Natural and Natural-like Phenolic Inhibitors of Type B Trichothecene

Article pubs.acs.org/JAFC

Natural and Natural-like Phenolic Inhibitors of Type B Trichothecene in Vitro Production by the Wheat (Triticum sp.) Pathogen Fusarium culmorum Giovanna Pani,†,‡ Barbara Scherm,† Emanuela Azara,‡ Virgilio Balmas,† Zahra Jahanshiri,†,§ Paola Carta,†,‡ Davide Fabbri,‡ Maria Antonietta Dettori,‡ Angela Fadda,∥ Alessandro Dessì,‡ Roberto Dallocchio,‡ Quirico Migheli,† and Giovanna Delogu*,‡ †

Dipartimento di Agraria, Sezione di Patologia Vegetale ed Entomologia and Unità di Ricerca Istituto Nazionale di Biostrutture e Biosistemi, Università degli Studi di Sassari, Viale Italia 39, I-07100 Sassari, Italy ‡ Istituto CNR di Chimica Biomolecolare, and ∥Istituto CNR di Scienze delle Produzioni Alimentari, UOS Sassari, Traversa La Crucca 3, I-07100 Sassari, Italy S Supporting Information *

ABSTRACT: Fusarium culmorum, a fungal pathogen of small grain cereals, produces 4-deoxynivalenol and its acetylated derivatives that may cause toxicoses on humans or animals consuming contaminated food or feed. Natural and natural-like compounds belonging to phenol and hydroxylated biphenyl structural classes were tested in vitro to determine their activity on vegetative growth and trichothecene biosynthesis by F. culmorum. Most of the compounds tested at 1.5 or 1.0 mM reduced 3acetyl-4-deoxynivalenol production by over 70% compared to the control, without affecting fungal growth significantly. Furthermore, several compounds retained their ability to inhibit toxin in vitro production at the lowest concentrations of 0.5 and 0.25 mM. Magnolol 27 showed fungicidal activity even at 0.1 mM. No linear correlation was observed between antioxidant properties of the compounds and their ability to inhibit fungal growth and mycotoxigenic capacity. A guaiacyl unit in the structure may play a key role in trichothecene inhibition. KEYWORDS: fungicide, deoxynivalenol, phenolic inhibitors, antioxidant activity, Fusarium head blight

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INTRODUCTION Fusarium culmorum (W.G. Smith) Sacc. is a soil-borne filamentous fungus that causes foot and root rot (FRR) and Fusarium head blight (FHB) diseases on small grain cereals, in particular, wheat and barley.1 This fungus is also reported as a post-harvest pathogen, especially on freshly harvested grain that has not been properly dried or stored.2,3 F. culmorum produces type B trichothecenes, namely, 4-deoxynivalenol and its acetylated forms 3-acetyl-4-deoxynivalenol and 15-acetyl-4deoxynivalenol, as well as nivalenol and its acetylated form 4acetylnivalenol or fusarenone X. These compounds may contaminate food and feed at high concentrations and cause serious poisoning in humans and animals.4−6 Moreover, trichothecenes play an important role as virulence factors by inhibiting defense mechanisms activated by the plant.1 Efficient control of disease and trichothecene contamination caused by F. culmorum is difficult and requires the application of an integrated management approach, including crop rotation, use of tolerant cultivars, plowing of crop residues, reduced use of nitrogen fertilizers, and seed dressing or spraying with fungicides. A wide array of fungicides, mainly belonging to the azole and strobin classes, can be used efficiently to control the pathogen in the field and to reduce grain contamination, especially at low disease pressure or on wheat genotypes that present a moderate level of resistance.7−10 However, an increase in mycotoxin contamination may occur when an inappropriate dosage of © 2014 American Chemical Society

fungicides is applied or when fungicide activity against different Fusarium pathogens is variable.8,11 Moreover, repeated use of fungicides sharing the same molecular target, such as in the case of sterol biosynthesis inhibitors, may induce a selective pressure on fungal populations, hence favoring the onset of resistant mutants. Therefore, there is an urgent need to identify new classes of compounds capable of limiting the pathogenic and mycotoxigenic potential of Fusarium spp. or enhancing natural resistance mechanisms of the host plant. For example, antioxidants play an important role in the natural defense response of the plant to the oxidative stress brought about by fungal invasion.12 The rich structural diversity of natural products endowed with multiple biological activities is of great interest in the search of agrochemical leads to be applied in the development of new selective and environmentally friendly mycotoxin inhibitors. Specific and strong inhibitory activities were demonstrated by phenolic and polyphenolic natural compounds against trichothecene-producing strains of Fusarium graminearum Schwabe and F. culmorum.13,14 There is a growing interest to exploring phenols as potential antimicrobial and fungicidal agents, where ketone, aldehyde, acid groups, or Received: Revised: Accepted: Published: 4969

January 15, 2014 May 9, 2014 May 12, 2014 May 12, 2014 dx.doi.org/10.1021/jf500647h | J. Agric. Food Chem. 2014, 62, 4969−4978

Journal of Agricultural and Food Chemistry

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Figure 1. Chemical structures of studied compounds.

