Article pubs.acs.org/JAFC
Synthesis, Characterization, and Antifungal Activities of Amphiphilic Derivatives of Diethylaminoethyl Chitosan against Aspergillus flavus Juliana dos Santos Gabriel, Marcio José Tiera, and Vera Aparecida de Oliveira Tiera* Department of Chemistry and Environmental Sciences, IBILCE, São Paulo State University − UNESP, São José do Rio Preto, São Paulo, Brazil S Supporting Information *
ABSTRACT: Amphiphilic derivatives of diethylaminoethyl chitosan (DEAE-CH) were synthesized using a two-step process involving initial substitution with diethylaminoethyl (DEAE) groups followed by reductive amination with dodecylaldehyde. The synthesized derivatives were characterized by 1H NMR, gel permeation, and FTIR. The associative behaviors of these compounds in aqueous solution were studied using fluorescence spectroscopy, whereas their antifungal activities against Aspergillus flavus were evaluated in terms of mycelial growth. The effects of deacetylated chitosans and their derivatives on the mycelial growth of A. f lavus were evaluated at several polymer concentrations (0.05−1.0 g/L), and the results were compared. The inhibition indices of the deacetylated chitosans increased with increasing Mw (16.9 kDa < 176 kDa < 517.7 kDa); however, derivatives with a combination of either a high molecular weight (Mw) and low hydrophobicity or a low Mw and high hydrophobicity were the most effective in inhibiting the in vitro radial growth of A. f lavus. KEYWORDS: Aspergillus flavus, chitosan, amphiphilic derivatives, antifungal activity
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INTRODUCTION It is well-known that chemical fungicides and pesticide residues are toxic to humans and animals and that many are not biodegradable, causing serious environmental problems, such as the contamination of water and soil.1 The search for more environmentally friendly antimicrobials has promoted research on natural products, including extracts from microbial sources,2 essential oils,3 and polysaccharides.4 Chitosan (CH) is among the most frequently studied polysaccharides due to its potential applications as a food preservative and as an adjuvant in agriculture to protect or promote the defense responses of various crops.5 CH exhibits many interesting functional properties, including biocompatibility, low toxicity, and natural antibacterial, antiviral, and antifungal activities.6 The antimicrobial properties of CH are mainly credited to its polycationic nature in acidic media, which enables it to interact with the cell walls of various microorganisms.4 In particular, CH and its oligomers have emerged as promising sources for application as biodegradable fungicides, for the regulation of plant growth and for the protection of seeds.7,8 The mechanism underlying the antifungal activity of CH has been extensively investigated. In the first step, its cationic chain interacts with negatively charged residues of macromolecules that are exposed on the fungal cell surface.9 CH is believed to affect cell wall morphogenesis, interfering directly with the activities of enzymes that are responsible for fungal growth.10,11 In the search for effective antimicrobials based on CH, a variety of strategies have been devised to increase its antifungal activity, and the control of molecular weight (Mw) and the hydrophobic/hydrophilic balance have both been recognized as being of major importance.12−15 We have recently shown that low-Mw quaternary derivatives of CH improve its in vitro antifungal activity against Aspergillus flavus, a pathogenic fungus that is found in tropical and © 2015 American Chemical Society
subtropical climates and that infects and contaminates preharvest and postharvest seed crops, generating carcinogenic aflatoxins.16 The derivatives were quaternized with propyltrimethylammonium bromide, followed by reductive amination with dodecylaldehyde. The results showed that the low-Mw derivatives containing dodecyl (Dod) groups exhibited enhanced inhibition with increasing proportions of hydrophobic groups and polymer concentrations.17 In this work, the synthesis, characterization, and antifungal activities of amphiphilic derivatives of CH against the fungus A. f lavus are described. We present a different approach based on modifications with diethylaminoethyl (DEAE) and Dod groups (Figure 1). The advantage of DEAE relies on its tertiary amine, the pKa of which is >7.0,18 which permits its solubility in neutral aqueous media. Moreover, DEAE chloride is produced on a large scale and is an important intermediary for the agrochemical, pharmaceutical, and polymer industries.19 We prepared three highly deacetylated CHs of different Mw values that were subsequently modified with DEAE and Dod groups. Their amphiphilic properties and antifungal activities were evaluated by examining the degree of mycelial growth of A. f lavus in the presence of increasing concentrations of the derivatives. The antifungal activities of CH and its amphiphilic derivatives are presented and discussed in relation to the degrees of substitution and Mw values of the derivatives.
