Antiaflatoxigenic and Antimicrobial Activities of Schiff Bases of 2

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Anti-aflatoxigenic and antimicrobial activities of Schiff bases of 2hydroxy-4-methoxybenzaldehyde, cinnamaldehyde and similar aldehydes Nanishankar V Harohally, Chris Cherita, Praveena Bhatt, and Anu appaiah K A J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02576 • Publication Date (Web): 24 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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Journal of Agricultural and Food Chemistry

254x190mm (96 x 96 DPI)

ACS Paragon Plus Environment


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Title:

2

Anti-aflatoxigenic

3

methoxybenzaldehyde, cinnamaldehyde and similar aldehydes

and

antimicrobial

activities

of

Schiff

bases

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of

2-hydroxy-4-

4 5

Author Names and affiliations

6

Nanishankar V. Harohally1*, Chris Cherita2, Praveena Bhatt2 Anu Appaiah K.A.2

7

1. Department of Spice and Flavour Science

8

CSIR-CFTRI

9

KRS Road,

10

Mysore 570020

11

Karnataka

12

India

13

2. Microbiology and Fermentation Technology

14

CSIR-CFTRI

15

KRS Road,

16

Mysore 570020

17

Karnataka

18

India

19

Corresponding Author

20

Nanishankar V. Harohally

21

Department of Spice and Flavour Science

22

CSIR-CFTRI

23

KRS Road,

24

Mysore 570020

25

Karnataka 1


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India

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phone: +91 821 2512352

28

Fax: +91 821 2517233

29

email:[email protected]

30

[email protected]

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 2



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Abstract:

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2-hydroxy-4-methoxybenzaldehyde (HMBA) is a non-toxic phenolic flavour from dietary

53

source Decalipus hamiltonii and Hemidesmus indicus. HMBA is an excellent antimicrobial

54

agent with additional anti-aflatoxigenic potency. On the other hand, cinnamaldehyde from

55

cinnamon is widely employed flavour with significant anti-aflatoxigenic activity. We have

56

attempted the enhancement of anti-aflatoxigenic and antimicrobial properties of HMBA,

57

cinnamaldehyde and similar molecules via Schiff base formation accomplished from

58

condensation reaction with amino sugar (D-glucamine). HMBA derived Schiff

59

exhibited commendable anti-aflatoxigenic activity at the concentration 0.1 mg/mL resulting

60

in 9.6±1.9 % growth of Aspergillus flavus and subsequent 91.4±3.9 % reduction of aflatoxin

61

B1 with respect to control.

62

Keywords:

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Aflatoxin, 2-hydroxy-4-methoxybenzaldehyde, cinnamaldehyde, Schiff base, Foodborne

64

pathogen,

65 66 67 68 69 70 71 72

3


bases

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Anti-aflatoxigenic and antimicrobial activities of Schiff bases of

74

2-hydroxy-4-methoxybenzaldehyde, cinnamaldehyde and similar

75

aldehydes

76

Nanishankar V. Harohally1*, Chris Cherita2, Praveena Bhatt2, Anu Appaiah, K.A2

77 78

Introduction

79

The 2-hydroxy-4-methoxybenzaldehyde (HMBA) is a significant and underutilized flavour

80

component from dietary sources including roots of Decalipus hamiltonii and Hemidesmus

81

indicus .1-2 It has shown an excellent antimicrobial property 3 due to presence of phenolic as

82

well as aldehyde group. Further, antioxidant potential of this compound has attracted lot of

83

attention possibly because of water solubility.4-6 In addition, due to unique structural

84

attributes, HMBA has been evaluated as tyrisinase inhibitor and also it has shown greater

85

activity against Helicobacter pylori.7 HMBA is also documented for its anti-aflatoxigenic

86

potency.8 On the other hand, cinnamaldehyde is a well explored anti-aflatoxigenic agent and

87

its mechanism has been recently evaluated.9

88

A diverse class of compounds composed of coumarins, flavonoids, alkaloids and terpenoids

89

have been evaluated as inhibitors for aflatoxin biosynthesis.10 In addition, plant derived as

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well as food derived array of essential oils have shown commendable anti-aflatoxigenic

91

activity.11-18. Apart from the essential oils, few extracts mainly tea and Garcinia have

92

exhibited substantial activity against Aspergillus flavus growth as well as inhibition of

93

aflatoxin production.19-20 Few natural products, in particular piperonal has been shown to

94

regulate even the biosynthesis of aflatoxin B1 production.21 Further, few synthetic compounds

95

have also displayed significant anti-aflatoxigenc activity.22 Schiff bases of amino acids in

96

particular L-serine, L-threonine, and L-tyrosine and also there Mn(III) complexes have 4



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shown excellent anti-aflatoxigenic activity.23 There is a need for development of anti-

98

aflatoxigenic agents derived from molecules exhibiting substantial activity as it encompasses

99

structural attributes responsible for activity and also provide opportunities for further

100

enhancement. In this context, we set out with a goal to utilize D-glucamine as an amino sugar

101

to transform food derived 2-hydroxy-4-methoxybenzaldehyde, cinnamaldehyde and similar

102

aldehydes including 2,4-dihydroxybenzaldehyde and 2-hydroxy-3-methoxybenzaldehyde to

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Schiff bases for the enhancement of antimicrobial and anti-aflatoxigenic properties. We

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discuss synthesis, anti-aflatoxigenic and antimicrobial activities of Schiff bases of HMBA,

105

cinnamaldehyde and similar aldehydes.