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unsaturated chain are embedded in the aromatic ring.15 Most of them are plant metabolites, generally present in food, spices, or food preservative or belonging to the generally recognized as safe (GRAS) list.16 The most abundant phenols extracted from maize grain pericarp and wheat bran are trans-ferulic acid and the corresponding dehydrodimers, namely, dehydrodiferulates.13,14,17 Hydroxycinnamic acids are known to be major components of the primary cell wall of cereals.13 The predominant feruloyl residues can also be dimerized under oxidative coupling mediated by peroxidases or form cross-links and/or dehydrodimers of ferulic acid that then lead to a reinforcement of the primary cell wall of the plant. Fungal esterases, which are overexpressed during growth on the host tissues, can release free forms of ferulic ester from cell wall tissues.18 Once released, free ferulate may inhibit the ability of Fusarium to produce mycotoxins. The objective of this study was to evaluate the inhibitory effect of a wide collection of natural and natural-like phenols and dimers on vegetative growth and mycotoxin in vitro production by a well-characterized 3-acetyl-4-deoxynivalenol producing strain of F. culmorum. We selected a series of compounds belonging to cinnamic acids, acetophenones, benzaldehydes, benzoic acids, phenylpropanoids, and hydroxylated biphenyls and compared their structural properties to the in vitro activity. A correlation between the inhibitory effect of the selected phenols and their lipophilicity, polarity, and antioxidant activity was also attempted.

MATERIALS AND METHODS

Fungal Strain and Culture Conditions. The highly virulent and well-characterized1 strain ISPaVe MCf21 (synthetic strain INRA 117) of F. culmorum (W.G. Smith) Sacc., isolated in 1989 from triticale grown in Foggia, Apulia, southern Italy, was used in all of the experiments. Under in vitro conditions, F. culmorum MCf21 mainly produces 3-acetyl-4-deoxynivalenol (3-ADON) and, to a lesser extent, deoxynivalenol.14,19 Strain cultures were set up on potato dextrose agar (PDA, at 25 °C in the dark). Short-term storage of PDA cultures was at 4 °C, while for long-term preservation, plugs of PDA cultures were stored in 50% aqueous glycerol at −80 °C. For subsequent experiments, F. culmorum MCf 21 was grown in V8 vegetable juice for 7 days at 25 °C and 170 rpm. Cultures were filtered, and spores were collected by centrifugation, adjusted to 1 × 106 colony-forming units (CFU)/mL in sterile water, and used as inoculum for Vogel’s liquid culture medium.20 All liquid and solid media were amended with streptomycin (50 μg/ mL) and tetracycline (50 μg/mL), respectively, to prevent bacterial growth. Tested Compounds (Figure 1 and Table 1). 2,5-Dimethoxycinnamic acid (1), 4-hydroxycinnamic acid (2), ferulic acid (3), caffeic acid (4), 3-hydroxycinnamic acid (5), apocynin (6), 4-bromoacetophenone (7), 3,4-dimethoxyacetophenone (8), 2-hydroxy-5-methoxyacetophenone (9), 2,4-dihydroxybenzaldehyde (10), veratraldehyde (11), vanillin (12), gallic acid (13), octyl gallate (14), propyl gallate (15), 3-methoxybenzoic acid (16), 3,4-dimethoxybenzoic acid (17), dodecyl gallate (18), trans-anethole (19), eugenol (20), isoeugenol (21), dehydrozingerone (22), Me-zingerone (23), Me-dehydrozingerone (24), zingerone (25), carvacrol (26), magnolol (27), dehydrodieugenol (eugenol dimer) (28), dehydrodiferulic acid (5,5′diferulic acid dimer) (29), 4,4′-dihydroxybiphenyl (30), and ellagic acid (31) were selected and used with a purity >98%. Dehydrozingerone (22), Me-zingerone (23), Me-dehydrozingerone (24), dehydrodieugenol (28), and dehydrodiferulic acid (29) were prepared 4970

dx.doi.org/10.1021/jf500647h | J. Agric. Food Chem. 2014, 62, 4969−4978


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Table 1. Compounds Listed According to Lipophilicity and Their Value of Log P, Radical Scavenging Activity, and Antioxidant Activity list of compounds according to the elution order by HPLC in reverse phase