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MATERIALS AND METHODS
Materials. CH (degree of deacetylation (DD) = 85%), which was used as the starting material, was purchased from Polymar Co. Received: Revised: Accepted: Published: 5725
January 15, 2015 May 28, 2015 June 3, 2015 June 3, 2015 DOI: 10.1021/acs.jafc.5b00278 J. Agric. Food Chem. 2015, 63, 5725−5731
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Figure 1. Synthesis of amphiphilic derivatives of diethylaminoethyl chitosan.
Table 1. Molar Ratios of Dodecyl/NH2 and Degrees of Substitution with Dodecyl Groups polymer CCH (DDA 86.0%) CHH (DDA = 98%) DEAE14-CH DEAE14-CH-Dod3 DEAE14-CH-Dod10 DEAE14-CH-Dod14 CHM (DDA = 98.0%) DEAE30-CH DEAE30-CH-Dod3 DEAE30-CH-Dod10 DEAE30-CH-Dod20 CHL (DDA = 98.0%) DEAE43-CH DEAE43-CH-Dod5 DEAE43-CH-Dod30 DEAE43-CH-Dod40
molar ratio dodecyl/NH2
DSDEAE (%)
0.05 0.15 0.36
14.0 14.0 14.0 14.0
0.05 0.15 0.37
30.0 30.0 30.0 30.0
0.06 0.45 0.65
43.0 43.0 43.0 43.0
DSDod (%)
Mw (kDa)
Mw/Mn
1068 517.7 548.6
4.8 3.4 3,9
2.6 10.0 14.0 176.0 151.0
5 28 39
I1/I3 (at 0.2 g/L)
7.0 × 10−3 6.0 × 10−3
1.50 1.22 1.13
1.14 × 10−2 7.0 × 10−3
1.80 1.21 1.18
9.0 × 10−3 6.2 × 10−3
1.79 1.24 1.15
3.35 2.90
2.7 10 19 16.9 15.7
CAC (g/L) pH 5.0
2.50 2.25
Peltier system. pH was measured using a DM-23 Digimed pH meter (Digimed, Campo Grande, Brazil). Synthesis of Amphiphilic Derivatives of DEAE-CH. CH derivatives were synthesized using deacetylated CH samples, prepared as previously described by Tiera et al.20 CH derivatives were obtained by reacting DEAE chloride with CH of different Mw values (517.7, 176, and 16.7 kDa) in aqueous media at pH 8.0 under an N2 atmosphere.21 The resulting derivatives (DEAE-CH) were purified by dialysis against water for 4 days and were then lyophilized. Subsequently, the derivatives were subjected to hydrophobic modifications through reductive amination with dodecylaldehyde, as shown in Figure 1. The procedure for generating DEAE30-CH-Dod10 with a sample containing 30% DEAE groups and 10% Dod groups is
(Fortaleza, Ceará, Brazil). Tween 80, sodium acetate, acetic acid, and sodium hydroxide were purchased from Synth (Diadema, São Paulo, Brazil). DEAE chloride, deuterium chloride (35%) in deuterium oxide, and deuterium oxide were purchased from Sigma-Aldrich Chemical Co. (São Paulo, Brazil). Potato dextrose agar (PDA) was purchased from Acumedia Manufacturers, Inc. (Lansing, MI, USA). Water was deionized using a Gehaka water purification system. Spectra/Pore membranes (Spectrum) were employed for dialysis. All solvents were of reagent grade and were used as received. Instrumentation. 1H NMR spectra were recorded using a Bruker ARX-500 500 MHz spectrometer (Bruker, Germany). UV−vis spectra were measured using a Cary 100 spectrophotometer equipped with a 5726
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colony, as measured using a caliper on the third, fifth, and seventh days of cultivation. The results obtained on the seventh day were used for comparative purposes. The antifungal index was calculated as
