Biophenols and Antioxidant Properties of Australian Canola Meal

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Biophenols and Antioxidant Properties of Australian Canola Meal Hassan K. Obied, Yi Song, Sonia Foley, Michael Loughlin, Ataur Rehman, Rodney Mailer, Tariq Masud, and Samson Agboola J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf4026585 • Publication Date (Web): 26 Jul 2013 Downloaded from http://pubs.acs.org on August 22, 2013

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Journal of Agricultural and Food Chemistry

Biophenols and Antioxidant Properties of Australian Canola Meal

1 2 3

Hassan K. Obied †*; Yi Song †; Sonia Foley †; Michael Loughlin ‡; Ata-u-Rehman §; Rodney

4

Mailer §; Tariq Masud ║; Samson Agboola ‡

5 6 7 8 9 10 11 12

†

Graham Centre for Agricultural Innovation, School of Biomedical Sciences, Charles Sturt

University, Wagga Wagga, NSW, Australia 2678 ‡

Graham Centre for Agricultural Innovation, School of Agriculture and Wine Sciences,

Charles Sturt University, Wagga Wagga, NSW, Australia 2678 §

Graham Centre for Agricultural Innovation; Industry and Investment NSW; Wagga Wagga,

NSW, Australia ║

Department of Food Technology, University of Arid Agriculture, Rawalpindi, Pakistan

13

1

ACS Paragon Plus Environment


14

ABSTRACT

15

During the extraction of canola oil, large quantities of meal are produced. Extracting

16

biophenols from Australian canola meal ACM adds value to otherwise a low value agro-

17

industrial by-product. We studied biophenol content and the antioxidant activity of ACM, the

18

impact of extraction conditions and varietal differences. Sinapine was the principal biophenol

19

in ACM. In crude and hydrolysed extracts, 31 compounds were identified: two dihexosides,

20

two organic acids, four glucosinolates, 17 sinapic acid derivatives, two cyclic spermidine

21

alkaloids, caffeic acid and its dihexoside, kaempferol and its C-glucoside. ACM showed

22

significant free radical scavenging activity in DPPH• and ABTS+• assays. Sinapine was the

23

chief contributor to ACM antioxidant activity while kaempferol sinapoyl triglucoside isomer

24

was the most potent antioxidant. Biophenol content ranged between 12.8 and 15.4 mg GAE/g

25

DW. Differences amongst studied cultivars were generally quantitative. Tarcoola cv. showed

26

the highest biophenol content and antioxidant activity.

27 28 29 30 31

Keywords: online ABTS, polyphenols, rapeseed, canola cake, Brassica napus, sinapine; HPLC; free

32

radical

33

2


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34

INTRODUCTION

35

Oil from rapeseed (Brassica napus L.) has been used for centuries as fuel and lubricant. The

36

cardiotoxic properties of rapeseed oil restricted its culinary use due to high erucic acid

37

content 1. Similarly, abundance of anti-nutrient components, mainly glucosinolates and

38

sinapine, limited the use of rapeseed cake as animal feed

39

introduced through hybridization to overcome rapeseed toxicities and present high quality

40

edible rapeseed varieties. In 1985, rapeseed and canola were recognized by the United States

41

Food and Drug Administration as different species 3. The name 'canola' is derived from

42

'Canadian oil, low acid' and was registered in Canada 4. It is used afterwards to denote any

43

cross-bred rapeseed variety that contained less than 2% erucic acid in its oil and less than 30

44

µmol/g of any one or any combination of the four aliphatic glucosinolates (gluconapin,

45

progoitrin, glucobrassicanapin and napoleiferin) in its defatted meal. This definition was

46

revised in 1997 to glucosinolates level of less than 18 µmol/g whole seeds and less than 1%

47

erucic acid in the oil 5.