log Pa

radical scavenging activity DPPH (TEb), EtOH abs, time = 30 min

ABTS (TEACc), EtOH abs, time = 6 min

gallic acid (13) caffeic acid (4) vanillin (12) 2,4-dihydroxybenzaldehyde (10) apocynin (6) 4-hydroxycinnamic acid (p-cumaric acid) (2) ellagic acid (31) 3,4-dimethoxybenzoic acid (17) ferulic acid (3) 3-hydroxcinnamic acid (5) 3-methoxybenzoic acid (16) veratraldehyde (11) 3,4-dimethoxyacetophenone (8) zingerone (25) propyl gallate (15) dehydrozingerone (22) 4,4′-dihydroxybiphenyl (30) Me-zingerone (23) 2,5-dimethoxycinnamic acid (1) 2-hydroxy-5-methoxyacetophenone (9) Me-dehydrozingerone (24) ferulic acid dimer (29) eugenol (20) 4-bromoacetophenone (7) isoeugenol (21) carvacrol (26) octyl gallate (14) trans-anethole (19) magnolol (27) eugenol dimer (28) dodecyl gallate (18)

0.47 1.15 1.26 1.00 0.83 1.54 1.05 1.34 1.42 1.54 1.46 1.53 1.09 1.95 1.51 1.27 2.93 2.21 1.70 0.83 1.53 2.47 2.57 2.51 2.52 3.37 3.60 2.91 5.03 4.78 5.27

3.20 ± 0.13 1.53 ± 0.04 0.029 ± 0.007 not determined 0.019 ± 0.002 no activity 2.62 ± 0.02 not determined 0.64 ± 0.04 not determined not determined 0.023 ± 0.03 not determined 0.38 ± 0.02 5.20 ± 0.08 0.60 ± 0.02 not determined no activity not determined not determined no activity 0.85 ± 0.02 028 ± 0.004 no activity 0.58 ± 0.005 0.03 ± 0.006 3.10 ± 0.048 not determined 0.023 ± 0.008 0.83 ± 0.02 2.99 ± 0.13

2.76 ± 0.07 1.25 ± 0.08 0.67 ± 0.12 not determined 0.41 ± 0.01 1.08 ± 0.04 3.05 ± 0.44 not determined 1.45 ± 0.18 not determined not determined 0.05 ± 0.02 not determined 1.34 ± 0.08 3.32 ± 0.10 1.10 ± 0.02 not determined no activity not determined not determined no activity 1.73 ± 0.03 0.32 ± 0.01 no activity 0.72 ± 0.03 0.29 ± 0.01 3.00 ± 0.13 not determined 0.55 ± 0.06 1.89 ± 0.02 2.90 ± 0.10

a

Log P = lipophilicity estimated by theoretical calculations, which express the partitioning of the phenols in a n-octanol/water system. bTE, Trolox equivalent; DPPH, 1,1′-diphenyl-2-picrylhydrazyl. cTEAC, Trolox equivalent antioxidant capacity; ABTS, 2,2-azinobis(3-ethylbenzothiazoline-6sulfonic acid).

according to known procedures.21−23 The other compounds were purchased from Sigma-Aldrich (Milan, Italy) and used as received. βCyclodextrin (β-CD, CAVAMAX W7 Pharma) was purchased from Wacker Chemie Italia (Peschiera Borromeo, Italy). Amendment with Phenolic Inhibitors and Extraction of Type B Trichothecenes. Petri plates (60 mm diameter) containing 8 mL of Vogel’s medium were amended with the compounds at different concentrations (ranging from 0.1 to 1.5 mM). β-CD (3.0 mM) was added to the Vogel’s medium to improve solubilization of phenolic compounds. The procedure was carried out in a way to exclude any stable inclusion of the compound into the βCD cavity. Each sample vial was sonicated at room temperature for 60 min and 60 Hz (Branson model 3510 OPTO-LAB). Sonicated media were inseminated with F. culmorum MCf 21 spore suspension to achieve a final concentration of 1 × 104 conidia/mL. Each compound was tested in triplicate, and cultures were incubated in the dark at 25 °C, without shaking. After 14 days of incubation, media and mycelia were collected for thin-layer chromatography (TLC) and liquid chromatography− mass spectrometry (LC−MS) analyses. Mycelia were separated from the medium by filtration through sterile filter paper, subsequently harvested from the filter papers with a sterile spatula, dried at 80 °C for 24 h, and weighed. Trichothecenes were extracted from 4 mL of harvested medium by adding 4 mL of ethyl acetate, followed by a shaking step at 150 rpm for 16−18 h at room temperature, and finally