106

Materials and Methods

107

Chemicals 2-hydroxy-4-methoxybenzaldehyde

108

(98%),

cinnamaldehyde

(98%)

and

2,4-

109

dihydroxybenzaldehyde (98%) and 2-hydroxy-3-methoxybenzaldehyde (99% ) and D2O were

110

procured from Sigma-Aldrich India. Methanol (99.8 %) and D-glucamine (> 95%) were

111

purchased from TCI chemicals. Solvents were used without any purification.

112

Spectroscopic measurements

113

NMR spectral data acquisition was accomplished on a Bruker Avance spectrometer

114

having frequency 400 MHz for 1H and 100 MHz for 13C. 1H chemical shift is referenced to

115

internal HOD signal (4.79 ppm) and

116

tetramethylsilane in D2O. NMR spectra assignments to accomplished compounds were based

117

1D techniques including

118

transfer), HSQC (heteronuclear single quantum coherence) and HMBC (heteronuclear

119

multiple bond correlation) experiments. Mass spectral data (ESI positive mode) was achieved

120

in the instrument Q-TOF ULTIMA of Waters Corporation. IR spectral data were recorded

121

in the Thermo FT–IR instrument.

1

H,

13

13

C chemical shift is referenced to external standard

C, DEPT (distortionless enhancement by polarization

5


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122


1-Deoxy-1-(2-hydroxy-4-methoxybenzylidene)amino-D-glucitol (1)

123

In an Erlenmeyer flask containing a magnetic stir bar, D-glucamine (1 g, 5.5 mmol)

124

was dissolved in water (7 mL). HMBA (0.837 g, 5.5 mmol) was dissolved in 4 mL of

125

methanol and then transferred to solution containing D-glucamine. Subsequently, reaction

126

mixture was allowed to stir at room temperature (30 ºC) till precipitation occurred (about

127

2h). Then, the precipitate was filtered and washed with water followed by acetone and diethyl

128

ether to get light yellow coloured solid. Yield=1.485 g, 85%. IR (KBr) υmax 3400-2900 (OH),

129

1657 (C=N), 1610, 1564 (Aryl), 1127,1087,1031 cm-1 (C-O). ESI-MS Positive mode (M+H)+

130

m/z 316.1390, exact mass (M+H)+ 316.1397. 1H NMR δH: 3.74 (s, 3H, H3C(14)), 7.22 (d,

131

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,

132

HC(10)), 8.28 (s, 1H, HC(7)), 3.58 (m, 1H, H2C(6)), 3.40 (m, 1H, H2C(6)), 3.47 (m, 1H,

133

HC(5)), 3.48 (m, 1H, HC(4)), 3.64 (m, 1H, HC(3)), 3.76 (m, 1H, HC(2)), 3.43 (m, 1H,

134

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

135

(m, 1H, OH). 13C NMR δC: 55.1 (C-14), 133.4 (C-13), 105.6 (C-12), 163.8 (C-11), 101.3 (C-

136

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),

137

72.2 (C-2), 58.4 (C-1).

138

1-Deoxy-1-(2,4-dihydroxybenzylidene)amino-D-glucitol (2)

139

Synthesis of this Schiff base (compound 2) was accomplished similar to compound 1

140

by employing D-glucamine (1 g, 5.5 mmol) and 2,4-dihydroxybenzaldehyde (0.760 g, 5.5

141

mmol). Yield=1.390 g, 84%. IR (KBr) υmax

142

(Aryl), 1095,1060 cm-1 (C-O). ESI-MS Positive mode (M+H)+ m/z 302.1289, exact mass

143

(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

144

= 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,

145

H2C(6)), 3.39 (m, 1H, H2C(6)), 3.45 (m, 1H, HC(5)), 3.47 (m, 1H, HC(4)), 3.63 (m, 1H,

146

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),

3400-2900 (OH), 1641 (C=N), 1624,1592

6



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147

4.36 (m, 1H, OH), 4.47 (m, 1H, OH), 4.83 (m, 1H, OH). 13C NMR δC: 133.5 (C-13), 106.4

148

(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

149

(C-5), 71.6 (C-4), 70.0 (C-3), 72.3 (C-2), 59.2 (C-1).

150

1-Deoxy-1-(2-hydroxy-3-methoxybenzylidene)amino-D-glucitol (3)

151


152

by employing D-glucamine (1 g, 5.5 mmol) and 2-dihydroxy-3-methoxybenzaldehyde (0.837

153

g, 5.5 mmol). Yield=1.480 g, 85%. IR (KBr) υmax 3400-2900 (OH), 1642 (C=N), 1601, 1540

154

(Aryl), 1125,1085,1046 cm-1 (C-O). ESI-MS Positive mode (M+H)+ m/z 316.1407, exact

155

mass (M+H)+ 316.1397. 1H NMR δH: 3.76 (s, 3H, H3C(14)), 6.98 (s, 1H, J = 8.0 Hz,

156

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

157

(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,

158

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,

159

H2C(1)), 4.35 (m, 1H, OH), 4.39 (m, 1H, OH), 4.49 (m, 1H, OH), 4.86 (m, 1H, OH).

