Article pubs.acs.org/JAFC
Antiaflatoxigenic and Antimicrobial Activities of Schiff Bases of 2‑Hydroxy-4-methoxybenzaldehyde, Cinnamaldehyde, and Similar Aldehydes Nanishankar V. Harohally,*,† Chris Cherita,‡ Praveena Bhatt,‡ and K. A. Anu Appaiah‡ †
Department of Spice and Flavour Science, CSIR-CFTRI, KRS Road, Mysuru 570020 Karnataka, India Microbiology and Fermentation Technology, CSIR-CFTRI, KRS Road, Mysuru 570020 Karnataka, India
‡
S Supporting Information *
ABSTRACT: 2-Hydroxy-4-methoxybenzaldehyde (HMBA) is a nontoxic phenolic flavor from dietary source Decalipus hamiltonii and Hemidesmus indicus. HMBA is an excellent antimicrobial agent with additional antiaflatoxigenic potency. On the other hand, cinnamaldehyde from cinnamon is a widely employed flavor with significant antiaflatoxigenic activity. We have attempted the enhancement of antiaflatoxigenic and antimicrobial properties of HMBA, cinnamaldehyde, and similar molecules via Schiff base formation accomplished from condensation reaction with amino sugar (D-glucamine). HMBA derived Schiff bases exhibited commendable antiaflatoxigenic activity at the concentration 0.1 mg/mL resulting in 9.6 ± 1.9% growth of Aspergillus flavus and subsequent 91.4 ± 3.9% reduction of aflatoxin B1 with respect to control. KEYWORDS: aflatoxin, 2-hydroxy-4-methoxybenzaldehyde, cinnamaldehyde, Schiff base, foodborne pathogen
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INTRODUCTION 2-Hydroxy-4-methoxybenzaldehyde (HMBA) is a significant and underutilized flavor component from dietary sources including roots of Decalipus hamiltonii and Hemidesmus indicus.1,2 It has shown excellent antimicrobial property3 due to the presence of phenolic as well as aldehyde groups. Further, the antioxidant potential of this compound has attracted lot of attention possibly because of water solubility.4−6 In addition, due to unique structural attributes, HMBA has been evaluated as a tyrisinase inhibitor and also it has shown greater activity against Helicobacter pylori.7 HMBA is also documented for its antiaflatoxigenic potency.8 On the other hand, cinnamaldehyde is a well explored antiaflatoxigenic agent and its mechanism has been recently evaluated.9 A diverse class of compounds composed of coumarins, flavonoids, alkaloids, and terpenoids have been evaluated as inhibitors for aflatoxin biosynthesis.10 In addition, plant derived as well as food derived arrays of essential oils have shown commendable antiaflatoxigenic activity.11−18 Apart from the essential oils, few extracts, mainly tea and Garcinia, have exhibited substantial activity against Aspergillus flavus growth as well as inhibition of aflatoxin production.19,20 Few natural products, in particular piperonal, has been shown to regulate even the biosynthesis of aflatoxin B1 production.21 Further, few synthetic compounds have also displayed significant antiaflatoxigenc activity.22 Schiff bases of amino acids, in particular Lserine, L-threonine, and L-tyrosine, and also the Mn(III) complexes have shown excellent antiaflatoxigenic activity.23 There is a need for development of antiaflatoxigenic agents derived from molecules exhibiting substantial activity as it encompasses structural attributes responsible for activity and also provides opportunities for further enhancement. In this context, we set out with a goal to utilize D-glucamine as an amino sugar to transform food derived 2-hydroxy-4-methox© 2017 American Chemical Society
ybenzaldehyde, cinnamaldehyde, and similar aldehydes including 2,4-dihydroxybenzaldehyde and 2-hydroxy-3-methoxybenzaldehyde to Schiff bases for the enhancement of antimicrobial and antiaflatoxigenic properties. We discuss synthesis and antiaflatoxigenic and antimicrobial activities of Schiff bases of HMBA, cinnamaldehyde, and similar aldehydes.