by recovering the organic phase. Each experiment was repeated at least 2 times. LC−MS Analysis of Type B Trichothecenes. Quantitation was performed with reference to external calibration using type B trichothecene standards (Sigma-Aldrich, Milan, Italy). Quantitative determinations were carried out as described previously,19 using a model HP 1100 liquid chromatography and mass spectrophotometric detector (Agilent Technologies, Palo Alto, CA). Estimation of Lipophilicity and Polarity. Retention of a compound in reversed-phase liquid chromatography is governed by its lipophilicity and shows significant correlation with the n-octanol/ water partition coefficient.24 Lipophilicity of all phenols diluted in pure methanol was estimated by high-performance liquid chromatography (HPLC) on HP 1100 Agilent Technologies, Palo Alto, CA, using a 15 × 4.6 mm inner diameter, 3 μm, Luna RP-8 (Phenomenex, Torrance, CA), at temperature of 25 °C and with a flow of 0.4 mL/min, as reported in the literature.25 Polarity of the compounds was estimated by TLC. Radical Scavenging and Antioxidant Activity of Phenols. For each compound, a 5 mM stock solution was prepared in absolute ethanol. The radical scavenging activity of the compounds was determined in vitro [2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)] by an electron paramagnetic resonance (EPR) spectrometer and ultraviolet/visible (UV/vis). 4971



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Table 2. In Vitro Effect of Different Phenolic Compounds (1.5 mM) on Vegetative Growth and 3-ADON Production by F. culmorum Strain ISPaVe MCf 21 (INRA 117) treatment 2,5-dimethoxycinnamic acid (1) apocynin (6) 4′-bromoacetophenone (7) veratraldehyde (11) 2,4-dihydroxybenzaldehyde (10) gallic acid (13) magnolol (27) trans-anethole (19) eugenol (20) isoeugenol (21) dehydrozingerone (22) Me-zingerone (23)

dry fungal biomass (relative yield ± SE)a

3-ADON (relative yield ± SE)b

Cinnamic Acids 59 ± 4c Acetophenones 70 ± 5 67 ± 5 Benzaldehydes 88 ± 7 25 ± 4c Benzoic Acids 78 ± 15 Hydroxylated Biphenyls 0c Phenylpropanoids 81 ± 3 47 ± 3c 47 ± 1c 54 ± 1c 54 ± 4c

1c 1c 0c 20 ± 6c 20 ± 10d ≤LODc,e 0c 1 ± 1c 0c ≤LODc,e 0c 0c

a Dry fungal biomass values are expressed as mean percent values (±standard error) relative to their level in the control culture (31.2 ± 2.4 mg). b3ADON yields are expressed as mean percent values (±standard error) relative to their level in the control culture (243 348 ± 42 326 ng/g of dry biomass). cValues are significantly different (p < 0.01) from the untreated control based on ANOVA followed by the Dunnett test. dValues are significantly different (p < 0.05) from the untreated control based on ANOVA followed by the Dunnett test. eBelow the limit of detection.

Table 3. In Vitro Effect of Different Phenolic Compounds (1 mM) on Vegetative Growth and 3-ADON Production by F. culmorum Strain ISPaVe MCf 21 (INRA 117) treatment 4-hydroxycinnamic acid (2) ferulic acid (3) caffeic acid (4) 3-hydroxycinnamic acid (5) apocynin (6) 4′-bromoacetophenone (7) 3,4-dimethoxyacetophenone (8) 2-hydroxy-5-methoxyacetophenone (9) 2,4-dihydroxybenzaldehyde (10) vanillin (12) octyl gallate (14) propyl gallate (15) magnolol (27) eugenol dimer (28) 4,4′-dihydroxybiphenyl (30) ellagic acid (31) eugenol (20) isoeugenol (21) dehydrozingerone (22) Me-zingerone (23) Me-dehydrozingerone (24) carvacrol (26)

dry fungal biomass (relative yield ± SE)a Cinnamic Acids 103 ± 15 111 ± 28 75 ± 1 41 ± 10c Acetophenones 66 ± 9 67 ± 2 67 ± 2 48 ± 3d Benzaldehydes 106 ± 40 77 ± 2 Benzoic Acids 0c 73 ± 1 Hydroxylated Biphenyls 9 ± 1c 101 ± 5 59 ± 0 79 ± 6 Phenylpropanoids 119 ± 22 135 ± 58 68 ± 2 134 ± 60 8 ± 2c 87 ± 26

3-ADON (relative yield ± SE)b 16 20 3 3

± ± ± ±

2c 10c 1c 1c

≤LODc,e 1c 84 ± 20 11 ± 5c 80 ± 32 30 ± 7d 0c 12 ± 4c 2 ± 1c 224 ± 41d 79 ± 9 1c ≤LODc,e 3 ± 1c 16 ± 2c 3 ± 1c 0c 1c

a Dry fungal biomass values are expressed as mean percent values (±standard error) relative to their level in the control culture (28.7 ± 2 mg). b3ADON yields are expressed as mean percent values (±standard error) relative to their level in the control culture (182 982 ± 40 725 ng/g of dry biomass). cValues are significantly different (p < 0.01) from the untreated control based on ANOVA followed by the Dunnett test. dValues are significantly different (p < 0.05) from the untreated control based on ANOVA followed by the Dunnett test. eBelow the limit of detection.