2, 3

. In the 1970s, canola was

48 49

From a minor crop in the late 1980s, Canola is now the largest oilseed crop and third largest

50

broad-acre crop (after wheat and barley) in Australia 6, which makes up to 91% of total

51

production. Australia produces around 3.2 million tonnes of canola 6. On average, Australian

52

canola seeds yield around 42% oil 7. Thus, about 1.9 million tonnes of canola meal is

53

produced annually. Canola meal, produced as a by-product during extraction of oil from

54

canola seed, is mainly used as a high protein source animal feed in Australia 8.

55 56

Studies of the biophenol composition of rapeseed meal can be traced back to early 1970s 9, 10.

57

The importance of phenolic compounds in canola seeds has since been continuously

58

recognised. During last two decades canola biophenols attracted more attention than ever 3



59

before in part due to the fact that canola production has grown faster than any other source of

60

vegetable oil. Most of the available literature on canola biophenols is derived from Canadian

61

canola 11-13. Health benefits (e.g. anticancer, anti-ageing, etc) of the canola oil/meal have been

62

recognised, with particular interest based on the antioxidant activities of the phenolic

63

compounds 14, 15.

64 65

No data have been reported on the phenolic composition and antioxidant activities of

66

Australian canola meal (ACM) to date. The unique environmental and ecological properties

67

of Australia are known to induce detectable changes in the phenolic composition of plants 16,

68 69

17

. Therefore, it is crucial to determine the phenolic content and antioxidant activity of ACM

for any future high technology utilisation.

70 71

MATERIALS AND METHODS

72

Chemicals and reagents. The following reagents were used without further purification:

73

Folin-Ciocalteu reagent, potassium phosphate monobasic, sodium phosphate dibasic

74

dihydrate, formic acid, sodium nitrite, ethylenediaminetetraacetic acid (EDTA), 2,2′-azino-

75

bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and 2,2′-diphenyl-1-

76

picrylhydrazyl radical (DPPH•) from Sigma-Aldrich (Sydney, Australia); hydrochloric acid

77

(32%) and HPLC grade methanol were obtained from Merck (Darmstadt, Germany); n-

78

hexane was purchased from J.T. Baker (USA); anhydrous acetonitrile from RCI Labscan

79

(Bangkok, Thailand); sodium chloride, glacial acetic acid and absolute ethanol from Merck

80

(Kilsyth, Australia); sodium carbonate from Biolab (Clayton, Australia); sodium molybdate

81

dihydrate, aluminium chloride, potassium persulfate were obtained from Univar (Seven Hills,

82

Australia); sodium hydroxide from Rowe Scientific (Lonsdale, Australia). Water used in all

83

analytical work was purified by Modulab Analytical model water system (Continental Water 4


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Systems Corporation, Australia). More than 30 phenolic standards were purchased from

85

Sigma-Aldrich (Australia) and Extrasynthese (France) and used without further purification

86

including sinapic acid, gallic acid, caffeic acid, ferulic acid, apigenin, kaempferol, quercetin

87

and rutin.

88 89

Sample collection and preparation. Seed samples from four different cultivars of canola

90

(Tarcoola, Rainbow, Warrior and Lantern) were obtained from Canola Breeding Program,

91

DTIRIS (NSW Department of Trade & Investment, Regional Infrastructure & Services), at

92

Wagga Wagga Agriculture Institute, Wagga Wagga, NSW. Portions of the canola seeds (50

93

g) were ground to fine powder in a coffee mill for 2 min. The powder was then defatted with

94

petroleum ether in a Soxhlet apparatus and air-dried overnight to produce the canola meal

95

samples.

96 97

Optimisation of biophenol extraction. Two different solvents (aqueous mixtures of 80%

98

methanol v/v and 70% acetone v/v) and two extraction conditions (stirring with a magnetic

99

stirrer and sonication in an ultrasonic bath) were investigated for extraction of canola phenols

100

using simple solid-liquid extraction as previously described 18.