160

NMR δC: 55.7 (C-14), 114.6 (C-13), 116.9 (C-12), 123.3 (C-11), 148.4 (C-10), 153.3 (C-9),

161

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-

162

1).

163

1-Cinnamylidenamino-1-deoxy-D-glucitol (4)

13

C

164


165

by employing D-glucamine (1 g, 5.5 mmol) and cinnamaldehyde (0.727 g, 5.5 mmol).

166

Yield=1.385 g, 85%. IR (KBr) υmax 3400-3000 (OH), 1634 (C=N),1556 (Aryl), 1089,1056

167

cm-1 (C-O). ESI-MS Positive mode (M+H)+ m/z, 296.1456, exact mass 296.1498. 1H NMR

168

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

169

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,

170

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,

171

HC(7)), 3.45 (m, 2H, H2C(6)), 3.59 (m, 1H, HC(5)), 3.38 (m, 1H, HC(4)), 3.50 (m, 1H, 7


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172

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

173

(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,

174

C(5)-OH), 4.32 (t, 1H, J = 5.6 Hz, C(6)-OH).

175

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-

176

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).

177

Evaluation of antimicrobial activity

178

Bacterial strains

13

C NMR δC: 127.2 (C-15), 128.8 (C-14),

179

The bacterial cultures employed in the current study consisted of Escherichia coli

180

(ATCC11775), Salmonella typhimurium (ATCC 25241), Staphylococcus aureus (ATCC

181

12600) and Listeria monocytogenes (MTCC 1143). The cultures were grown in Brain Heart

182

Infusion Broth (BHIB, HiMedia Laboratories Pvt Ltd, India) under aerobic conditions at 37

183

°C. For the antimicrobial assays, Mueller Hinton broth and agar media (MH, HiMedia

184

Laboratories Private Limited, India) was utilized. Aspergillus flavus 68 strain of CFTRI was

185

used for the production of aflatoxin. The strain was revived and sub-cultured on Potato

186

Dextrose Agar (PDA). The culture was maintained on PDA.

187

Antimicrobial assay

188

The antimicrobial assay for the synthesized compounds was carried out by the agar

189

well diffusion method described by Owais et al. 24 with slight modification. Briefly, a culture

190

of the pathogens(overnight grown) was subjected to centrifugation (4000 rpm, 20 mins).

191

Subsequently, the pellets obtained were suspended in saline and adjusted to 0.5 Mc Farland

192

standard (3 X 105 cells/mL). The bacterial inoculum was then spread onto Mueller Hinton

193

agar media. Wells having diameter of 7 mm were punched in the agar and subsequently

194

packed with the compounds(to be tested) at an array of concentrations (5-50 mg/ml). Control

195

wells were also made in a similar fashion and subsequently filled with neat DMSO as

196

negative control and streptomycin sulphate (1000 U/mL) as positive control in the same 8



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197

plates. The plates were incubated at 37°C for a period of 24 h. Antimicrobial activity was

198

determined via measurement of diameter of the zone of inhibition for all the compounds.

199

Measurement was done in triplicate.

200

Assay was also carried out to determine the minimum inhibitory concentration (MIC)

201

and the minimum bactericidal concentration (MBC) by the broth micro dilution method.25

202

MIC and MBC measurement was done in triplicate. Briefly, serial 2-fold dilutions of the test

203

compounds were prepared in DMSO, and 100 µL of each dilution was added to 96 well

204

microtitre plates containing 100 µL of MH medium with inoculum of 1 X 105 cells/mL. The

205

plates were incubated at 37°C for a period of 24 h and MIC was evaluated as the least

206

concentration of the compound that demonstrated no visible growth. Subsequent to the

207

determination of MIC, 10 fold dilutions from each well showing no turbidity were added to

208

fresh MH broth (5000 µL) taken in tubes and incubated at 37°C for 48 h. An aliquot of this

209

was also plated onto nutrient agar plates and incubated for a period of 24 h at 37°C. The

210

MBC was the least concentration of the compound that displayed no visible growth in the

211

broth as well as absence of bacterial colonies on the agar plates.

212

Anti-aflatoxigenic activity of compounds The

213

compounds

2-hydroxy-4-methoxybenzaldehyde

(HMBA)

and

2,4-

214

dihydroxybenzaldehyde (DHB) were screened for their anti-aflatoxigenic activity in the

215

following way. Potato dextrose broth was prepared and autoclaved at 121˚C. The media was

216

allowed to equilibrate to room temperature and 3 mL was dispensed into each well in a 6-well

217

sterile cell culture plates. The experiment was conducted by modifying the broth micro

218

dilution method. The test compound was tested from 1 mg/mL to 0.0625 mg/mL

219

concentration. Experiments were conducted in triplicate. Appropriate positive control was

220

included in the experiment. Aspergillus flavus 68 (106 spores /ml) was inoculated into all the

9


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221

wells in the plate and incubated at 28˚C for 7 days. The cell culture plates were observed each

222

day for the changes in the growth pattern and spore formation.

223

Weight of the biomass

224

After 7 days of growth period, the fungal mat was separated from the media, washed

225

with distilled water. The mat was then heated overnight at 100o C to obtain the dry biomass.

226

The dry fungal mat was weighed to determine the change in the growth of the fungi

227

Aspergillus flavus.