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MATERIALS AND METHODS
Chemicals. 2-Hydroxy-4-methoxybenzaldehyde (98%), cinnamaldehyde (98%), 2,4-dihydroxybenzaldehyde (98%), 2-hydroxy-3methoxybenzaldehyde (99%), and D2O were procured from SigmaAldrich India. Methanol (99.8%) and D-glucamine (>95%) were purchased from TCI chemicals. Solvents were used without any purification. Spectroscopic Measurements. NMR spectral data acquisition was accomplished on a Bruker Avance spectrometer having a frequency of 400 MHz for 1H NMR and 100 MHz for 13C NMR. 1 H chemical shift is referenced to internal HOD signal (4.79 ppm), and 13 C chemical shift is referenced to external standard tetramethylsilane in D2O. NMR spectra assignments to accomplished compounds were based on 1D techniques including 1H, 13C, DEPT (distortionless enhancement by polarization transfer), HSQC (heteronuclear single quantum coherence), and HMBC (heteronuclear multiple bond correlation) experiments. Mass spectral data (ESI positive mode) was acquired using the Q-TOF ULTIMA instrument from Waters Corporation. IR spectral data were recorded in the Thermo FT-IR instrument. 1-Deoxy-1-(2-hydroxy-4-methoxybenzylidene)amino-D-glucitol (1). In an Erlenmeyer flask containing a magnetic stir bar, Dglucamine (1 g, 5.5 mmol) was dissolved in water (7 mL). HMBA (0.837 g, 5.5 mmol) was dissolved in 4 mL of methanol and then Received: Revised: Accepted: Published: 8773
June 3, 2017 September 19, 2017 September 24, 2017 September 24, 2017 DOI: 10.1021/acs.jafc.7b02576 J. Agric. Food Chem. 2017, 65, 8773−8778
Article
Journal of Agricultural and Food Chemistry
Evaluation of Antimicrobial Activity. Bacterial Strains. The bacterial cultures employed in the current study consisted of Escherichia coli (ATCC11775), Salmonella typhimurium (ATCC 25241), Staphylococcus aureus (ATCC 12600), and Listeria monocytogenes (MTCC 1143). The cultures were grown in Brain Heart Infusion Broth (BHIB, HiMedia Laboratories Pvt Ltd., India) under aerobic conditions at 37 °C. For the antimicrobial assays, Mueller Hinton broth and agar media (MH, HiMedia Laboratories Private Limited, India) were utilized. A. flavus 68 strain of CFTRI was used for the production of aflatoxin. The strain was revived and subcultured on Potato Dextrose Agar (PDA). The culture was maintained on PDA. Antimicrobial Assay. The antimicrobial assay for the synthesized compounds was carried out by the agar well diffusion method described by Owais et al.24 with slight modification. Briefly, a culture of the pathogens (overnight grown) was subjected to centrifugation (4000 rpm, 20 min). Subsequently, the pellets obtained were suspended in saline and adjusted to 0.5 McFarland standard (3 × 105 cells/mL). The bacterial inoculum was then spread onto Mueller Hinton agar media. Wells having diameter of 7 mm were punched in the agar and subsequently packed with the compounds (to be tested) at an array of concentrations (5−50 mg/mL). Control wells were also made in a similar fashion and subsequently filled with neat DMSO as negative control and streptomycin sulfate (1000 U/mL) as positive control in the same plates. The plates were incubated at 37 °C for a period of 24 h. Antimicrobial activity was determined via measurement of diameter of the zone of inhibition for all the compounds. Measurement was done in triplicate. An assay was also carried out to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by the broth microdilution method.25 MIC and MBC measurements were done in triplicate. Briefly, serial 2-fold dilutions of the test compounds were prepared in DMSO, and 100 μL of each dilution was added to 96-well microtiter plates containing 100 μL of MH medium with inoculum