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Table 4. In Vitro Effect of Different Phenolic Compounds (0.5 mM) on Vegetative Growth and 3-ADON Production by F. culmorum Strain ISPaVe MCf 21 (INRA 117) treatment ferulic acid (3) apocynin (6) octyl gallate (14) propyl gallate (15) 3-methoxybenzoic acid (16) 3,4-dimethoxybenzoic acid (17) dodecyl gallate (18) magnolol (27) eugenol dimer (28) ferulic acid dimer (29) ellagic acid (31) trans-anethole (19) dehydrozingerone (22) Me-zingerone (23) Me-dehydrozingerone (24) zingerone (25)



Cinnamic Acids 84 ± 1 Acetophenones 85 ± 5 Benzoic Acids 79 ± 1 82 ± 3 92 ± 3 85 ± 5 115 ± 1d Hydroxylated Biphenyls 15 ± 12c 97 ± 4 67 ± 16 84 ± 3 Phenylpropanoids 101 ± 3 72 ± 2 86 ± 4 72 ± 1 83 ± 6

45 ± 6c 17 ± 9c 106 17 129 107 91

± ± ± ± ±

41 5c 10 11 41

0c 451 ± 22c 68 ± 44 101 ± 19 57 12 35 22 33

± ± ± ± ±

8d 1c 14d 11c 10c

a Dry fungal biomass values are expressed as mean percent values (±standard error) relative to their level in the control culture (26.3 ± 1.3 mg). b3ADON yields are expressed as mean percent values (±standard error) relative to their level in the control culture (242 692 ± 41 029 ng/g of dry biomass). cValues are significantly different (p < 0.01) from the untreated control based on ANOVA followed by the Dunnett test. dValues are significantly different (p < 0.05) from the untreated control based on ANOVA followed by the Dunnett test.

Radical scavenging activity was determined by the DPPH method,26 with few modifications. Briefly, 100 μL of a solution of 1.0 mM DPPH was mixed with 1900 μL of the appropriately diluted sample solution. The mixture was incubated in the dark for 30 min and subsequently analyzed by an EPR spectrophotometer. The antioxidant activity determination was carried out as reported previously.27 Statistical Analysis. Data on mycelium growth (milligrams of dry weight per Petri plate) and 3-acetyl-4-deoxynivalenol production (ng/ mL) obtained from separate experiments were expressed as percent values of the relative control treatment and pooled to perform a oneway analysis of variance (ANOVA), followed by multiple comparisons by Dunnett test, using Minitab for Windows, release 16.1.

Table 5. In Vitro Effect of Different Phenolic Compounds (0.25 mM) on Vegetative Growth and 3-ADON Production by F. culmorum Strain ISPaVe MCf 21 (INRA 117) treatment dodecyl gallate (18) Me-dehydrozingerone (24) carvacrol (26) magnolol (27) ferulic acid dimer (29)

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78 ± 0 94 ± 5

11 ± 1c 344 ± 71c

99 ± 32 21 ± 2d 73 ± 6

11 ± 3c 0c 33 ± 8d

a

Dry fungal biomass values are expressed as mean percent values (±standard error) relative to their level in the control culture (25.3 ± 2.4 mg). b3-ADON yields are expressed as mean percent values (±standard error) relative to their level in the control culture (202 873 ± 66 153 ng/g of dry biomass). cValues are significantly different (p < 0.01) from the untreated control based on ANOVA followed by the Dunnett test. dValues are significantly different (p < 0.05) from the untreated control based on ANOVA followed by the Dunnett test.

RESULTS AND DISCUSSION Screening of Type B Trichothecene Inhibitors. In a first screening, performed at the concentration of 1.5 mM, representatives of each class of compounds were selected according to their functional and structural features (Table 2). The results obtained from the initial screening were used to design further assays at the lower 1.0 mM concentration (Table 3). When their concentration in the medium was reduced, it was possible to test other phenolic compounds that were not soluble at higher concentrations. On the basis of the results achieved at 1.0 mM, a limited set of compounds were further assayed at the concentration of 0.5 mM (Table 4) and 0.25 mM (Table 5), respectively. The strategy allowed us to investigate the efficacy of compounds that are poorly soluble in water and may inhibit mycotoxin in vitro production at low concentrations without affecting fungal growth. Cinnamic Acids. Vegetative growth was reduced to 59% compared to the untreated control, and 3-acetyl-4-deoxynivalenol production was almost completely inhibited when F. culmorum was grown in the presence of 1.5 mM 2,5dimethoxycinnamic acid (1) (Table 2). At the concentration