described below. Deacetylated CH (0.8 g, 176 kDa) was solubilized in a mixture of 90 mL of 2% acetic acid and 50 mL of ethanol. The pH was adjusted to 5.0, and while the reaction mixture was stirred, 0.12 mL of dodecylaldehyde dissolved in 10 mL of ethanol was added to it in a dropwise manner. The reaction mixture was continuously stirred for 25 h at room temperature, and sodium cyanoborohydride (3:1, NaCNBH3/NH2, mol/mol) was added after the first hour. Thereafter, the reaction mixture was dialyzed (using a membrane with a MWCO of 6000−8000 g/mol), first against water for 2 days, then against aqueous NaOH (0.05 M) for 1 day, and finally against water for 3 days. The derivative DEAE30-CH-Dod10 was lyophilized. Other DEAECH-Dod derivatives were prepared under identical conditions using different initial NH2/dodecylaldehyde molar ratios (Table 1). The derivatives were subsequently purified with a Soxhlet system using chloroform to remove nonreacted dodecylaldehyde. The derivatives were characterized by 1H NMR, FTIR and gel permeation chromatography (GPC) measurements. GPC Analysis. GPC measurements were performed using an LC20 Shimadzu liquid chromatograph (Shimadzu, Japan) equipped with a refractive index detector (model RID-10) using OHpak SB-803 HQ and OHpak SB-805 HQ columns (Shodex) and pullulan standards from 805 to 6.2 kDa (Supporting Information, Supplementary Figure S1). A mixture of acetic acid (0.3 M)/sodium acetate (0.2 M) (pH 4.5) was used as the mobile phase at a flow rate of 0.8 mL/min at 35 °C. Solutions were prepared for GPC analysis by dissolving the polymer in an acetic acid (0.3 M)/sodium acetate (0.2 M) buffer to achieve concentrations of between 0.5 and 1.0 mg/mL. The polymer solutions were stirred for 3 days and then filtered through 0.45 μm membranes before analysis. Self-Association Study: Determination of Critical Aggregation Concentrations (CACs). The self-association of the amphiphilic derivatives was evaluated at 25.0 ± 0.1 °C using a Hitachi 4500 fluorescence spectrometer (Hitachi, Japan). The temperature of the water-jacketed cell holder was controlled using a circulating bath. Pyrene fluorescence was assessed to monitor the solution properties of the modified CHs. One microliter of a stock solution of pyrene (1 mM) in methanol was added to quartz cuvettes, and the solvent was removed by blowing N2. To each of these pyrene-containing cuvettes was added 2 mL of an acetic acid/sodium acetate buffer, pH 5.0, followed by sonication for 4−5 min. Measured amounts (5 μL) of concentrated stock solutions of the derivatives (2.0 g/L) were added to buffered aqueous solutions of pyrene (5.0 × 10−7 M) under magnetic stirring, and fluorescence spectra were recorded after each addition. The ratio between the fluorescence intensities of peaks I (372.4 nm) and III (384 nm) of the emission spectrum of pyrene (I1/ I3 ratio) was used to evaluate the polarity of the local environment22 and to determine the CAC. Pyrene was excited at 310 nm, and its emission was recorded from 350 to 650 nm. Fungal Strains and Culture Conditions. The antifungal activities of CH and its derivatives were tested against A. f lavus and Aspergillus parasiticus. The strains used were kindly provided by the Brazilian Collection of Microorganisms from the Environment and Industry (CBMAI; Campinas, São Paulo, Brazil) and were maintained on PDA (200 g/L potato infusion, 20 g/L dextrose, and 15 g/L agar) in the dark at 25 ± 2 °C. Effects of CH Derivatives on Mycelial Growth of Aspergillus flavus. To compare the antifungal potentials of deacetylated and commercial CH to those of the synthesized amphiphilic DEAE-CHDod derivatives, an in vitro mycelial growth inhibition assay was conducted. The different CH solutions were previously prepared in an aqueous solution of acetic acid (1% m/m) and adjusted to pH 5.5. Next, 10 mL of the derivatives was added aseptically to 10 mL of sterile melted culture medium (PDA, 10%) to obtain 20 mL of culture medium at the desired polymer concentration (0−1.0 g L−1), which was then transferred to Petri dishes. After solidification, the mixtures were inoculated with fragments of A. f lavus mycelium (1 mm in diameter), which were placed at the center of the Petri dishes. The plates were then incubated in an oven for 7 days at 25 ± 2 °C. The inhibition index, which is a measure of the inhibition of the fungus by the derivatives, was determined on the basis of the radial growth of the