228

Extraction of aflatoxin

229

The media was extracted for aflatoxin after 7 days and further analysed. The mycelial

230

mass was separated and the media was extracted with equal volume of chloroform (media to

231

chloroform ratio 1:1) during the course of overnight. The chloroform layer was stripped

232

under nitrogen and followed by vacuum. About 100 µL of methanol (HPLC grade) was

233

added to dilute the sample. The extracted sample was subjected to HPLC analysis. The

234

concentration of aflatoxin was calculated based on the standard graph with appropriate

235

experimental corrections.

236

Quantification of aflatoxin using HPLC The HPLC procedure employed for the quantification of aflatoxin B1 was carried out

237

26

238

according to Ferreira et al

with slight modifications. The samples obtained were filtered

239

through 0.2 µm filter before injecting into HPLC. The concentration of aflatoxin B1 was

240

determined by HPLC (SCL-10 AVP Shimadzu with fluorescence detector) connected to a

241

reverse phase C-18 column (15 cm and 150mm). The mobile phase consisted of water-

242

methanol (60:40, v/v) and a flow rate of 1 mL/min was employed. Detection was achieved by

243

excitation at 365 nm and followed by emission at 425 nm. The retention time was 24.5 min.

244

Evaluation of anti-aflatoxigenic activity compounds 1-4

10



245

The compounds 1, 2, 3 and 4 were screened for their anti-aflatoxigenic activity in the

246

following way. Potato dextrose broth was prepared and 10 mL media was dispensed into 50

247

mL flasks. The media was autoclaved at 121˚C and was allowed to cool to room temperature.

248

0.1 mg/mL and 1 mg/mL concentration of each of the compounds were dissolved in separate

249

flasks containing the media. One flask was kept as positive control without the compound.

250

Experiment was conducted in duplicates. Aspergillus flavus 68 was inoculated into all the

251

five flasks and incubated at 28˚C for 7 days. The flasks were observed each day for the

252

changes in the growth pattern and spore formation.

253

Extraction of aflatoxin

254

The media was extracted for aflatoxin after 7 days and further analysed. The mycelial

255

mass was separated and the media was extracted with equal volume of chloroform (media to

256

chloroform ratio 1:1) during the course of overnight. The chloroform layer was stripped

257

under nitrogen and followed by vacuum. About 100 µL of methanol (HPLC grade) was

258

added to dilute the sample. The extracted sample was subjected to HPLC analysis. The

259

concentration of aflatoxin was calculated based on the standard graph with appropriate

260

experimental corrections.

261

Quantification of aflatoxin B1 using HPTLC

262

The experiment was performed on either 5 cm × 10 cm or 20 cm × 20 cm HPTLC

263

plates pre coated with silica gel 60 having thickness of 0.2 mm (E. Merck, Darmstadt, FRG).

264

Aliquots of 0.5 µL of sample extracts were spotted onto a 5 cm × 10 cm HPTLC plate

265

adjacent to aliquots of standards ranging from 0.1 mg/mL to 1 mg/mL of aflatoxin B1. The

266

plates were developed in a TLC glass chamber which was pre saturated with chloroform:

267

ethyl acetate (8:2 v/v) for 30 mins at room temperature. After chamber saturation, the plates

268

were developed to a distance of 70 mm. Subsequent to development; the plate were air dried.

11


Page 12 of 25

Page 13 of 25


269

The plates were scanned using a CAMAG TLC scanner III in reflectance fluorescence mode

270

at 366 nm for all measurements and operated by the winCATS software.

271

Quantification of aflatoxin using HPLC The HPLC procedure employed for the quantification of aflatoxin B1 was carried out

272

26

273

according to Ferreira et al

with slight modifications. The samples obtained were filtered

274

through 0.2 µm filter before injecting into HPLC. The concentration of aflatoxin B1 was

275

determined by HPLC (SCL-10 AVP Shimadzu with fluorescence detector) connected to a

276

reverse phase C-18 column (15 cm and 150mm). The mobile phase consisted of water-

277

methanol (60:40, v/v) and a flow rate of 1 mL/min was employed. Detection was achieved by

278

excitation at 365 nm and followed by emission at 425 nm. The retention time was 24.5 min.

279

Statistics

280

All the data in tables except table 3 (consisting of MIC and MBC determination) are

281

represented as mean ± standard deviation. Mean, standard deviation determination and

282

student t test were carried out utilizing Microsoft excel. ANOVA and Tukey-Kramer post-

283

hoc test was carried out in GraphPad Prism 6 software. The data on Aspergillus flavus growth

284

and as well as percentage reduction of aflatoxin B1 were subjected to one way analysis of

285

variance (ANOVA) followed by Tukey-Kramer post-hoc test to separate means on the basis

286

of P < 0.05. Student t test was carried out for statistical evaluation of effect of DHB and

287

HMBA for aflatoxin B1 reduction. Student t test was also employed for statistical evaluation

288

of effect of Schiff bases (compounds 1-4) for aflatoxin B1 reduction. Biological and technical

289

replicates for each experiment is mentioned in the appropriate paragraph and also in the

290

caption for the table describing the experiments.

