Accepted Manuscript A comprehensive study on the phenolic profile of widely used culinary herbs and spices: rosemary, thyme, oregano, cinnamon, cumin and bay Anna Vallverdú-Queralt, Jorge Regueiro, Miriam Martínez-Huélamo, José Fernando Rinaldi Alvarenga, Leonel Neto Leal, Rosa M. Lamuela-Raventos PII: DOI: Reference:

S0308-8146(14)00004-1 http://dx.doi.org/10.1016/j.foodchem.2013.12.106 FOCH 15223

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

1 October 2013 16 December 2013 31 December 2013

Please cite this article as: Vallverdú-Queralt, A., Regueiro, J., Martínez-Huélamo, M., Alvarenga, J.F., Leal, L.N., Lamuela-Raventos, R.M., A comprehensive study on the phenolic profile of widely used culinary herbs and spices: rosemary, thyme, oregano, cinnamon, cumin and bay, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/ j.foodchem.2013.12.106

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1

A comprehensive study on the phenolic profile of widely used culinary herbs and

2

spices: rosemary, thyme, oregano, cinnamon, cumin and bay.

3 4

Anna Vallverdú-Queralta,b, Jorge Regueiroc, Miriam Martínez-Huélamoa,b, José

5

Fernando Rinaldi Alvarengad, Leonel Neto Leale and Rosa M. Lamuela-Raventosa,b*

6 7 8 9

a

Nutrition and Food Science Department, XaRTA, INSA. Pharmacy School, University

of Barcelona, Spain. b

CIBER Fisiopatología de la Obesidad y la Nutrición (CIBERobn), Instituto de Salud

Carlos III, Spain.

10

c

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Faculty of Food Science and Technology, University of Vigo, Spain.

12 13 14

Nutrition and Food Science Group, Department of Analytical and Food Chemistry,

d

Department of Food and Nutrition. School of Pharmaceutical Science. São

Paulo State University, Brazil. e

Nutreco Research and Development, The Netherlands.

15 16 17

*

18

Pharmacy School, University of Barcelona, Spain. Telephone +34-934034843. Fax +34-

19

934035931; e-mail [email protected]

20

Corresponding author: Nutrition and Food Science Department, XaRTA, INSA

21

ABSTRACT

22

Herbs and spices have long been used to improve the flavour of food without being

23

considered as nutritionally significant ingredients. However, the bioactive phenolic

24

content of these plant-based products is currently attracting interest.

25

In the present work, liquid chromatography coupled to high-resolution/accurate mass

26

measurement LTQ-Orbitrap mass spectrometry was applied for the comprehensive

27

identification of phenolic constituents of six of the most widely used culinary herbs

28

(rosemary, thyme, oregano and bay) and spices (cinnamon and cumin). In this way, up

29

to 52 compounds were identified in these culinary ingredients, some of them, as far as

30

we know, for the first time. In order to establish the phenolic profiles of the different

31

herbs and spices, accurate quantification of the major phenolics was performed by

32

multiple reaction monitoring in a triple quadrupole mass spectrometer. Multivariate

33

statistical treatment of the results allowed the assessment of distinctive features among

34

the studied herbs and spices.

35 36 37

Key words: Culinary herbs, spices, polyphenols, Rosemary, Thyme, Oregano,

38

Cinnamon, Bay, Cumin, LC–ESI-LTQ-Orbitrap,

39 40 41 42 43 44

45

1. Introduction

46

Since ancient times, herbs and spices have been used all over the world to enhance or

47

improve the flavour of food due to their sensory properties, and also as preservative

48

agents (Kivilompolo & Hyotylainen 2007; Park 2011; Shan, Cai, Sun, & Corke 2005).

49

However, most of their potential health-promoting properties have received little

50

attention. Recent research has shown culinary herbs and spices to be a dietary source of

51

bioactive polyphenols (Hinneburg, Damien Dorman, & Hiltunen, 2006; Wojdyło,

52

Oszmiański, & Czemerys, 2007), which has stimulated the study of their phenolic

53

composition and antioxidant properties. Several culinary herbs and spices are now

54

known to have beneficial effects for human health, including digestive stimulant, anti-

55

inflammatory, antimicrobial, antioxidant and anticarcinogenic activities (Shobana &

56

Akhilender Naidu 2000; Velioglu, Mazza, Gao, & Oomah 1998; Zheng & Wang 2001),

57

which are attributed to the predominant polyphenol compounds in these plant materials.

58

Moreover, the volatile constituents (essential oils) that are the main cause for use of

59

these plants can significantly contribute to biological activity (Inouye, Takizawa, &

60

Yamaguchi, 2001).