antifungal index (%) = [1 − (Da /D b)] × 100 where Da is the diameter of the growth zone on the test plates and Db is the growth zone on the control plate.23 All experiments were performed in quadruplicate, and the data were averaged. Student’s t test and the Kruskal−Wallis test with Dunn’s multiple-comparison test were used to evaluate differences in the antifungal index obtained using the antifungal tests. Results with P < 0.05 were considered statistically significant.
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RESULTS AND DISCUSSION Synthesis and Characterization of Amphiphilic Derivatives. Deacetylation was carried out under continuous nitrogen bubbling to prevent degradation of the polymer. The degrees of deacetylation, expressed as −NH2 mol % and determined on the basis of the 1H NMR spectra of commercial chitosan (CCH) and the deacetylated samples (CH), were 86.0 and 98.0 mol %, respectively. These values were calculated on the basis of the area of the singlet at 3.7 ppm, which was assigned to the proton linked to carbon 2 of the glucosamine ring (H2), and on the singlet at 2.5 ppm, which was assigned to the acetamido methyl protons (Support Information, Supplementary Figure S2a). The degrees of substitution by DEAE groups (DS) were calculated on the basis of the areas (ICH3) of the signal at 1.72 ppm, which were assigned to the methyl protons NH−(CH2−CH3)2, and on the signals at 5.1−5.35 ppm, which were assigned to the anomeric protons of substituted and unsubstituted glucosamine residues, H1s and H1, respectively (Figure S2b). The degrees of substitution were calculated using eq 1: DSDEAE =
ICH3/6 (IH1 + IH1s)
(1)
Derivatives substituted with DEAE (DEAE30-CH and DEAE43-CH) were subsequently grafted with increasing proportions of Dod groups. The 1H NMR spectra of the derivatives DEAE43-CH and DEAE43-CH-Dod30 are shown in the Support Information (Supplementary Figures S2b and S2c, respectively). The 1H NMR spectra of the amphiphilic derivatives exhibited three new signals after substitution with Dod, including a singlet at 1.47 ppm, corresponding to the protons of the methyl group on the Dod chain, and signals at 1.94 and 2.35 ppm (Supplementary Figures S3−S5). The latter signals corresponded to protons in the hydrocarbon chain and the methylene protons of the carbon atom linked to the amino group of CH, respectively. The attachment of Dod groups to the DEAE-CH framework further altered the 1H NMR spectrum of DEAE43-CH, most notably with respect to the resonances of the methyl protons of the DEAE groups and the anomeric protons of substituted and unsubstituted glucosamine units. The former underwent a downfield shift from 1.72 to 1.88 ppm, and the latter underwent a shift from 5.15 to 5.3 to 5.5−5.7 ppm, which might have resulted from aggregation of the amphiphilic chains (Supplementary Figures S2b and S2c). The integrated areas of the signal at 1.47 ppm and those assigned to the anomeric protons of CH chains at 5.5−5.7 ppm were used to calculate the degree of substitution by Dod (DSDod) using eq 2:24 5727
DOI: 10.1021/acs.jafc.5b00278 J. Agric. Food Chem. 2015, 63, 5725−5731
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ICH3 IH1 + IH1s
