyl parabens were not effective against all the fungal strains tested in this study. As for gallates,6 an increase in the chain length of linear alkyl parabens led to enhanced antifungal activity. Among the linear alkyl parabens tested (1–7), nonyl paraben (7), which was less active than nonyl gallate (11), had the highest antifungal activity. Consistent with the antifungal activity of alkyl gallates,6 branched alkyl parabens showed stronger activity than linear alkyl parabens with similar
Table 1 MIC values (lM) of alkyl parabens and derivatives against plant pathogenic fungi
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Benomyl Boscalid
F. solani
C. dematium
C. acutatum
C. acutatum (CAB03)
>50 >50 >50 >50 >50 50 50 25 >50 25 6.25 12.5 >50 25 50 3.13* 3.13
>50 >50 >50 >50 >50 50 50 50 >50 25 12.5 25 >50 6.25 50 25* nd
>50 >50 >50 >50 >50 50 25 25 50 25 6.25 12.5 >50 25 >50 12.5* nd
>50 >50 >50 >50 >50 >50 50 25 50 25 12.5 12.5 >50 25 >50 50 nd
nd: not determined. * The values of the previously described.6
log P values. Compounds 10 and 12, which have 4-hydroxyl group, showed the increased antifungal activities in comparison with the compounds 13 and 15, which do not have 4-hydroxyl group, respectively. This result suggests that the introduction of 4-hydroxyl group is important for these derivatives to show the antifungal activities. In addition, all A. brassicicola strains were more sensitive to branched alkyl parabens than the other fungal strains tested (Table 2). Notably, 3,3-dimethylbutyl paraben (10) had the highest antifungal activity against A. brassicicola.8 Interestingly, only monohydroxy benzoates (10 and 14) showed higher antifungal activity against A. brassicicola than against the other fungi. In addition, some alkyl parabens and gallates (8, 10, 11, 12 and 14) were effective against benomyl-resistant C. acutatum (CAB03). Though benomyl is a fungicide used worldwide, the rapid development of resistant strains has limited its use. These results suggest the possibility of using alkyl parabens as antifungal agents. It has been proposed that the mode of antimicrobial action of alkyl parabens depends on their ability to disrupt the native membrane due to their hydrophobicity.9 Though mechanosensitive channels are one of the targets of short-chain alkyl parabens (methyl to butyl), the target of longer-chain alkyl parabens has not been investigated. As previously described, the structurally similar linear alkyl gallates inhibit the mitochondrial complex II activity. In addition, such inhibitory activity against complex II was correlated with the antifungal property of linear alkyl gallates.6 These findings helped to determine the effects of alkyl parabens on oxygen consumption and activity of complex II in fungi.10,11 The inhibitory action on oxygen consumption was enhanced by increasing the chain length of linear alkyl parabens in A. brassicicola and F. solani (Fig. 3A and B). Especially, nonyl paraben (7) showed the highest activity consistent with the antifungal activities of linear alkyl parabens. Branched alkyl paraben (10) also inhibited the oxygen consumption, although its inhibition was slightly weaker than that of the nonyl parabens (7). In addition, nonyl and 3,3-dimethylbutyl parabens (7 and 10, respectively)
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S. Ito et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1774–1777 Table 2 MIC values (lM) of alkyl parabens and derivatives against A. brassicicola
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Benomyl
A. brassicicola
A. brassicicola (726527)
A. brassicicola (726705)
A. brassicicola (237450)
A. brassicicola (305011)
>50 >50 >50 >50 >50 25 25 12.5 25 6.25 12.5 12.5 >50 6.25 50 6.25*
>50 >50 >50 >50 >50 50 25 12.5 50 12.5 12.5 25 >50 12.5 25 nd
>50 >50 >50 >50 50 50 25 25 12.5 6.25 6.25 12.5 >50 12.5 50 nd
>50 >50 >50 >50 50 25 25 50 50 12.5 6.25 25 >50 25 25 nd
>50 >50 >50 >50 50 50 50 25 12.5 12.5 12.5 25 >50 12.5 50 nd
nd: not determined. * The values of the previously described.6
Figure 4. Inhibition of mitochondrial complex II by alkyl parabens. Values in parentheses indicate the concentration of the chemicals (lM). The data are means ± SD of three samples. ⁄⁄ indicates significant differences from control (DMSO treatment) (t-test, P <0.01).