101 102

Optimised extraction method. All extractions were performed at ambient temperature (21 ±

103

2 °C). Two grams of canola meal samples were extracted with 10 mL solvent (aqueous

104

methanol 80% v/v) for 30 minutes with continuous stirring using a magnetic stirrer at low

105

speed. The extract was then filtered through Advantec grade No. 1 filter paper. The raffinate

106

was re-extracted with 10 mL solvent for 15 minutes and filtered over the first filtrate. The

107

combined filtrate was defatted twice with n-hexane (2 × 10 mL) and filtered through GF/F

5



108

filter paper and re-filtered using 0.45 µm nylon syringe filter to produce canola meal crude

109

extract (CE). CE was stored at − 20 °C until further analysis.

110 111

Acid and base hydrolyses of CE. Acid and base hydrolyses of CE were performed as

112

described earlier 17 with some modification.

113

Acid hydrolysis. 5 mL of 12.0 N HCL were added to 5 mL CE. The mixture was refluxed at

114

80 °C for 30 min under a condenser. The hydrolysed solution was allowed to cool and 1 g

115

NaCl was added. The mixture was extracted with ethyl acetate (30 mL × 3). The ethyl

116

acetate combined extract was dried over anhydrous sodium sulphate and ethyl acetate was

117

evaporated under vacuum at 35 °C. The residue was reconstituted in methanol (10 mL) and

118

filtered through 0.45 µm nylon syringe filter. Acid hydrolysed crude extract AHCE was

119

stored at − 20 °C as above.

120

Base Hydrolysis. 5 mL of 2.0 N NaOH was added to 5 mL CE. The mixture was refluxed

121

under condenser for 30 min at 80 °C. The hydrolysed extract was allowed to cool and

122

adjusted to pH 2 with 4.0 N HCL. NaCl (1 g) was added to the solution and the solution was

123

extracted with ethyl acetate (30 mL × 3). The ethyl acetate combined fraction was dried over

124

anhydrous sodium sulphate before removing ethyl acetate under vacuum at 35 °C. The

125

residue was reconstituted in methanol (10 mL) and filtered through 0.45 µm nylon syringe

126

filter. Base hydrolysed crude extract BHCE was stored at − 20 °C as above.

127 128

Acidic and alkaline extraction. Two grams of the canola meal samples were extracted with

129

16 mL 80% aqueous methanol and 4 mL of concentrated HCl or 4.0 N NaOH respectively

130

with refluxing under condenser at 80 °C for 30 min, with or without 15 mL protection

131

solution (10 mM EDTA and 1% ascorbic acid or 10 mM EDTA and 1% sodium

132

metabisulfite). The extracts were filtered through Advantec grade No. 1 filter paper. Extracts 6


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133

were adjusted to pH 2 by 4.0 N HCl and volumes made up to 20 mL using ultrapure water.

134

The extracts were then filtered through GF/F filter paper. The extracts were divided into two

135

equal portions. First portion, 10 mL of the filtrate was re-filtrated using 0.45µm nylon syringe

136

filter and stored at − 20 °C till analysed. Second portion of the extracts, remaining 10 mL,

137

was further extracted with ethyl acetate as described above.

138 139

Spectrophotometric assays. All spectrophotometric measurements were performed on Cary

140

50 spectrophotometer, using Cary WinUV ‘version 3’ software (Varian, Australia).

141 142

UV-Vis spectrum. Spectra of diluted CE (1:500 v/v in water) were recorded between 200–800

143

nm.

144 145 146 147

Folin Ciocalteu (FC) total phenols. Total phenol content was determined as described earlier 18

. CE was diluted (1:5) with 80% aqueous methanol. Results were expressed as mg of gallic

acid equivalents per gram dry weight (mg GAE/g DW).

148 149

Total Flavonoids. Total flavonoid content was determined as described previously 17. Results

150

were expressed as mg of quercetin equivalent per gram dry weight (mg QE/g DW).