291

Results and Discussion

292

Anti-aflatoxigenic activity of HMBA and DHB

12



The

293

compounds


(HMBA)

Page 14 of 25

and

2,4-

294

dihydroxybenzaldehyde (DHB) were subjected to evaluation of anti-aflatoxigenic activity

295

primarily to understand variation of functional group on anti-aflatoxigenic activity.

296

Compound DHB is similar in structure with only variation being the OH group instead of

297

methoxy group at the C-2 position. During the experiments, we witnessed dose-dependent

298

inhibition of mycelial growth of Aspergillus flavus by HMBA and DHB compounds. The

299

growth of Aspergillus flavus was observed for HMBA concentration of 0.125 and 0.0625

300

mg/mL and for DHB at 0.25, 0.125 and 0.0625 mg/mL (Table 1). Based on these results

301

MIC of HMBA was calculated at 0.25 mg/mL and that of DHB at 0.5 mg/mL. Evaluation of

302

anti-aflatoxigenic activity quantified as percentage reduction of aflatoxin B1 is represented

303

in the Table 1. HMBA and DHB exhibited about 50 % reduction of aflatoxin B1 compared

304

to control (where no compound is added) at concentration 0.125 mg/mL (Table 1) indicating

305

similar effect with respect to aflatoxin B1 reduction. However, for the concentration of 0.25

306

mg/mL, DHB displayed 61.0±3.9 % reduction of aflatoxin B1 whereas, HMBA at this

307

concentration exhibited no growth of Aspergillus flavus and subsequent complete reduction

308

of aflatoxin B1 indicating the superior effect of HMBA compared to DHB. Further, the

309

effectiveness of HMBA over DHB as anti-aflatoxigenic compound is also substantiated by

310

the observation of MIC of 0.25 mg/mL for HMBA and 0.5 mg/mL for DBH with respect to

311

Aspergillus flavus growth. Hence, these results indicate that HMBA is better anti-

312

aflatoxigenic compound compared to DHB.

313

Synthesis of Schiff bases of D-glucamine with HMBA and other aldehydes

314

Reaction

of

D-glucamine

with

2-hydroxy-4-methoxybenzaldehyde,

2,4-

315

dihydroxybenzaldehyde, 2-hydroxy-3-methoxybenzaldehyde and cinnamaldehyde, in solvent

316

combination MeOH and H2O was smooth and resulted in the formation of Schiff bases

317

(Scheme 1). The reaction of D-glucamine with 2,4-dihydroxybenzaldehyde and 13


Page 15 of 25


318

cinnamaldehyde has been reported earlier.27

319

Figure 1

320

All the four compounds were characterized by employing multinuclear and multi-

321

dimensional NMR, IR and mass spectrometric techniques. The IR spectra clearly revealed

322

presence of imino group attributed to an absorption band at 1657 cm-1 for compound 1, 1641

323

cm-1 for compound 2, 1642 cm-1 for the compound 3 and 1635 cm-1 for compound 4. All the

324

compounds displayed in the mass spectra peak patterns corresponding to (M+H)+

325

316.3569, 302.3997, 316.3785 and 296.1456 respectively for the compounds 1, 2, 3 and 4.

at

326

In 1H NMR, the compounds 1, 2, 3 & 4 exhibited for the imino proton a singlet peak in the

327

chemical shift range 8.25–8.40 ppm. The hydroxyl groups displayed peaks in the region

328

4.10–4.9 ppm, whereas, the peaks due to glucamine moiety showed up in the region 3.40–

329

3.80 ppm. The 13C NMR exhibited the imino carbon in the range 165.1–166.8 ppm, whereas,

330

the glucamine carbon attached to imino group displayed chemical shift in the range 58.4–63.4

331

ppm for compounds 1–3. The

332

methylene carbons in all the four compounds, there by confirming the acyclic form of these

333

compounds. The three Schiff bases ( compounds 1, 2, and 3) have O-hydroxyl group (2-

334

hydroxyl) and a broad peak around ~14 ppm seen in 1H NMR spectra accounted for it,

335

clearly indicated the existence of the intramolecular hydrogen bonding. Further, it is

336

substantiated by the

337

compound 2 and 153.3 ppm for compound 3 ascertained to the carbon attached to hydroxyl

338

group. The O-hydroxy Schiff bases of D-glucamine occur either in the imine form or

339

enamino form. The enamino form is identified by signature downfield chemical shift of

340

carbon attached to hydroxyl group (about 180ppm) (Avalos et al.,2008)17. The

341

chemical shift values of accomplished compounds by us clearly indicate that these

342

compounds occur in the imine form. In addition, the stretching absorption bands at 1657,

13

13

C NMR DEPT-135 experiment clearly demonstrated two

C down field peaks at 168.5 ppm for compound 1, 167.7 ppm for

14


13

C NMR


343

1641, 1642 & 1635 cm-1 for compounds 1, 2, & 3 in IR spectra also confirm imino group

344

instead of enamino group as enamino is expected appear much lower than 1635 cm-1((Avalos

345

et al.,2008).27

346

Antimicrobial activity of Schiff bases

347

The antimicrobial activity of compounds 1, 2, 3 and 4 were tested against four food

348

borne pathogens namely E. coli, S. typhimurium, S. aureus and L. monocytogenes by agar

349

well diffusion method. The results are presented in Table 2. Results revealed that compound

350

1 had significant antimicrobial activity against all tested bacterial cultures. Compound 4 was

351

highly effective against E. coli and L. monocytogenes. Compounds 2 and 3 displayed

352

antimicrobial activity against the tested organisms but their inhibitory effect was less

353

significant compared the compounds 1 and 4. All compounds exhibited poor inhibitory

354

activity against S. aureus. The results of the agar well diffusion method correlated well with

355

results obtained by the microbroth dilution technique (Table 3). The MIC and MBC of

356

compound 1 and 4 against L. monocytogenes was 0.16 and 0.32 mg/ml, respectively,

357

signifying a considerable antimicrobial activity of these compounds against the organism.