of 1.0 mM, the in vitro production of 3-acetyl-4-deoxynivalenol was reduced to 16−20% without affecting fungal growth by 4hydroxycinnamic acid (2) and ferulic acid (3) (Table 3), with the latter maintaining a significant inhibitory activity also at 0.5 mM (Table 4), as reported previously.14 When tested at 1.0 mM, caffeic acid (4) caused almost complete 3-acetyl-4deoxynivalenol inhibition but did not significantly affect mycelial growth. On the contrary, 3-hydroxycinnamic acid (5) reduced fungal growth to 41% and 3-acetyl-4-deoxynivalenol production to 3% with respect to the untreated control (Table 3). Acetophenones. Fungal growth was slightly, albeit not significantly, reduced to 67−70% compared to the untreated control upon treatment with apocynin (6) or 4-bromo4973



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Almost no fungal growth and complete inhibition of trichothecene production were observed when Me-dehydrozingerone (24) was added at 1.0 mM to liquid culture (Table 3). When its concentration in the medium was reduced to 0.5 mM, Me-dehydrozingerone (24) had no significant effect on fungal growth, whereas 3-acetyl-4-deoxynivalenol was reduced to 22% of the untreated control (Table 4). When tested at 0.25 mM, this compound behaved as a strong inducer of 3-acetyl-4deoxynivalenol in vitro production (Table 5). Only 33% 3-acetyl-4-deoxynivalenol production was detected with zingerone (25) tested at 0.5 mM without significantly affecting fungal growth (Table 4). Almost complete 3-acetyl-4-deoxynivalenol inhibition, with no significant inhibition of fungal growth, was observed when carvacrol (26), an alkyl phenol, was added at 1.0 mM in the liquid medium (Table 3). At 0.25 mM, carvacrol (26) reduced 3-acetyl-4-deoxynivalenol production to 11% without affecting vegetative growth (Table 5). In the present work, the trichothecene inhibitory activity of carvacrol (26) was not concentration-dependent. Hydroxylated Biphenyls. Magnolol (27), a symmetrical hydroxylated biphenyl with two free phenolic −OH groups and two side chains para to each other, displayed a marked fungicide activity when tested at 1.5 mM (Table 2). Despite not being perceivable, the progressive decline in its inhibitory effect on fungal growth as the dosage decreased to 1.0, 0.5, and 0.25 mM, this compound retained the capacity to inhibit 3-acetyl-4deoxynivalenol production by the fungus even at the lowest concentration (Tables 3 and 5). Dehydrodieugenol (28), structurally equivalent to magnolol (27) but having a methoxyl group ortho to each phenolic −OH group, strongly induced 3acetyl-4-deoxynivalenol overproduction, with no reduction of fungal growth at both 1.0 and 0.5 mM (Tables 3 and 5). No significant inhibitory activity on vegetative growth or trichothecene in vitro production were observed upon amendment of the substrate with ferulic acid dimer 29 at 0.5 mM, whereas at 0.25 mM, a significant reduction of 3-acetyl-4deoxynivalenol production was detected (Tables 4 and 5). This effect might be due to a different pH of the environment, which would influence the interactions between inhibitor and enzyme. Amendment with 4,4′-dihydroxylbiphenyl (30) produced a 40% reduction of the fungal growth, with no significant effect on 3-acetyl-4-deoxynivalenol production when tested at 1.0 mM (Table 3). Almost complete 3-acetyl-4-deoxynivalenol inhibition with no significant reduction of vegetative growth was observed with ellagic acid (31) tested at 1.0 mM (Table 3), while no significant effect was detected when the substrate was amended with 0.5 mM ellagic acid (31) (Table 4). Antioxidant Activity and Lipophilicity. The cinnamic acids (2−4), which display poor lipophilic properties, presented radical scavenging and antioxidant activity comparable to that of Trolox (Table 1). No radical scavenging activity nor antioxidant activity were detected for acetophenones and aldehydes, whose lipophilicity values, estimated by a computational program (log P) and HPLC, differ significantly only for 4-Br-acetophenone (7) (Table 1). Lipophilicity values of all benzoic acids analyzed are welldistributed in the list of compounds, ranging between 0.47 and 5.27 log P according to the trend observed by reverse-phase (RP)-HPLC (13 < 17 < 16 < 15 < 14 < 18). High radical scavenging and antioxidant activities were detected for gallic acid (13) and its esters, compounds 18, 14, and 15, with the