(2)
The DEAE-CH derivatives were synthesized at pH 8.0 under an N2 atmosphere. As shown in Table 1, GPC analysis confirmed that the molecular masses of the DEAE derivatives (DEAE 14 -CH, DEAE 30 -CH, and DEAE 43 -CH) did not significantly differ from those of the starting CHs (CHH, CHM, and CHL). Although degradation can occur during the first step of the reaction, the Mw values of the DEAEx-CH derivatives remained similar to those of the starting CHs (Table 1). However, the obtained Mw values of the DEAE-CH derivatives may be rationalized by considering that the attachment of DEAE can induce conformational changes, thus affecting retention time. Moreover, the Mw distribution is also affected by extensive dialysis. Thus, the polydispersities (Mw/Mn) of the DEAE derivatives were smaller than those of the starting CHs. The reductive amination reaction carried out during the second synthesis step was performed at a low temperature and pH 5.0; thus, significant degradation was not expected to have occurred during the reaction. Amphiphilic Properties. The self-association of the amphiphilic derivatives was studied in aqueous buffered solutions at pH 5.0. At this pH, CH is positively charged, and medium-Mw samples might aggregate at concentrations >3.0 g/L.25 Hence, it is expected that at low concentrations, hydrophobically modified CHs will self-associate in aqueous solutions to form intra- and intermolecular aggregates. This intrinsic property might affect not only the conformation of the polymer chains but also their interactions with the cell wall. Hydrophobic modifications might strengthen the interaction with the cell membrane but could also introduce intra- and intermolecular interactions that affect antimicrobial activity. Therefore, amphiphilic polycations, such as small amphiphilic molecules, may be active as aggregate assemblies rather than as discrete polymer chains.26 The CAC values were determined on the basis of fluorescence experiments using pyrene as a probe. Figure 2 shows typical graphs of I1/I3 based on the emission spectra of pyrene in the presence of increasing concentrations of CH and its derivatives. The obtained I1/I3 ratios reflect the microenvironments probed by pyrene and indicate the concentration range at which self-association begins. The less substituted derivatives, DEAE30-CH-Dod3 and DEAE43-CH-Dod5, exhibited no clear breaks in the curves (I1/I3 vs concentration) at concentrations of >0.2 g/L, and the I1/I3 ratio values remained at approximately 1.7−2.0, indicating that the derivatives were of hydrophilic character. Electrostatic repulsion between positively charged chains overcame the hydrophobic effects of Dod groups, hindering intermolecular aggregation at this concentration range (Figure 2). The more substituted derivatives exhibited clear decreases in the I1/I3 ratios in the range from 5.0 × 10−3 to 1.2 × 10−2 g/L, indicating that aggregates were formed due to intermolecular interactions. The CACs decreased modestly with the degree of substitution with Dod groups (Table 1); however, the CAC values indicate the onset of aggregation in the polydisperse samples. The amphiphilic derivatives prepared from DEAE14CH (Mw = 548.6 kDa), the highest Mw derivative, exhibited a more gradual decrease in the I1/I3 ratio (Support Information, Supplementary Figure S6), which might have resulted from the concurrent formation of inter- and intramolecular aggregates. Overall, amphiphilic derivatives of increasing hydrophobicity
Figure 2. I1/I3 ratio versus DEAE30-CH-Dodx concentration at pH 5.0.