of 1 × 105 cells/mL. The plates were incubated at 37 °C for a period of 24 h, and MIC was evaluated as the least concentration of the compound that demonstrated no visible growth. Subsequent to the determination of MIC, 10-fold dilutions from each well showing no turbidity were added to fresh MH broth (5000 μL) taken in tubes and incubated at 37 °C for 48 h. An aliquot of this was also plated onto nutrient agar plates and incubated for a period of 24 h at 37 °C. The MBC was the least concentration of the compound that displayed no visible growth in the broth as well as absence of bacterial colonies on the agar plates. Antiaflatoxigenic Activity of Compounds. The compounds 2hydroxy-4-methoxybenzaldehyde (HMBA) and 2,4-dihydroxybenzaldehyde (DHB) were screened for their antiaflatoxigenic activity in the following way. Potato dextrose broth was prepared and autoclaved at 121 °C. The media was allowed to equilibrate to room temperature, and 3 mL was dispensed into each well in a 6-well sterile cell culture plates. The experiment was conducted by modifying the broth microdilution method. The test compound was tested from 1 to 0.0625 mg/mL concentration. Experiments were conducted in triplicate. Appropriate positive control was included in the experiment. A. flavus 68 (106 spores/mL) was inoculated into all the wells in the plate and incubated at 28 °C for 7 days. The cell culture plates were observed each day for the changes in the growth pattern and spore formation. Weight of the Biomass. After 7 days of growth period, the fungal mat was separated from the media, washed with distilled water. The mat was then heated overnight at 100 °C to obtain the dry biomass. The dry fungal mat was weighed to determine the change in the growth of the fungi A. flavus. Extraction of Aflatoxin. The media was extracted for aflatoxin after 7 days and further analyzed. The mycelial mass was separated, and the media was extracted with equal volume of chloroform (media to chloroform ratio 1:1) during the course of overnight. The chloroform layer was stripped under nitrogen and followed by vacuum. About 100 μL of methanol (HPLC grade) was added to dilute the sample. The extracted sample was subjected to HPLC analysis. The concentration
transferred to solution containing D-glucamine. Subsequently, the reaction mixture was allowed to stir at room temperature (30 °C) until precipitation occurred (about 2 h). Then, the precipitate was filtered and washed with water followed by acetone and diethyl ether to get a light yellow colored solid. Yield = 1.485 g, 85%. IR (KBr) υmax 3400− 2900 (OH), 1657 (CN), 1610, 1564 (aryl), 1127, 1087, 1031 cm−1 (C−O). ESI-MS positive mode (M + H)+ m/z 316.1390, exact mass (M + H)+ 316.1397. 1H NMR δH: 3.74 (s, 3H, H3C(14)), 7.22 (d, 1H, J = 8.6 Hz, HC(13)), 6.28 (dd, 1H, J = 8.6 Hz, J = 2.3 Hz, HC(12)), 6.22 (s, 1H, HC(10)), 8.28 (s, 1H, HC(7)), 3.58 (m, 1H, H2C(6)), 3.40 (m, 1H, H2C(6)), 3.47 (m, 1H, HC(5)), 3.48 (m, 1H, HC(4)), 3.64 (m, 1H, HC(3)), 3.76 (m, 1H, HC(2)), 3.43 (m, 1H, H2C(1)), 3.73 (m, 1H, H2C(1)), 4.35 (m, 1H, OH), 4.40 (m, 1H, OH), 4.49 (m, 1H, OH), 4.88 (m, 1H, OH). 13C NMR δC: 55.1 (C14), 133.4 (C-13), 105.6 (C-12), 163.8 (C-11), 101.3 (C-10), 168.5 (C-9), 111.6 (C-8), 165.1 (C-7), 63.4 (C-6), 71.4 (C-5), 71.5 (C-4), 70.0 (C-3), 72.2 (C-2), 58.4 (C-1). 1-Deoxy-1-(2,4-dihydroxybenzylidene)amino-D-glucitol (2). Synthesis of this Schiff base (compound 2) was accomplished in a similar manner as compound 1 by employing D-glucamine (1 g, 5.5 mmol) and 2,4-dihydroxybenzaldehyde (0.760 g, 5.5 mmol). Yield = 1.390 g, 84%. IR (KBr) υmax 3400−2900 (OH), 1641 (CN), 1624, 1592 (aryl), 1095, 1060 cm−1 (C−O). ESI-MS positive mode (M + H)+ m/z 302.1289, exact mass (M + H)+ 302.1240. 