151 152

Total phenols, total hydroxycinnamic acids and total flavonols. Analysis was performed as

153

described earlier

154

The absorbance was measured at 280 nm to determine total phenols using gallic acid as a

155

standard; at 320 nm to determine hydroxycinnamic acid derivatives using caffeic acid as a

156

standard; and at 360 nm to estimate total flavonols using quercetin as a standard.

18

. The crude extract was diluted (1:20 v/v) with 80% aqueous methanol.

157 7



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158

Ortho-diphenol content. The o-diphenol content was determined as described earlier 18. The

159

crude extract was diluted (1:20) with 80% aqueous methanol. Results were expressed as

160

milligrams of caffeic acid equivalents per gram dry weight of freeze-dried material (mg

161

CAE/g).

162 163

Antioxidant Activity. CE was diluted (1:5) with 80% aqueous methanolic solution.

164

ABTS assay. ABTS radical scavenging activity of canola meal extracts was determined as

165

described earlier

166

capacity). TEAC is defined as mg of canola meal dry weight giving the same percentage

167

ABTS+• scavenging activity as that of 1 mM Trolox.

17

. Results were expressed as TEAC value (Trolox equivalent antioxidant

168 169

DPPH assay. DPPH radical scavenging activity of canola meal extracts was determined as

170

described previously

171

antioxidant capacity). TEAC is defined as mg of canola meal dry weight giving the same

172

percentage DPPH• scavenging activity as that of 1 mM Trolox.

17

. Results were expressed as TEAC value (Trolox equivalent

173

Characterization

of

Extracts.

High

Performance

Liquid

174

Chromatographic

175

Chromatography-Diode Array Detection with online ABTS+• scavenging HPLC-DAD-ABTS.

176

Analysis was performed as described earlier 17, 19. Sample analysis was performed by gradient

177

elution on a 250 mm × 4.6 mm i.d., 5 µm, Gemini C-18 column with a SecurityGuardTM

178

guard cartridges (Phenomenex, Australia). Solvent A was a 0.2% formic acid solution in

179

water, and solvent B was a 0.2% formic acid solution in methanol. Initial condition was 5%

180

solvent B and then solvent B increased to 80% over 65 min. The system was allowed to

181

equilibrate at initial conditions for 15 min between runs.

182 8


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High Performance Liquid Chromatography-Diode Array Detection-Tandem Mass (HPLC-

184

DAD-MS/MS). Samples were analysed on an Agilent 1200 series liquid chromatograph

185

(Agilent technologies, Germany) by gradient elution. The flow rate was 0.7 mL/min and the

186

injection volume was 5 µL. Mobile phase used was as described above. A three-step gradient

187

elution for a total run time of 70 min was used as follows: initial conditions 5% solvent B;

188

solvent B increased to 80% over 65 min; then solvent B increased to 100% over 2 min; and

189

finally back to initial conditions in 3 min. The effluent from the DAD was directed to a 6410

190

triple quad LC-MS (Agilent Technologies, USA) equipped with electrospray ionization (ESI)

191

interface. MS analysis was performed in the negative and positive ion mode (m/z 100-1200)

192

under the following conditions: nitrogen gas; gas temperature 350 °C and gas flow rate 9

193

L/min; the nebulizer pressure was 40 psi; capillary voltage was 4 kV; and cone voltage 100

194

V. Data analysis was performed using Agilent MassHunter workstation version B.01.04 2008

195

(Agilent Technologies, Germany).

196 197

Quantitative determination of sinapic acid and sinapine. Sinapine and sinapic acid

198

concentrations in our samples were determined by quantitative HPLC-DAD analysis. Peak

199

areas from triplicate extracts were integrated at 280 nm and averaged. Sinapic acid reference

200

compound (Sigma-Aldrich) was used to construct a seven-point calibration curve for

201

concentrations between 0.1-1000 µg/mL; regression coefficient R2 = 0.993. Recoveries were

202

determined as sinapic acid equivalent.