acetophenone (7), resulting in complete inhibition of 3-acetyl4-deoxynivalenol in vitro production when tested at 1.5 and at 1.0 mM (Tables 2 and 3), respectively. Apocynin (6) maintained its ability to significantly reduce 3-acetyl-4deoxynivalenol at the lower concentration of 0.5 mM without affecting fungal growth (Table 4). When tested at 1.0 mM compounds, 3,4-dimethoxyacetophenone (8) and 2-hydroxy-5methoxyacetophenone (9) caused 67 and 48% vegetative growth, respectively, whereas only acetophenone (9) induced a significant inhibition (11% compared to the untreated control) of 3-acetyl-4-deoxynivalenol production (Table 3). Benzaldehydes. Fungicide activity (25% vegetative growth compared to the control) and 3-acetyl-4-deoxynivalenol inhibition (20% trichothecene production) were observed when 1.5 mM 2,4-dihydroxybenzaldehyde (10) was added to F. culmorum growth medium, whereas at the same concentration, veratraldehyde (11), a benzaldehyde with two phenolic −OH groups protected by methylation, reduced 3-acetyl-4deoxynivalenol production to 20%, with no significant inhibition of mycelium growth (Table 2). The same effects were observed with vanillin (12) tested at 1.0 mM (Table 3). Benzoic Acids. Gallic acid (13) was selected as a prototype of benzoic acids. The production of 3-acetyl-4-deoxynivalenol was below the limit of detection ( 21 > 20 > 24 > 23 > 22 > 25. Approximately, the same trend was maintained in the score of lipophilicity estimated by theoretical calculations that ranged between 3.37 and 1.27 log P. Both radical scavenging and antioxidant activities increased in the hydroxylated biphenyls with the following trend: 31 > 28 > 29 > 27. Ellagic acid (31) showed radical scavenging and antioxidant activities more than doubled and tripled compared to Trolox units (Table 1). Although ellagic acid (31) is conformationally hindered, a large electronic delocalization occurs in the rings, hence providing strong radical scavenging and antioxidant activities. TLC Chromatography. Mycotoxins cannot be detected under UV at 254 nm and are poorly detectable in common TLC visualization reagents.28 Although a F. culmorum strain producing mainly 3-acetyl-4-deoxynivalenol has been used, a fast and reliable TLC detection protocol was optimized for the identification of five trichothecene mycotoxins (3-acetyl-4deoxynivalenol, 15-acetyl-4-deoxynivalenol, nivalenol, deoxynivalenol, and fusarenone X) by a single chromatographic run. In vitro assays indicated that 31 of the tested compounds belonging to the classes of cinnamic acids, acetophenones, benzaldehydes, benzoic acids, phenylpropanoids, and hydroxylated biphenyls are inhibitors of mycotoxin type B trichothecene (3-acetyl-4-deoxynivalenol) in vitro production by F. culmorum MCf 21 without significant reduction of fungal growth. Four compounds showed effective fungicidal activity, hence providing new opportunities for application in crop protection. In an attempt to improve bioavailability of the tested compounds, β-CD was added to the growth medium. Although previous studies have demonstrated a synergistic effect of β-CD on the antimicrobial activity of plant-derived phenols,29 no significant effect was observed in the present study. It is worth doing further comments on results achieved with some compounds. Carvacrol (26), whose fungicidal activity at 1.0 mM was previously demonstrated30 turned out to be a good type B trichothecene inhibitor, whereas inhibition of fungal growth was determined only at higher concentrations (data not shown). Zingerone (25) and dehydrozingerone (22), both constituents of Zingiber officinale and Curcuma longa, are structurally and biologically related to curcumin. Both compounds possess antioxidant, anti-inflammatory, and anti-tumor-promoting activities.31,32 Recently, the in vitro inhibitory effect and fungicide activity of some curcumin derivatives were observed in F. proliferatum, a fumonisin B1 producer.33 Fungitoxic properties of dehydrozingerone (22) and zingerone (25) were reported toward F. oxysporum and Aspergillus sp., respectively.34,35 In our study, dehydrozingerone (22), zingerone (25), and its natural-like Me-zingerone (23) tested at 1.0 and 0.5 mM showed a strong inhibitory effect on trichothecene production by F. culmorum, without significantly affecting its vegetative growth in vitro. Protection of the phenolic −OH 4975