1H NMR δH: 7.15 (d, 1H, J = 8.4 Hz, HC(13)), 6.19 (dd, 1H, J = 8.6 Hz, J = 1.8 Hz, HC(12)), 6.09 (d, 1H, J = 1.6 Hz, HC(10)), 8.25 (s, 1H, HC(7)), 3.58 (m, 1H, H2C(6)), 3.39 (m, 1H, H2C(6)), 3.45 (m, 1H, HC(5)), 3.47 (m, 1H, HC(4)), 3.63 (m, 1H, HC(3)), 3.74 (m, 1H, HC(2)), 3.42 (m, 1H, H2C(1)), 3.70 (m, 1H, H2C(1)), 4.10 (m, 1H, OH), 4.36 (m, 1H, OH), 4.47 (m, 1H, OH), 4.83 (m, 1H, OH). 13C NMR δC: 133.5 (C-13), 106.4 (C-12), 162.0 (C-11), 102.9 (C-10), 166.8 (C-9), 111.1 (C-8), 165.3 (C-7), 63.4 (C-6), 71.5 (C-5), 71.6 (C-4), 70.0 (C-3), 72.3 (C-2), 59.2 (C-1). 1-Deoxy-1-(2-hydroxy-3-methoxybenzylidene)amino-D-glucitol (3). Synthesis of this Schiff base (compound 3) was accomplished in a similar manner as compound 1 by employing Dglucamine (1 g, 5.5 mmol) and 2-dihydroxy-3-methoxybenzaldehyde (0.837 g, 5.5 mmol). Yield = 1.480 g, 85%. IR (KBr) υmax 3400−2900 (OH), 1642 (CN), 1601, 1540 (aryl), 1125, 1085, 1046 cm−1 (C− O). ESI-MS positive mode (M + H)+ m/z 316.1407, exact mass (M + H)+ 316.1397. 1H NMR δH: 3.76 (s, 3H, H3C(14)), 6.98 (s, 1H, J = 8.0 Hz, HC(13)), 6.73 (dd, 1H, J = 7.9 Hz, J = 7.9 Hz, HC(12)), 6.98 (s, 1H, J = 8.0 Hz, HC(11)), 8.4 (s,1H, HC(7)), 3.59 (m, 1H, H2C(6)), 3.40 (m, 1H, H2C(6)), 3.46 (m, 1H, HC(5)), 3.49 (m, 1H, HC(4)), 3.66 (m, 1H, HC(3)), 3.80 (m, 1H, HC(2)), 3.52 (m, 1H, H2C(1)), 3.81 (m, 1H, H2C(1)), 4.35 (m, 1H, OH), 4.39 (m, 1H, OH), 4.49 (m, 1H, OH), 4.86 (m, 1H, OH). 13C NMR δC: 55.7 (C14), 114.6 (C-13), 116.9 (C-12), 123.3 (C-11), 148.4 (C-10), 153.3 (C-9), 118.1 (C-8), 166.6 (C-7), 63.4 (C-6), 71.5 (C-5), 72.0 (C-4), 70.1 (C-3), 72.2 (C-2), 60.4 (C-1). 1-Cinnamylidenamino-1-deoxy-D-glucitol (4). Synthesis of this Schiff base (compound 4) was accomplished in a similar manner as compound 1 by employing D-glucamine (1 g, 5.5 mmol) and cinnamaldehyde (0.727 g, 5.5 mmol). Yield = 1.385 g, 85%. IR (KBr) υmax 3400−3000 (OH), 1634 (CN), 1556 (aryl), 1089, 1056 cm−1 (C−O). ESI-MS positive mode (M + H)+ m/z, 296.1456, exact mass 296.1498. 1H NMR δH: 7.60 (d, 1H, J = 7.2. Hz, HC(15)), 7.39 (t, 1H, J = 7.3 Hz, HC(14)), 7.34 (t, 1H, J = 7.1 Hz, HC(13)), 7.39 (t, 1H, J = 7.3. Hz, HC(12)), 7.60 (d, 1H, J = 7.2 Hz, HC(11)), 7.10 (d, 1H, J = 16.3. Hz, HC(9)), 6.90 (dd, 1H, J = 16.1 Hz, J = 8.7 Hz, HC(8)), 8.04 (d, 1H, J = 8.7 Hz, HC(7)), 3.45 (m, 2H, H2C(6)), 3.59 (m, 1H, HC(5)), 3.38 (m, 1H, HC(4)), 3.50 (m, 1H, HC(3)), 3.80 (m, 1H, HC(2)), 3.66 (m, 2H, H2C(1)), 4.66 (d, 1H, J = 4.8 Hz, C(2)-OH), 4.26 (d, 1H, J = 6.6 Hz, C(3)-OH), 4.55 (d, 1H, J = 5.4 Hz, C(4)-OH),), 4.48 (d, 1H, J = 5.4 Hz, C(5)-OH), 4.32 (t, 1H, J = 5.6 Hz, C(6)-OH). 13C NMR δC: 127.2 (C-15), 128.8 (C-14), 129.0 (C-13), 128.8 (C-12), 127.2 (C-11), 135.6 (C-10), 128.2 (C-9), 141.1 (C-8), 163.49 (C-7), 63.6 (C-6), 71.5 (C-5), 72.1 (C-4), 69.72. (C-3), 72.5 (C-2), 63.4 (C-1). 8774
DOI: 10.1021/acs.jafc.7b02576 J. Agric. Food Chem. 2017, 65, 8773−8778
Journal of Agricultural and Food Chemistry
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of aflatoxin was calculated based on the standard graph with appropriate experimental corrections. Quantification of Aflatoxin Using HPLC. The HPLC procedure employed for the quantification of aflatoxin B1 was carried out according to Ferreira et al.26 with slight modifications. The samples obtained were filtered through 0.2 μm filter before injecting into HPLC. The concentration of aflatoxin B1 was determined by HPLC (SCL-10 AVP Shimadzu with fluorescence detector) connected to a reverse phase C-18 column (15 cm and 150 mm). The mobile phase consisted of water−methanol (60:40, v/v), and a flow rate of 1 mL/ min was employed. Detection was achieved by excitation at 365 nm and followed by emission at 425 