Figure 3. Inhibition of oxygen consumption by alkyl parabens. Oxygen consumption in (A) A. brassicicola and (B) F. solani was determined using an oxygen electrode. Alkyl parabens were added at a concentration of 200 lM. The data are means ± SD of three samples. ⁄ and ⁄⁄ indicate significant differences from control (DMSO treatment) (t-test, P <0.05 and P <0.01, respectively).
inhibited the activity of complex II in F. solani (Fig. 4). Furthermore, boscalid, which is a mitochondrial complex II inhibitor, showed about 10-fold higher in the antifungal activity and complex II inhibition than compound 7 (Table 1, Fig. 4). These results strongly indicate that the antifungal activity of linear alkyl parabens against plant pathogenic fungi is due to the inhibition of complex II.
However, there may be other mechanisms of antifungal action for branched alkyl paraben, because the inhibitory activity of 3,3-dimethylbutyl paraben (10) against oxygen consumption was less effective than its antifungal activity. A. brassicicola is a necrotrophic fungal pathogen that causes black leaf spot disease in many cruciferous crops including cabbage (Brassica oleracea L.). Branched alkyl parabens were more effective against A. brassicicola than against other plant pathogenic fungi. Thus, we estimated the effect of alkyl parabens on A. brassicicola infection of detached cabbage leaves. The infection test was performed according to a previously described method, with slight modifications.12,13 The results showed that paraben-treated plants were more resistant to A. brassicicola than untreated plants (Fig. 5A). Quantification of infection levels of A. brassicicola in the plants was carried out by measuring the infective gene copy numbers in the fungal genome by real-time PCR.14,15 Alkyl paraben treatment reduced fungal genomic DNA levels in a dose-dependent manner (Fig. 5B). These results indicate that alkyl parabens have the potential to reduce crop losses due to A. brassicicola infection. In this Letter, we demonstrated that alkyl parabens showed the antifungal activity against plant fungal pathogens. In particular, branched alkyl parabens (8–10) were more potent in inhibiting fungal growth than the linear alkyl parabens with the similar log P values. In addition, linear alkyl parabens inhibited oxygen consumption, which was linked to the increase in the chain length
S. Ito et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1774–1777
1777
although the specific mode of action was poorly understood. Our findings provide a new perspective for the antifungal properties of alkyl parabens against plant fungal pathogens, via the inhibition of oxygen consumption, especially in mitochondrial complex II. Acknowledgment We thank Dr. Ryoji Nakaune (National Agricultural and Food Research Organization, Japan) for kindly providing C. acutatum (CAB03). References and notes
Figure 5. Effect of alkyl parabens on infection of cabbage with A. brassicicola. (A) The appearance of cabbage leaf after three days of fungal inoculation. (B) Fungal DNA was quantified using AbSCD1 gene specific primer.12 AbSCD1 forward: 50 -GCA GAC AGC TAC GAT AGC AA-30 , AbSCD1 reverse: 50 -GAT GCA TTT GCG GAG AC-30 . Genomic DNA was quantified using Beckman DU530 UV/Vis Spectrophotometer (CA, USA). ⁄⁄ indicates significant differences from control (DMSO treatment) (t-test, P <0.01). Values in parentheses indicate the concentration of the chemicals (lM).
of alkyl paraben. Moreover, these parabens inhibited A. brassicicola infection of plants. Parabens had been used as preservatives in food and cosmetics because of their broad antimicrobial spectrum,
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