358

Compound 1 was bactericidal against E. coli and S. typhimurium at a lower concentration

359

(0.32 mg/mL) compared to other compounds. All the compounds showed poor antimicrobial

360

activity against S. aureus with MIC and MBC ≥ 0.64 mg/mL. Although, two gram positive

361

and two gram negative bacteria were selected as candidates for testing the antimicrobial

362

efficacy of the compounds, statistically significance between gram positive and gram

363

negative bacteria was not observed.

364

Anti-aflatoxigenic activity of Schiff bases Anti-aflatoxigenic activity was evaluated against Aspergillus flavus 68 using a

365 366

modified method and results are presented in Table 4.

15


Page 16 of 25

Page 17 of 25


367

Initially anti-aflatoxigenic activity was evaluated utilizing the concentration 0.1

368

mg/mL of the compounds wherein, all the compounds exhibited lower growth and different

369

levels of reduction of aflatoxin B1

370

increased to 1 mg/mL growth was observed only in the case of compound 3 (Table 4).

371

Compound 3 at the concentration of 1mg/mL did show some growth of the organism,

372

however, resulted in none production of aflatoxin B1. This observation is not new and it has

373

been earlier shown that growth of Aspergillus flavus not necessarily lead to production of

374

aflatoxin B1.28

compared to control. When the concentration was

375

On the comparison of compounds at the concentration 0.1 mg/mL for the effect on

376

reduction aflatoxin B1, compound 1 exhibited statistically significant 91.4±3.9 % reduction of

377

aflatoxin B1 and it was the most effective compound among the four Schiff bases.

378

Cinnamaldehyde derived Schiff base (compound 4) did not show much activity compared to

379

other three O-hydroxy Schiff bases indicating that at least one hydroxyl group (Phenolic OH)

380

might be crucial factor. In the case of pure compound cinnamaldehyde, it has been suggested

381

by earlier studies that the alleviation of oxidative stress is the primary factor responsible for

382

the reduction of aflatoxin B1.9 We propose that compound 1 (most effective compound)

383

characterized by presence of hydroxyl group at C-2 and methoxy group at C-4 position

384

imparts better inhibitor effect for the reduction of aflatoxin B1 compared to cinnamaldehyde

385

derived Schiff base.

386

In conclusion, we accomplished Schiff bases of food derived aldehydes including 2-

387

hydroxy-4-methoxybenzaldehyde, cinnamaldehyde and other aldehydes. The Schiff base of

388

2-hydroxy-4-methoxybenzaldehyde displayed moderate antimicrobial and anti-aflatoxigenic

389

activity.

390

Acknowledgments

16



391

This work was supported from an in-house CSIR project. We acknowledge Mr.

392

Mukund and Bhargava for helping in acquisition of mass and IR spectral data. We thank

393

Prof.Ram Rajsekharan, Director CSIR-CFTRI for the support and encouragement.

394

Supporting information The supporting information consisting of IR, Mass and 1H,13C NMR spectral data of

395 396

all the Schiff bases is available free of charge in the website.

397

References

398

(1) Nagarajan, S.; Rao, L.J.M.; Gurudutt, K.N. Chemical composition of the volatiles of

399

Decalepis hamiltonii (Wight & Arn). Flavour Frag J. 2001, 16, 27–29.

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(2) Nagarajan, S.; Rao, L.J.M. Determination of 2-hydroxy-4-methoxybenzaldehyde in roots

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of Decalepis hamiltonii (Wight & Arn.) and Hemidesmus indicus R.Br. J. AOAC int

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2003, 86, 564–567.

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(3) Wang, J.; Liu, H.; Zhao, J.; Gao, H.; Zhou, L.; Liu, Z.; Chen, Y.; Sui, P. Antimicrobial

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and antioxidant activities of the root bark essential oil of Periploca sepium and its main

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component 2-Hydroxy-4-methoxybenzaldehyde. Molecules 2010, 15, 5807–5817.

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(4) Murthy, K.N.; Rajasekaran, T.; Giridhar, P.; Ravishankar. G.A. Antioxidant activity

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property of Decalepis hamiltonii (Wight & Arn). Indian J Exp Biol 2006, 44, 832–837.

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(5) Srivastava, A.; Harish, S.R.; Shivanandappa, T. Antioxidant activity of roots of Decalepis

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hamiltonii (Wight & Arn). LWT-Food Science and Technology 2006, 39, 1059–1065.

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(6) Srivastava, A.; Rao, L.J.M.; Shivanandappa, T. Isolation of ellagic acid from the aqueous

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extract of the roots of Decalepis hamiltonii: Antioxidant activity and cytoprotective effect.

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Food Chem. 2007, 103, 223–233.