nm. The retention time was 24.5 min. Evaluation of Antiaflatoxigenic Activity Compounds 1−4. The compounds 1−4 were screened for their antiaflatoxigenic activity in the following way. Potato dextrose broth was prepared, and 10 mL of media was dispensed into 50 mL flasks. The media was autoclaved at 121 °C and was allowed to cool to room temperature. Next 0.1 and 1 mg/mL concentrations of each of the compounds were dissolved in separate flasks containing the media. One flask was kept as positive control without the compound. The experiment was conducted in duplicates. A. flavus 68 was inoculated into all the five flasks and incubated at 28 °C for 7 days. The flasks were observed each day for the changes in the growth pattern and spore formation. Extraction of Aflatoxin. The media was extracted for aflatoxin after 7 days and further analyzed. The mycelial mass was separated, and the media was extracted with equal volume of chloroform (media to chloroform ratio 1:1) during the course of overnight. The chloroform layer was stripped under nitrogen and followed by vacuum. About 100 μL of methanol (HPLC grade) was added to dilute the sample. The extracted sample was subjected to HPLC analysis. The concentration of aflatoxin was calculated based on the standard graph with appropriate experimental corrections. Quantification of Aflatoxin B1 Using HPTLC. The experiment was performed on either 5 cm × 10 cm or 20 cm × 20 cm HPTLC plates pre coated with silica gel 60 having thickness of 0.2 mm (E. Merck, Darmstadt, FRG). Aliquots of 0.5 μL of sample extracts were spotted onto a 5 cm × 10 cm HPTLC plate adjacent to aliquots of standards ranging from 0.1 to 1 mg/mL of aflatoxin B1. The plates were developed in a TLC glass chamber which was pre saturated with chloroform: ethyl acetate (8:2 v/v) for 30 min at room temperature. After chamber saturation, the plates were developed to a distance of 70 mm. Subsequent to development; the plate were air-dried. The plates were scanned using a CAMAG TLC scanner III in reflectance fluorescence mode at 366 nm for all measurements and operated by the winCATS software. Quantification of Aflatoxin Using HPLC. The HPLC procedure employed for the quantification of aflatoxin B1 was carried out according to Ferreira et al.26 with slight modifications. The samples obtained were filtered through 0.2 μm filter before injecting into HPLC. The concentration of aflatoxin B1 was determined by HPLC (SCL-10 AVP Shimadzu with fluorescence detector) connected to a reverse phase C-18 column (15 cm and 150 mm). The mobile phase consisted of water−methanol (60:40, v/v) and a flow rate of 1 mL/ min was employed. Detection was achieved by excitation at 365 nm and followed by emission at 425 nm. The retention time was 24.5 min. Statistics. All the data in tables except Table 3 (consisting of MIC and MBC determination) are represented as mean ± standard deviation. Mean, standard deviation determination and Student’s t test were carried out utilizing Microsoft excel. ANOVA and Tukey− Kramer posthoc test was carried out in GraphPad Prism 6 software. The data on A. flavus growth and as well as percentage reduction of aflatoxin B1 were subjected to one way analysis of variance (ANOVA) followed by Tukey−Kramer posthoc test to separate means on the basis of P < 0.05. Student’s t test was carried out for statistical evaluation of effect of DHB and HMBA for aflatoxin B1 reduction. Student’s t test was also employed for statistical evaluation of effect of Schiff bases (compounds 1−4) for aflatoxin B1 reduction. Biological and technical replicates for each experiment is mentioned in the appropriate paragraph and also in the caption for the table describing the experiments.