may be obtained by increasing the proportion of dodecylaldehyde in the feed. In Vitro Antifungal Activity against A. f lavus. The antimicrobial activity of CH has been mainly attributed to its cationic nature in acidic media, which enables its interaction with fungal cell walls and triggers its mechanisms of action. To evaluate the effect of Mw on antifungal activity, we tested three highly deacetylated CHs (16.9, 176, and 517.7 kDa). Figure 3a shows the effects of deacetylated and commercial CHs at various polymer concentrations (0.1 to 1.0 g/L) on the mycelial growth of A. f lavus after an incubation period of 7 days at 25 ± 2 °C. As can be inferred from Figure 3a, the inhibitory effects of both the deacetylated and commercial samples increased with increasing polymer concentrations and Mw values (16.9 kDa < 176 kDa < 517.7 kDa). The effect of Mw on the antifungal activity of CH has been reported to depend on the type of fungus; for example, fungal growth has been reported to decrease with increasing Mw for Fusarium oxysporum and with decreasing DA for Alternaria solani, but no dependence on MW or DA has been observed for Αspergillus niger.27 Seyfarth et al. reported that antifungal activity against Candida species decreases with declining molecular mass;28 however, oligochitosans with Mw values ranging from 2 to 20 kDa have been recently reported to efficiently inhibit the growth of Candida species and clinical isolates of Candida albicans.13 Although the mechanism of antifungal activity remains controversial, reports of cell wall alterations and membrane damage have been shown for oligochitosans against Candida species.13 In the case of A. parasiticus, the mechanism has been reported to be mainly fungistatic and has been attributed to the high Mw values (170− 300 kDa) of the CH samples employed.29 Therefore, the increase in activity with increasing Mw may be due to improved fungistatic activity because higher Mw CHs do not permeate the 5728
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hypha but may affect nutrition through the cell wall.11 CCH exhibited a lower level of activity (5% at 1.0 g/L), possibly due to the lower availability of amino groups (DDA 85%). The greater antifungal activities exhibited by the CHH and CHM samples compared with CCH is attributed to stronger interactions with the cell membrane. The hydrophobic modification of CH has been reported to effectively enhance antibacterial15 and antifungal activities by strengthening its interaction with the cell wall.16,29,30 To investigate the effect of the hydrophobic modification itself, 517.7 kDa deacetylated chitosan (CHH) was grafted with only Dod (5.0%) (Figure 3b). At a concentration of 1.0 g/L, the inhibition produced was 43%, which was higher than that provided by CHH (30%). A similar result has also been obtained against A. parasiticus in a previous study showing 32% inhibition of radial growth, indicating the importance of hydrophobic interactions in causing further activity.17 Conversely, the attachments of both groups (DEAE and Dod) appeared to be synergistic, and a marked increase in antifungal activity was noted. A representative experiment comparing the effect of DEAE43-CH-Dod5 concentration on the radial growth of A. f lavus after the seventh day of inoculation is shown in Figure 3c, revealing that at a concentration of 1.0 g/L, fungal growth was completely inhibited. At 0.5 g/L, the inhibition indices obtained for the less substituted derivatives of the three series (DEAE14-CHDod2.5, DEAE30-CH-Dod3, and DEAE43-CH-Dod5) were 15, 51, and 27%, respectively. Overall, amphiphilic compounds synthesized from DEAE43-CH (Mw = 15.7 kDa) exhibited the highest activities; however, complete inhibition of A. flavus was achieved with the most hydrophobic derivatives (DEAE43-CHDod28 and DEAE43-CH-Dod39; Figure 3d). Despite their different degrees of substitution, all amphiphilic derivatives exhibited higher activities than those of the starting CHs and their hydrophilic DEAE-CH derivatives. At 1.0 g/L, the most substituted derivatives of the three series (DEAE43-CH-Dod39, DEAE30-CH-Dod19, and DEAE14-CH-Dod14) were able to reduce the mycelial growth of A. f lavus by 100, 29, and 34%, respectively, indicating that the antifungal activities decreased with the increasing Mw values of the starting DEAE-CH derivatives (15.7, 176.0, and 548.6 kDa, respectively). The importance of hydrophobic effects on the antimicrobial activities of CH derivatives has been recently reported by our group16,17 and by others.15,31 The improved activities of hydrophobized CH derivatives have been attributed to the reinforcement of hydrophobic