203 204

Statistical Analyses. All measurements were done in triplicates at least and results were

205

presented as means ± standard deviations. Statistical comparisons were made using one-way

206

ANOVA and post-hoc LSD test. Data analyses were performed by Microsoft Excel and

9



207

PASW Statistics package version 17.0 (SPSS Inc., Chicago, IL). Results were considered

208

statistically significant at p < 0.05.

209 210

RESULTS AND DISCUSSION

211

UV-Vis spectrum of ACM extract. UV-Vis spectra of extracts were investigated to provide

212

a preliminary idea about the chemical nature of canola meal biophenols and help selecting

213

suitable detection wavelengths for HPLC monitoring. Scanning the UV-Vis spectrum within

214

the range 200-800 nm, two peaks of equal intensities were observed at 225 nm and 325 nm

215

with a shoulder at 235 (data not shown). This is indicative of the presence of

216

hydroxycinnamic acids

217

simple phenols and flavonols are not major constituents of ACM. The findings are consistent

218

with previous literature where sinapic acid and its derivatives were identified as the main

219

constituents in canola meal 12, 13, 21. Sinapic acid has a λ max = 322 nm while sinapine has a λ

220

max

20

; the absence of prominent peaks at 280 nm and 360 nm suggests

=329 nm 12.

221 222

Recovery of biophenols from canola meal. Optimisation of extraction conditions. In a

223

preliminary pilot study, we found that methanol and acetone based solvents were superior to

224

ethanol and acetonitrile based solvents; and dynamic extraction conditions were more

225

efficient than static extraction. Hence, an optimisation study was conducted to compare the

226

phenol recovery efficiencies between 80% aqueous methanol and 70% aqueous acetone with

227

stirring or sonication. Results are given in Table 1.

228 229

TABLE 1

230

10


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Results show that 70% aqueous acetone is a more efficient extraction solvent compared with

232

80% aqueous methanol. While the total phenol content showed a 70% increased recovery, o-

233

diphenol content was doubled and total flavonoid recovery was more than tripled. Acetone

234

superiority in extraction of biophenols from plant material has been frequently reported.

235

However, the compatibility of methanolic extracts with HPLC and the high UV cut off of

236

acetone at 330 nm 18 makes it more appealing to use methanolic extracts for further assays to

237

simplify sample preparation procedures. Furthermore, comparison of the fingerprint

238

chromatograms of methanol and acetone extractions showed no qualitative differences (data

239

not shown).

240 241

In the comparison between stirring and sonication methods, overall stirring was slightly more

242

efficient than sonication yet there was no statistically significant difference apart from o-

243

diphenols recovery. On the other hand, it was observed that sonication promoted micro-

244

emulsion formation. Hence, stirring was considered in preference to stirring for recovery of

245

ACM biophenols.

246 247

Biophenolic composition of ACM. The use of hyphenated liquid chromatography especially

248

tandem mass spectroscopy has increased the number of biophenols reported in canola meal

249

and facilitated the detection of complex biophenol derivatives

250

MS/MS, we could distinguish twenty nine peaks in ACM extracts (Table 2) (Figure 1). We

251

could tentatively identify twenty seven compounds. Two peaks, 14 and 20, remained

252

unidentified. Both 14 and 20 have absorption maxima around 325 nm and ABTS scavenging

253

activity suggesting that they may be sinapic or caffeic acid derivatives. Compound 20 has an

254

odd molecular weight, 727 Dalton; most likely to be a mono-choline ester.

255 11


12, 22

. By means of LC-DAD-


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256

Identification of peaks was carried out via comparing their UV-Vis and mass spectra with

257

biophenol standards and literature values as described earlier

258

ABTS scavenging chromatograms (Figure 3) provided valuable insight into not only the

259

antioxidant activity but also the chemical structures of ACM biomolecules. We reported

260

earlier that in online ABTS scavenging assay, o-diphenols entity is essential for ABTS+•

261

scavenging activity and compounds devoid of this structural feature such as monophenols

262

failed to scavenge ABTS+•

263

to assist in identifying aglycones and unconjugated structures (Table 2) (Figure 2).