RESULTS AND DISCUSSION Antiaflatoxigenic Activity of HMBA and DHB. The compounds 2-hydroxy-4-methoxybenzaldehyde (HMBA) and 2,4-dihydroxybenzaldehyde (DHB) were subjected to evaluation of antiaflatoxigenic activity primarily to understand variation of functional group on antiaflatoxigenic activity. Compound DHB is similar in structure with the only variation being the OH group instead of the methoxy group at the C-2 position. During the experiments, we witnessed dose-dependent inhibition of mycelial growth of A. flavus by HMBA and DHB compounds. The growth of A. flavus was observed for HMBA concentrations of 0.125 and 0.0625 mg/mL and for DHB at 0.25, 0.125, and 0.0625 mg/mL (Table 1). Based on Table 1. Effect of Treatment of Compounds on the Growth of A. flavus and Reduction of Aflatoxin B1a compds positive control HMBA
DHB
concn of compd in mg/mL
growth (%) of A. flavus wrt control 100
0.25 0.125 0.0625 0.5 0.25 0.125 0.0625
ngb 71 ± 2 18.3 ± 1 ngb 67 ± 1 69.8 ± 1 29.8 ± 2.8
% of reduction of aflatoxin B1 0
47.3 ± 3.1 38.4 ± 3.5 61.0 ± 3.9 52.2 ± 2.1 41.4 ± 4.5
a Measurement of growth of Aspergillus f lavus in duplicate and aflatoxin reduction study in triplicate. bng = no growth.
these results, MIC of HMBA was calculated at 0.25 mg/mL and that of DHB at 0.5 mg/mL. Evaluation of antiaflatoxigenic activity quantified as percentage reduction of aflatoxin B1 is represented in the Table 1. HMBA and DHB exhibited about 50% reduction of aflatoxin B1 compared to control (where no compound is added) at a concentration 0.125 mg/mL (Table 1), indicating similar effect with respect to aflatoxin B1 reduction. However, for the concentration of 0.25 mg/mL, DHB displayed 61.0 ± 3.9% reduction of aflatoxin B1, whereas HMBA at this concentration exhibited no growth of A. flavus and subsequent complete reduction of aflatoxin B1, indicating the superior effect of HMBA compared to DHB. Further, the effectiveness of HMBA over DHB as antiaflatoxigenic compound is also substantiated by the observation of MIC of 0.25 mg/mL for HMBA and 0.5 mg/mL for DBH with respect to A. flavus growth. Hence, these results indicate that HMBA is better antiaflatoxigenic compound compared to DHB. Synthesis of Schiff Bases of D-Glucamine with HMBA and Other Aldehydes. Reaction of D-glucamine with 2hydroxy-4-methoxybenzaldehyde, 2,4-dihydroxybenzaldehyde, 2-hydroxy-3-methoxybenzaldehyde, and cinnamaldehyde in asolvent combination MeOH and H2O was smooth and resulted in the formation of Schiff bases (Figure 1). The reaction of D-glucamine with 2,4-dihydroxybenzaldehyde and cinnamaldehyde has been reported earlier.27 All four compounds were characterized by employing multinuclear and multidimensional NMR, IR, and mass spectrometric techniques. The IR spectra clearly revealed presence of imino group attributed to an absorption band at 1657 cm−1 for compound 1, 1641 cm−1 for compound 2, 1642 cm−1 for the compound 3, and 1635 cm−1 for compound 4. All the compounds displayed in the mass spectra peak patterns 8775
DOI: 10.1021/acs.jafc.7b02576 J. Agric. Food Chem. 2017, 65, 8773−8778
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Journal of Agricultural and Food Chemistry
Figure 1. Synthesis of Schiff bases 1−4. Solvent and conditions: MeOH and H2O, RT, 2 h.