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(7) Srikanta, B.M.; Harish Nayaka, M.A.; Dharmesh, S.M. Inhibition of Helicobacter

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pylori growth and its cytotoxicity by 2-hydroxy 4-methoxy benzaldehyde of Decalepis

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hamiltonii (Wight & Arn); a new functional attribute. Biochimie, 2011, 93, 678–688. 17


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(8) Mohana, D.C.; Raveesha, K.A.; Antimycotic, antibiodeteriorative and anti-aflatoxigenic

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potency of 2-hydroxy-4-methoxybenzaldehyde isolated from Decalepis hamiltonii on fungi

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causing bio deterioration of maize and sorghum grains. J Mycol Pl Pathol 2010, 40(2), 197–

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(9) Sun, Q.; Wang, L.; Lu, Z.; Liu, Y. In vitro anti-aflatoxigenic effect and mode of action of

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cinnamaldehyde against aflatoxin B1. Int Biodeter Biodegr 2015, 104, 419–425.

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(10) Holmes, R.A.; Boston, R.A.; Payne, G.A. Diverse inhibitors of aflatoxin biosynthesis.

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Appl Microbiol Biotechnol 2008, 78, 559–572.

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(11) Prakash, B.; Singh, P.; Goni, R.; Raina, A.K.P.; Dubey, N.K. Efficacy of Angelica

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archangelica essential oil, phenyl ethyl alcohol and α- terpineol against isolated molds from

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walnut and their antiaflatoxigenic and antioxidant activity. J Food Sci Technol 2015, 52(4),

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2220–2228.

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(12) Shukla, R.; Singh, P.; Prakash, B.; Dubey, N.K. Efficacy of Acorus calamus L. essential

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oil as a safe plant-based antioxidant, Aflatoxin B1 suppressor and broad spectrum

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antimicrobial against food-infesting fungi. Int J Food Sci Tech 2013, 48, 128–135.

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(13) Prakash, B.; Singh, P.; Mishra, P.K.; Dubey, N.K. Safety assessment of Zanthoxylum

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alatum Roxb. essential oil, its antifungal, antiaflatoxin, antioxidant activity and efficacy as

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antimicrobial in preservation of Piper nigrum L. fruits. Int Food Microbiol 2012, 153, 183–

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(14) Ali, E.M. Phytochemical composition, antifungal, antiaflatoxigenic, antioxidant, and

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anticancer activities of Glycyrrhiza glabra L. and Matricaria chamomilla L. essential oils. J.

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Med. Plant Res. 2013, 7(129), 2197–2207.

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(15) Ferreira, F.D.; Kemmelmeier, C.; Arrotéia, C.C.; Da Costa C.L.; Mallmann, C.C.;

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Janeiro V.; Dias Ferreira, F.M.; Galerani Mossini, S.A.; Silva, E.L.; Machinski Jr, M.

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440

Inhibitory effect of the essential oil of Curcuma longa L. and curcumin on aflatoxin

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production by Aspergillus flavus Link. Food Chem 2013, 136, 789–793.

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(16) Gibriel, Y.A.Y.; Hamza, A.S.; Gibriel, A.Y.; Mohsen, S.M. In vivo effect of Mint

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(Mentha virdis) essential oil on growth and Aflatoxin production by Aspergillus flavus

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isolated from stored corn. J. Food Saf 2011, 31, 445–451.

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(17) Prakash, B.; Shukla , R.; Singh, P.; Mishra, P.K.; Dubey, N.K.; Kharwar, R.N. Efficacy

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of chemically characterized Ocimum gratissimum L. essential oil as an antioxidant and a safe

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plant based antimicrobial against fungal and aflatoxin B1 contamination of spices. Food Res

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Int 2011, 44, 385–390.

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(18) Kedia, A.; Prakash, B.; Mishra, P.K.; Dubey, N.K.; Antifungal and antiaflatoxigenic

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properties of Cuminum cyminum (L.) seed essential oil and its efficacy as a preservative in

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stored commodities. Int J Food Microbiol 2014, 168-169, 1–7

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(19) Mo, H.Z.; Zhang, H.; Wu, Q.H.; Hu, L.B. Inhibitory effects of tea extract on aflatoxin

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production by Aspergillus flavus. Lett Appl Microbiol 2013, 56, 462–466

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(20) Joseph G.S.; Jayaprakasha, G.K.; Selvi, A.T.; Jena, B.S.; Sakaraiah, K.K.

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Antiaflatoxigenic and antioxidant activities of Garcinia extracts. Int J Food Microbiol 2005,

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101, 153–160

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(21) Park, E.S.; Bae, I.K.; Kim, H.J.; Lee, S.E. Novel regulation of aflatoxin B1 biosynthesis

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in Aspergillus flavus by piperonal. Nat. Prod. Res 2016, 30(16), 1854–1857

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(22) Moon, Y.S.; Choi, W.S.; Park, E.S.; Bae, I.K.; Choi, S.D.; Paek, O.; Kim, S.H.; Chun

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H.S.; Lee, S.E. Antifungal and antiaflatoxigenic methylenedioxy containing compounds and

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piperine-like synthetic compounds. Toxins 2016, 8(240), 2–10

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(23) Anar, M.; Ozcan, E. H.; Oğutcu, H.; Agar, G.; Sakiyan, I.; Sari, N. Useful agents against

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aflatoxin B1–antibacterial azomethine and Mn(III) complexes involving L-Threonine, L-

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Serine, and L-Tyrosine. Artificial cells, Nanomedicine and Biotechnology, 2016, 44(3), 678–

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(24) Owais, M.; Sharad, K. S.; Shehbaz, A.; Saleemuddin, M. Antibacterial efficacy of

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Withania somnifera (ashwagandha), an indigenous medicinal plant against experimental

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murine salmonellosis. Phytomedicine, 2005, 12, 229–235.