23

. Furthermore, the study of

17

. Acid and base hydrolyses of the extracts were also performed

264 265

CE contained biophenols and non-biophenolic components (Table 2). Early eluting peaks, 1-

266

4, in CE chromatogram (Figure 1) showed no UV absorbance around 280 nm and did not

267

have ABTS+• scavenging activity. Based on their mass spectrum, peak 1 and 2 were assigned

268

as dihexoses, most probably gentiobiose and sophorose

269

presence of peaks 3 and 4 in other plants 17. Based on their molecular ions [M−H]− at m/z 133

270

and 191, UV spectra and literature data 25, peaks 3 and 4 were identified as malic and citric

271

acids, respectively. Farag et al. (2012) detected malic and citric acids in all canola tissues

272

apart from seeds 25. This can be ascribed to their extraction under liquid nitrogen of ground

273

seeds versus hexane refluxing prior to extraction in our procedures.

22, 24

. We previously reported the

274 275

Four major glucosinolates (Table 2) (Figure 1) were tentatively identified in ACM, namely

276

progoitrin 5, gluconapin 8, 4-hydroxyglucobrassicin 17 and glucobrassicanapin 18 based on

277

matching their UV and mass spectra with literature

278

biophenolic canola extracts has been encountered before 28. Using ion trap mass spectroscopy

279

in the negative ionization mode, Millán et al. (2009) could identify eight glucosinolates

280

While Fang et al. (2012) reported eleven glucosinolates by applying positive ionization mode

26, 27

. Detection of glucosinolates in

12


26

.

Page 13 of 31

281 282


29

. Hence it is not our aim to profile canola glucosinolates, only glucosinolates showing

prominent peaks in our chromatograms are reported.

283 284

In agreement with the UV-Vis spectra of the extracts, sinapic acid derivatives were the most

285

prevalent biophenols (Figure 1 and Figure 3). Sinapic acid derivatives are estimated to

286

constitute 99% of the canola meal biophenols 4. Upon base hydrolysis, trans-sinapic acid

287

peak 21 constituted 90% of the total detected peak area at 280 nm (Figure 2 C). In accord

288

with most available literature sinapoyl choline, sinapine 6, was the principal sinapic acid

289

derivative in ACM (Figure 1). Cis- and trans-sinapic acids were the only free phenolic acid

290

detected in ACM (Table 2). The cis-isomer 19 is more polar and elutes earlier than trans-

291

isomer 21. Literature on the presence of free phenolic acids in canola meal is contradictory 11,

292

25, 30

. By means of biophenol standards, we confirmed the absence of vanillic, p-

293

hydroxybenzoic, gentisic, protocatechuic, syringic, p-coumaric, and ferulic acids in our

294

samples. After acid hydrolysis, we could detect minute amounts of caffeic acid 31 (Figure 2).

295

FIGURE 1

296

FIGURE 2

297

TABLE 2

298

FIGURE 3

299

Few researchers have reported a broad/skewed peak for sinapine on reversed phase HPLC

300

chromatograms

301

split into two peaks [6a and 6b] (Figure 3.A) on the Varian HPLC-DAD-ABTS

302

chromatograph, though the same column and mobile phases were used. We could rule out the

303

possibility of being an artefact (double peak) via injecting standards, catechuic acid and

304

catechin, with similar retention time to sinapine. Both compounds showed a single sharp

305

peak. In addition, two peaks [6a and 6b] were observed in the ABTS chromatogram (Figure

13, 31

. The sinapine peak 6 detected by Agilent HPLC-DAD-MS (Figure 1)

13



Page 14 of 31

306

3.B) though a different UV-detector was used. Peaks, 6a and 6b, both have the same

307

molecular weight yet they have slightly different UV-Vis spectra (Table 2). Hence, 6a and 6b

308

can be two sinapine stereoisomers. Examining sinapine structural formula (Figure 3), the two

309

peaks can be either cis/trans isomers or sterically hindered conformers. Cis-sinapine has

310

never been reported so far although cis-sinapic acid was frequently reported in canola meal 32,

311

33

.