corresponding to (M+H)+ at 316.3569, 302.3997, 316.3785, and 296.1456, respectively, for the compounds 1−4. In 1H NMR, the compounds 1−4 exhibited for the imino proton a singlet peak in the chemical shift range 8.25−8.40 ppm. The hydroxyl groups displayed peaks in the region 4.10− 4.9 ppm, whereas the peaks due to glucamine moiety showed up in the region 3.40−3.80 ppm. The 13C NMR exhibited the imino carbon in the range 165.1−166.8 ppm, whereas the glucamine carbon attached to imino group displayed a chemical shift in the range 58.4−63.4 ppm for compounds 1−3. The 13C NMR DEPT-135 experiment clearly demonstrated two methylene carbons in all the four compounds, thereby confirming the acyclic form of these compounds. The three Schiff bases (compounds 1−3) have an O-hydroxyl group (2hydroxyl) and a broad peak around ∼14 ppm seen in 1H NMR spectra, clearly indicating the existence of the intramolecular hydrogen bonding. Further, it is substantiated by the 13C downfield peaks at 168.5 ppm for compound 1, 167.7 ppm for compound 2, and 153.3 ppm for compound 3 ascertained to the carbon attached to hydroxyl group. The O-hydroxy Schiff bases of D-glucamine occur in either the imine form or enamino form. The enamino form is identified by a signature downfield chemical shift of carbon attached to hydroxyl group (about 180 ppm).27 The 13C NMR chemical shift values of compounds accomplished by us clearly indicate that these compounds occur in the imine form. In addition, the stretching absorption bands at 1657, 1641, 1642, and 1635 cm−1 for compounds 1−3 in IR spectra also confirm imino group instead of enamino group as enamino is expected appear much lower than 1635 cm−1.27
Antimicrobial Activity of Schiff Bases. The antimicrobial activity of compounds 1−4 were tested against four food borne pathogens, namely, E. coli, S. typhimurium, S. aureus, and L. monocytogenes by agar well diffusion method. The results are presented in Table 2. Results revealed that compound 1 had Table 2. Antimicrobial Activity of D-Glucamine Schiff Bases against Food Borne Organismsa zone of Inhibition (mm) compd 1 2 3 4 a
E. coli 6 5 5 12
± ± ± ±
S. typhimurium
0.5 1 1.2 0.3
12 9 8 9
± ± ± ±
1.3 0.3 1.2 0.6
S. aureus 2 3 3 9
± ± ± ±
0.2 1 0.5 1.2
L. monocytogenes 12 9 9 10
± ± ± ±
1.0 2 2.5 0.3
Measurement in triplicate.
significant antimicrobial activity against all tested bacterial cultures. Compound 4 was highly effective against E. coli and L. monocytogenes. Compounds 2 and 3 displayed antimicrobial activity against the tested organisms, but their inhibitory effect was less significant compared the compounds 1 and 4. All compounds exhibited poor inhibitory activity against S. aureus. The results of the agar well diffusion method correlated well with results obtained by the microbroth dilution technique (Table 3). The MIC and MBC of compound 1 and 4 against L. monocytogenes was 0.16 and 0.32 mg/mL, respectively, signifying considerable antimicrobial activity of these compounds against the organism. Compound 1 was bactericidal against E. coli and S. typhimurium at a lower concentration (0.32 8776
DOI: 10.1021/acs.jafc.7b02576 J. Agric. Food Chem. 2017, 65, 8773−8778
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Journal of Agricultural and Food Chemistry Table 3. Determination of MIC and MBC for D-Glucamine Schiff Bases against Foodborne Pathogensa MIC (mg/mL)
a
MBC (mg/mL)
organism
1
2
3
4
1
2
3
4
E. coli S. typhimurium S. aureus L. monocytogenes
0.32 0.32 0.64 0.16
0.64 0.64 1.28 0.32
0.64 0.64 1.28 0.64
0.16 0.32 0.32 0.16
0.32 0.32 1.28 0.32
0.64 0.64 1.28 1.28
1.28 1.28 2.56 2.56
0.64 1.28 2.56 0.32
Measurement in triplicate.