interactions with the cell membrane.16,32 Therefore, the attachment of Dod groups is considered important for the antifungal activities of these derivatives. However, the lower inhibition indices observed for the most substituted derivatives of this series indicate that the hydrophilic/hydrophobic balance must be adjusted to obtain higher antifungal activities. As shown in Figure 4a, the attachment of increasing numbers of Dod groups initially increased antifungal activity, but it was decreased for the more substituted derivatives (DEAE14-CH-Dod14 and DEAE30-CHDod20). To confirm this trend, we tested the DEAE14-CH-Dod derivatives against A. parasiticus and found that inhibition increased with increasing derivative concentration; however, less substituted derivatives were again more effective in inhibiting fungal growth (Figure 4b). It is postulated that the discrete amphiphilic chains of less substituted derivatives might interact with cell membranes more effectively than aggregates
Figure 3. (a) Effect of chitosan molecular weight (CHH, CHM, and CHL) on the mycelial growth of A. f lavus. (b) Effect of CH-Dod5 concentration on the inhibition index of A. f lavus. (c) Effect of DEAE43-CH-Dod30 concentration on the radial growth of A. f lavus on the seventh day after inoculation. From left to right: 0.05, 0.1, and 1.0 g/L. (d) Effect of DEAEx-CH-Dody derivative concentration on the inhibition index of A. f lavus. From left to right: 0.05, 0.1, 0.5, and 1.0 g/L. Vertical bars with different letters indicate significant differences according to the Kruskal−Wallis test (P < 0.05). 5729
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however, no significant reduction in the mycelial growth of A. f lavus was observed as a result of these modifications, indicating that small changes in the polymer structure may significantly affect activity. Hydrophobic modifications with Dod groups strengthened the interaction with the fungal cell wall and markedly increased the antifungal activity of CH against A. f lavus. These results clearly show that Mw and hydrophobicity must both be controlled to increase antifungal activity. Overall, higher inhibition can be achieved using amphiphilic compounds with high Mw values and low hydrophobicities or using compounds with low Mw values and high hydrophobicities. The synthesis and characterization of new derivatives containing an appropriate hydrophilic/hydrophobic balance are ongoing in our laboratory to optimize their antifungal activities.
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ASSOCIATED CONTENT
S Supporting Information *
Figures S1−S9. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jafc.5b00278.
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AUTHOR INFORMATION
Corresponding Author
*(V.A.O.T.) E-mail:
[email protected]. Phone: +55 17 32212358. Fax: +55 17 32248692. Funding
We thank FAPESP (Grant 2012/03619-9) for their financial support. J.S.G. thanks FAPESP (2010/13942-6) for providing an undergraduate fellowship. Notes
The authors declare no competing financial interest.
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REFERENCES
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Figure 4. (a) Effects of changes in hydrophobicity: inhibitory effects of DEAEx-CH-Dody derivatives (1.0 g/L) on the mycelial fungal growth of A. f lavus. (b) Inhibition of in vitro growth of A. parasiticus in the presence of various concentrations of DEAE14-CH-Dodx derivatives. Vertical bars with different letters indicate significant differences according to the Kruskal−Wallis test (P < 0.05). The arrows on the top indicate the trend for inhibition indices with increasing hydrophobe content.
that are formed due to the self-association of more hydrophobic derivatives, thus decreasing the antifungal activities of these two types of derivatives. Overall, the results confirm the importance of hydrophobic groups for the antifungal activities of CH derivatives and may enable the development of more effective biofungicides based on natural polysaccharides. Conclusions. Novel amphiphilic derivatives of CH were synthesized and characterized, and their antifungal activities against A. f lavus were tested. Our results showed that deacetylated, medium-Mw (200−500 kDa) CHs were the most effective in inhibiting the mycelial growth of A. f lavus, achieving an inhibition index of >25%. Attaching DEAE groups increased the solubilities of these derivatives at a neutral pH; 5730
DOI: 10.1021/acs.jafc.5b00278 J. Agric. Food Chem. 2015, 63, 5725−5731
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DOI: 10.1021/acs.jafc.5b00278 J. Agric. Food Chem. 2015, 63, 5725−5731