312 313

Further, sinapic acid esters with hexoses, dihexoses, kaempferol, malic acid and methanol

314

were found (Table 2). In agreement with Liu et al. (2012), two sinapic acid hexoses 9 & 13

315

were detected in ACM

316

derivative after sinapine is sinapoyl glucoside, 1-O-β-D-glucopyranosyl sinapate

317

compounds 9 & 13 are most likely glucoside derivatives

318

scavenging activity as expected from an ester of sinapic acid with glucose. Thus, we assigned

319

13 as 1-O-β-D-glucopyranosyl sinapate. Absence of ABTS peak for peak 9 (Figure 3.B) can

320

be attributed to blocking of the p-hydroxy group of sinapic acid via ether/glycoside linkage

321

with glucose, thus 9 was tentatively assigned as 4'-glucosyl sinapic acid. Two isomers of

322

sinapoyl glucoside were discovered in Chinese canola

323

sinapic acid were found. Di-sinapoyl hexoside 27 was detected in ACM. In accordance with

324

Baumert et al. (2005), we identified caffeoyl dihexoside 7 but did not detect any mono-

325

sinapoyl dihexoside derivatives 22. Meanwhile, di-sinapoyl 25 and tri-sinapoyl 26 dihexosides

326

were found in ACM. For the first time, we report the presence of tetra-sinapoyl dihexoside

327

28 (Table 2). More complex sinapoyl glucoside derivatives were also reported in canola

328

meal where the glucoside is further linked to a flavonoid, most commonly kaempferol

329

Three isomers of kaempferol sinapoyl trihexose, 15, 16 & 22, were detected in our sample;

330

similar to the earlier findings of Baumert et al. (2005) 22. Quercetin sinapoyl trihexoside was

32

. Literature shows that the second most abundant sinapic acid

32

5, 12

. Hence,

32

. Peak 13 showed ABTS

but no earlier reports of 4'-glucosyl

14


22, 29

.

Page 15 of 31


331

previously identified in canola inflorescence but not in canola seed 25. Peak 23 and 24 were

332

tentatively assigned as sinapoyl malate isomers. While many reports were found for trans-

333

sinapoyl malate in Brassica

334

malate 34. Methyl sinapate 34 was only detectable in acid hydrolysed extracts (Figure 2. A &

335

B), supporting earlier findings

336

methanol in the extraction solvent under acidic conditions.

25

, only few studies have documented presence of cis-sinapoyl

35

about being an artefact formed via esterification with

337 338

Absence of free flavonoids such as apigenin, catechin, luteolin, naringenin and quercetin was

339

confirmed in ACM. Upon hydrolysis, the only flavonoid aglycone detected in ACM was

340

kaempferol 35 (Table 2) (Figure 2). Metabolic profiling of Egyptian canola revealed presence

341

of isorhamnetin, isoquercetrin, quercetin, naringenin and kaempferol and their glycosides,

342

while only kaempferol glycosides were detected in canola seeds 25.

343 22, 29

344

A cyclic spermidine alkaloid was identified in German canola seeds

, and a hexoside

345

derivative was identified in transgenic canola

346

spermidine alkaloid were identified, 10 and 11. This is the first time to report two isomers for

347

this alkaloid in canola meal.

24

. In ACM, two isomers of the cyclic

348 349

Upon performing acid and base hydrolyses, seven more peaks, 30-36, were observed (Table

350

2), in addition to the main peak of trans-sinapic acid 21 (Figure 2). While base hydrolysis

351

produced principally trans-sinapic acid, acid hydrolysis resulted in generation of a new major

352

peak, 30, which was present neither in the un-hydrolysed nor the base hydrolysed extracts.