a methoxy group at C-4 position imparts a better inhibitor effect for the reduction of aflatoxin B1 compared to cinnamaldehyde derived Schiff base. In conclusion, we accomplished Schiff bases of food derived aldehydes including 2-hydroxy-4-methoxybenzaldehyde, cinnamaldehyde, and other aldehydes. The Schiff base of 2-hydroxy4-methoxybenzaldehyde displayed moderate antimicrobial and antiaflatoxigenic activity.
mg/mL) compared to other compounds. All the compounds showed poor antimicrobial activity against S. aureus with MIC and MBC ≥ 0.64 mg/mL. Although two Gram-positive and two Gram-negative bacteria were selected as candidates for testing the antimicrobial efficacy of the compounds, statistical significance between Gram-positive and Gram-negative bacteria was not observed. Antiaflatoxigenic Activity of Schiff Bases. Antiaflatoxigenic activity was evaluated against A. flavus 68 using a modified method and results are presented in Table 4.
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ASSOCIATED CONTENT
* Supporting Information S
Table 4. Effect of Treatment of Compounds on the Growth of A. flavus and Reduction of Aflatoxin B1a compd positive control 1 2 3 4
concn of compd in mg/mL
growth (%) of A. flavus wrt control 100
0.1 1 0.1 1 0.1 1 0.1 1
9.6 ± 1.9 ngb 15.2 ± 1 ngb 8.9 ± 1.8 36.6 ± 8 10.9 ± 0.3 ngb
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b02576. IR, mass and 1H,13C NMR spectral data of all the Schiff bases (PDF)
% of reduction of aflatoxin B1
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0 91.4 ± 3.9
AUTHOR INFORMATION
Corresponding Author
83.7 ± 4.7
*Phone: +91 821 2512352. Fax: +91 821 2517233. E-mail:
[email protected];
[email protected].
66.2 ± 5
ORCID
Nanishankar V. Harohally: 0000-0003-2306-5897
73.0 ± 2.6
Funding
This work was supported from an in-house CSIR project.
Measurement of growth of A. flavus in duplicate and aflatoxin reduction study in triplicate. bNo growth. a
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge Mr. Mukund and Bhargava for helping in acquisition of mass and IR spectral data. We thank Prof. Ram Rajsekharan, Director CSIR-CFTRI for the support and encouragement.
Initially antiaflatoxigenic activity was evaluated utilizing the concentration 0.1 mg/mL of the compounds wherein, all the compounds exhibited lower growth and different levels of reduction of aflatoxin B1 compared to control. When the concentration was increased to 1 mg/mL growth was observed only in the case of compound 3 (Table 4). Compound 3 at the concentration of 1 mg/mL did show some growth of the organism; however, this resulted in no production of aflatoxin B1. This observation is not new, and it has been shown earlier that growth of A. flavus does not necessarily lead to production of aflatoxin B1.28 On the comparison of compounds at the concentration 0.1 mg/mL for the effect on reduction aflatoxin B1, compound 1 exhibited a statistically significant 91.4 ± 3.9% reduction of aflatoxin B1 and it was the most effective compound among the four Schiff bases. Cinnamaldehyde derived Schiff base (compound 4) did not show much activity compared to other three O-hydroxy Schiff bases, indicating that at least one hydroxyl group (phenolic OH) might be crucial factor. In the case of pure compound cinnamaldehyde, it has been suggested by earlier studies that the alleviation of oxidative stress is the primary factor responsible for the reduction of aflatoxin B1.9 We propose that compound 1 (most effective compound) characterized by the presence of a hydroxyl group at C-2 and
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