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compounds bearing a methoxy group in the ortho position to phenolic −OH, namely, a guaiacyl unit, usually form an intramolecular hydrogen bond, which is energetically favorable.41 The guaiacyl unit exhibits antioxidant proprieties because it acts as a H donor to scavenge radicals.42 It is wellacknowledged that antioxidant activity of the guaiacyl unit acts as a free radical scavenger by H atom transfer from the phenolic OH group.43,44 Phenoxy radicals formed from a guaiacyl moiety do not favor quinone formation, as happens when radicals are generated by a catechol moiety (e.g., compound 4). When the corresponding phenoxy radical ArO• formed in guaiacyl moiety is also delocalized in the side chain para to the phenolic −OH group, the radical is easier to form, e.g., in compounds 3, 4, 22, 25, 28, and 29, as compared to eugenol (20), isoeugenol (21), vanillin (12), veratraldehyde (11), and apocynin (6), all having a less extended electronic conjugation. As a result, the antioxidant activity of compounds 6, 11, 12, 20, and 21 is lower than in compounds 3, 4, 22, 28, and 29. Except for biphenyl (28), all compounds bearing a guaiacyl moiety caused a significant inhibition of mycotoxin production without affecting fungal growth. It is therefore reasonable to assume that the presence of a guaiacyl unit with an electronwithdrawing substituent in the para position may be a key structure in trichothecene inhibition. The inhibitory activity of a guaiacyl unit, with a phenolic −OH methyl protected, was not strictly associated with the antioxidant activity in the present work (e.g., compounds 11, 23, and 24). In solution, the guaiacyl unit presenting an extended conjugation is expected to be in plane or to deviate only marginally from the plane. Consequently, any difference in the activity of the molecules should be correlated to electronic phenomena rather than steric effects. On the contrary, in dimer form (e.g., hydroxylated biphenyls), steric effects would strongly influence the molecular properties (e.g., capacity to form stable radicals, interaction with proteins, increased lipophilicity, Lewis acidity enhancement, and polarizability), mainly when the two phenolic −OH groups are ortho to the C−C bond of the two aromatic rings. Nonplanar arrangement of the four groups along the C−C coupling bond occurs, providing steric influence, even when free rotation around the C−C single bond is expected.45 Therefore, a steric effect may drive the ability of hydroxylated biphenyls to inhibit mycotoxin production. In 2,2′-dihydroxybiphenyls lacking a guaicyl unit and being conformationally more flexible, such as magnolol (27), marked antifungal activity was observed also at 0.1 mM (data not shown). The relative order to partitioning in the organic phase (lipophilicity, determined by RP-HPLC) is in agreement with that determined for the polarity of each compound, as determined by HPLC. In principle, the least polar compound should present higher partitioning in the lipid phase, and this behavior should reflect an inhibitory property. Nonetheless, fungal matrices are multi-component systems, where the medium can be considered as both lipidic and emulsion systems. Thus, correlating theoretical calculations and experimental data is not a straightforward approach. The balance among lipophilicity, antioxidant activity, and capacity to activate weak interactions with enzymes is a key factor to estimate a good phenolic mycotoxin inhibitor.46,47 In addition to the known inhibitory activity of ferulic acid (3) and its derivatives, common natural compounds, such as compounds 6, 15, 20, 22, 25, and 26, exhibited a strong

inhibitory activity within the range of the tested concentrations. Although some of these compounds have already been known for their fungicidal or antimicrobial activity on different fungi, this is the first time such molecules have been evaluated as mycotoxin inhibitors in F. culmorum. It has not escaped our notice that the remarkable fungicide activity detected for magnolol (27) is worth further application in different fields. The inhibitory activity of selected compounds is being tested in planta to evaluate potential application for the control of Fusarium affecting wheat and other small grain cereals.

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ASSOCIATED CONTENT

S Supporting Information *

Description of TLC and LC−MS methods, HPLC of the tested compounds in one chromatographic run, materials for detection of mycotoxins by TLC, estimation of lipophilicity (log P) of studied compounds, and detection of polarity of studied compounds by TLC. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +39-079-2841220. Fax: +39-079-2841299. E-mail: [email protected]. Present Address §

Zahra Jahanshiri: Department of Mycology, Faculty of Medical Science, Tarbiat Modares University, Teheran 14115-331, Iran. Funding

The authors acknowledge support by the Regione Autonoma della Sardegna (Legge Regionale 7 Agosto 2007, no. 7 “Promozione della Ricerca Scientifica e dell’Innovazione Tecnologica in Sardegna”), the Ministry of University and Research (PRIN 2011 “Cell Wall Determinants To Improve Durum Wheat Resistance to Fusarium Diseases”), and the University of Sassari (P.O.R. SARDEGNA F.S.E. 2007-2013Obiettivo Competitività Regionale e Occupazione, Asse IV Capitale Umano, Linea di Attività l.3.1. “Identification of Natural and Natural-like Molecules Inhibiting Mycotoxin Biosynthesis by Fusaria Pathogen on Cereals”). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Corrado Dimauro for assisting in statistical analysis. REFERENCES

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Natural and Natural-like Phenolic Inhibitors of Type B Trichothecene

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