353

Peak 30 was the most abundant peak in the acid hydrolysed extract followed by trans-sinapic

354

acid 21 (Figure 2. A). Therefore, compound 30 is either a degradation product of sinapic acid

355

or an artefact of the acid hydrolysis process. The compound failed to ionize under negative 15



Page 16 of 31

356

ESI conditions while a peak was generated under positive ionization conditions with an m/z

357

of 149. This compound is highly polar with a retention time, 14.2 min, less than sinapine,

358

17.6 min, and after gallic acid, 13 min. The intense UV absorption at 285 nm suggests a

359

hydroxybenzoic acid derivative. Lack of ABTS scavenging activity (Table 2) negates the

360

presence of dihydroxy or trihydroxy phenolic groups. Thus, high polarity is most probably

361

due to a charged molecule.

362 363

Peak 33 was assigned as kaempferol-C-hexoside. C-glycosides are known for their resistance

364

to hydrolysis under acidic conditions. Though peak 32 was detected under acidic conditions,

365

both 32 and 36 were prominent under base hydrolysis conditions. Autoxidation of alkaline

366

solution of sinapic acid results in formation of lignan derivatives such as thomasidioic acid,

367

molecular weight 446 Dalton 36. Seven lignan derivatives were reported in Brassica napus 29.

368 369

Antioxidant activity of ACM. 1-O-β-D-glucopyranosyl sinapate was reported as the most

370

active antioxidant in canola meal in a lipid model system

371

scavenging assay, sinapic acid was more potent scavenger than sinapine followed by 1-O-β-

372

D-glucopyranosyl sinapate. Examining the ABTS scavenging chromatogram (Figure 3 B), we

373

can ascertain that the total antioxidant activity of ACM is chiefly due to sinapine 6a and 6b,

374

kaempferol sinapoyl triglucoside isomer 15, disinapoyl glucoside 27,

375

glucopyranosyl sinapate 13, unknown sinapoyl derivative 20, tetrasinapoyl dihexoside 28,

376

kaempferol sinapoyl triglucoside isomer 22 and disinapoyl dihexoside 25. In quantitative

377

terms, sinapine was the most abundant antioxidant. Meanwhile, the most potent antioxidant

378

in ACM was kaempferol sinapoyl triglucoside isomer 15. Three different kaempferol

379

sinapoyl triglucosides were detected in ACM, in accord with earlier findings

37

. Meanwhile, in DPPH radical

16


22

1-O-β-D-

(Figure 3).

Page 17 of 31


380

The ABTS scavenging activities of the three isomers vary based on the way kaempferol,

381

sinapic acid and glucose are bound to each other.

382 383

Biophenol content and antioxidant activity: Impact of cultivar. Total phenols,

384

hydroxycinnamic acids, flavonols, o-diphenols, flavonoids and free radical scavenging

385

activities were significantly affected by cultivar (Table 3). Total phenol content was 12.8-

386

15.4 (FC) and 7.3-9.5 (280 nm) mg GAE/g DW. Total phenol content in canola by-products

387

in literature varied from 6.4 to 18.4 mg /g DW 12. o-Diphenols were the most abundant class

388

of biophenols, ranging from 9.2 to 12.0 mg CAE/g DW. We found that sinapic acid gives

389

positive reaction with the o-diphenol assay. Flavonoids were the least abundant in canola

390

meal, ranging from 2.3 to 3.1 mg QE/g DW. The relative difference between the highest total

391

phenol content, Tarcoola cv., and the lowest phenol content, Rainbow cv., was approximately

392

20%. This comes in parallel to o-diphenols content, 23%, and hydroxycinnamic acids

393

content, 30%.

394

TABLE 3

395 396 397

Cultivars can be ranked according to their total phenol content as follows: Tarcoola > Warrior

398

> Lantern = Rainbow. Consistently, Tarcoola showed the highest phenol content in all

399

employed assays, while Rainbow was the lowest. There was no statistically significant

400

difference between Lantern and Rainbow samples in all assays (p

Biophenols and Antioxidant Properties of Australian Canola Meal

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