469

(25) CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow

470

Aerobically. Approved Standard, CLSI document 8th Ed, Clinical and Laboratory Standards

471

Institute 2009, 29, M07–A8.

472

(26) Ferreira, I. M.; Mendes, E.; Oliveira, M. B. Quantification of aflatoxins B1, B2, G1, and

473

G2 in pepper by HPLC/Fluorescence. J of Liq. Chromatogr. Related Technol 2004, 27, 325–

474

334.

475

(27) Avalos, M.; Babiano, R.; Cintas, P.; Jimenez, J. L.; Light, M. E.; Palacios, J.C.; Perez,

476

E.M. Chiral N-Acyloxazolidines: synthesis, structure, and mechanistic Insights. J. Org. Chem

477

2008, 73, 661–672.

478

(28) Klich A.M. Environmental and developmental factors influencing aflatoxin production

479

in Aspergillus flavus and Aspergillus paraciticus. Mycoscience 2007, 48, 71–80.

480 481 482 483 484 485 486 487 488 20



Page 22 of 25

489 490 491 492 493

H2C

N OH H OH OH

HO

CH2OH

4 Cinnamaldehyde

H2C HO

N OH H OH OH

H2C OMe HO

HO


NH2 OH H OH OH O-vanillin

H2C HO

CH2OH

CH2OH

N OH H OH OH

CH2OH

HO MeO

3

D-glucamine 2,4-dihydroxybenzaldehyde

1 H2C HO

N OH H OH OH

OH HO

CH2OH

2 494 495 496

Figure 1 : Synthesis of Schiff bases 1-4. Solvent and conditions: MeOH and H2O, RT, 2h

497 21


Page 23 of 25


498 499 500 501 502

Table 1: Effect of treatment of compounds on the growth of Aspergillus flavus and

503

reduction of aflatoxin B1 Compounds

Concentration of the

Growth ( %) of

compound in mg/mL

Aspergillus flavus

% of reduction of Aflatoxin B1

w.r.t control Positive control

-

100

0

HMBA

0.25

ng*

-

0.125

71±2

47.3±3.1

0.0625

18.3±1

38.4±3.5

0.5

ng*

-

0.25

67±1

61.0±3.9

0.125

69.8±1

52.2±2.1

0.0625

29.8±2.8

41.4±4.5

DHB

504

Measurement of growth of Aspergillus flavus in duplicate and aflatoxin reduction study in

505

triplicate

506

ng* = no growth

507

Table 2:

508

organisms

Antimicrobial activity of

Compounds

D-glucamine Schiff bases against

Escherichia Salmonella coli typhimurium

Staphylococc us aureus

Listeria monocytogenes

Zone of Inhibition (mm) Compound 1

6 ± 0.5

12 ± 1.3

2 ± 0.2

12 ± 1.0

Compound 2

5±1

9 ± 0.3

3±1

9±2

22


food borne


509

Compound 3

5 ± 1.2

8 ± 1.2

3 ± 0.5

9 ± 2.5

Compound 4

12 ± 0.3

9 ± 0.6

9 ± 1.2

10 ± 0.3

Page 24 of 25

Measurement in triplicate

510 511

Table 3 :Determination of MIC and MBC for D-glucamine Schiff bases against

512

foodborne pathogens Organisms

MIC (mg/mL)

MBC (mg/mL)

Compounds

Compounds

1

2

3

4

1

2

3

4

0.32

0.64

0.64

0.16

0.32

0.64

1.28

0.64

Salmonella typhimurium 0.32

0.64

0.64

0.32

0.32

0.64

1.28

1.28

Staphylococcus aureus

0.64

1.28

1.28

0.32

1.28

1.28

2.56

2.56

Listeria monocytogenes

0.16

0.32

0.64

0.16

0.32

1.28

2.56

0.32

Escherichia coli

513

Measurement in triplicate

514

Table 4: Effect of treatment of compounds on the growth of Aspergillus flavus and

515

reduction of aflatoxin B1 Compounds

Positive

Concentration of

Growth(%) of

the compound in

Aspergillus flavus w.r.t

mg/mL

control

% of reduction of Aflatoxin B1

-

100

0

0.1

9.6±1.9

91.4±3.9

1

ng*

-

0.1

15.2±1

83.7±4.7

1

ng*

-

0.1

8.9±1.8

66.2±5

1

36.6±8

-

control Compound 1

Compound 2

Compound 3

23


Page 25 of 25


Compound 4

0.1

10.9±0.3

73.0±2.6

1

ng*

-

516

Measurement of growth of Aspergillus flavus in duplicate and aflatoxin reduction study in

517

triplicate

518

* no growth

24


Antiaflatoxigenic and Antimicrobial Activities of Schiff Bases of 2

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