61

Recently, there has been growing awareness of the importance of a high dietary content

62

of phenolic compounds, such as flavonoids and hydroxycinnamic acids, because of their

63

apparent multiple biological effects, including metal chelation, free-radical scavenging,

64

inhibition of cellular proliferation, modulation of enzymatic activity and signal

65

transduction pathways (Del Rio, Rodriguez-Mateos, Spencer, Tognolini, Borges, &

66

Crozier, 2013).

67

Although the contribution of several widely-used culinary herbs and spices to the total

68

intake of dietary polyphenols has been previously investigated (Halvorsen et al. 2002;

69

(Halvorsen et al. 2002; Hinneburg et al., 2006; Wojdyło et al., 2007), a comprehensive

70

identification of their phenolic profile is still lacking, mainly due to the wide variety of

71

structures of these natural compounds and unavailability of commercial standards. In

72

this context, high-resolution/accurate mass measurement (HR/AM) mass spectrometry

73

techniques have been demonstrated to be a reliable tool for the structural elucidation of

74

unknown compounds in complex samples (Vallverdú-Queralt, Jáuregui, Medina-

75

Remón, Andrés-Lacueva & Lamuela-Raventós, 2010). Among the HR/AM systems,

76

linear ion trap quadrupole-Orbitrap-mass spectrometry (LTQ-Orbitrap-MS) delivers

77

single-stage mass analysis providing molecular mass information, two-stage mass

78

analysis (MS/MS) and multi-stage mass analysis (MSn) with useful structural

79

information. Zhou et al. have recently identified the phenolics of Sarcandra glabra by

80

non-targeted high-performance liquid chromatography fingerprinting and targeted

81

electrospray ionisation tandem quadrupole mass spectrometry/time-of-flight mass

82

spectrometry analyses (Zhou, Liang, Lv, Hu, Zhu, Si , & Wu, 2013).

83

The objective of this work was therefore to extensively study the phenolic profile of

84

several widely-used culinary herbs (rosemary, thyme, oregano and bay) and spices

85

(cinnamon and cumin) by liquid chromatography coupled to electrospray ionisation

86

LC–ESI-LTQ-Orbitrap mass spectrometry. The high-resolution MS analyses revealed

87

the presence of 51 phenolic compounds, some of them hitherto unreported in culinary

88

herbs and spices. Quantification of major compounds was also carried out by LC

89

coupled to triple quadrupole mass spectrometry (LC–ESI-QqQ) using multiple reaction

90

monitoring (MRM) mode. The quantification levels of phenolic compounds allowed the

91

identification of distinguishing features among Lamiaceae, Lauraceae and Apiaceae

92

botanical families.

93 94

95

2. Materials and methods

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2.1. Standards and reagents

97

All samples and standards were handled without exposure to light. Caffeic, ferulic, p-

98

coumaric, protocatechuic, syringic, rosmarinic, p-hydroxybenzoic and chlorogenic acid

99

(5-O-caffeoylquinic acid), quercetin, catechin, epicatechin, ABTS: 2,2’azino-bis(3-

100

ethylbenzothiazoline-6-sulfonic

acid),

Trolox:

(±)-6-hydroxy-2,5,7,8-

101

tetramethylchromane-2-carboxylic acid 97% and manganese dioxide were purchased

102

from Sigma-Aldrich (Madrid, Spain); DPPH: 2,2-diphenyl-1-picrylhydrazyl from

103

Extrasynthèse (Genay, France). Ethanol, methanol and HPLC-grade formic acid were

104

obtained from Scharlau (Barcelona, Spain) and ultrapure water (Milli-Q) from Millipore

105

(Billerica, MA). Samples were stored at 4 ºC and protected from light until analysis.

106 107

2.2. Extraction and analysis of polyphenols

108

2.2.1. Samples

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Dried and ground rosemary (Rosmarinus officinalis), oregano (Origanum vulgare),

110

thyme (Thymus vulgaris), bay leaf (Laurus nobilis), cumin (Cuminum cyminum) and

111

cinnamon (Cinnamomum zeylanicum) were sourced by Nutreco B.V., Amersfoort, the

112

Netherlands. According to product specifications, the countries of origin were China

113

(oregano, cumin, cinnamon and bay) and Spain (rosemary and thyme). Spices were

114

extracted with a hydroalcoholic solvent, centrifuged, concentrated and dried. The dried

115

spices were ground (particle size range: 500 to 600 µm) and stored at ‒20 °C in

116

darkness.

117 118 119

2.2.2. Extraction of polyphenols

120

Each dried spice was divided into 3 portions, each extracted, and each extract analysed

121

twice in a darkened room with a red safety light to avoid photodegradation of the

122

analytes following a previously reported procedure (Vallverdú-Queralt et al., 2010) with

123

minor modifications. Briefly, samples (1 g) were extracted with 5 mL of 50 % ethanol

124

in ultrapure water with 0.1 % formic acid, sonicated for 5 min and centrifuged at 3000 g

125

for 10 min at 4 ºC. The extraction procedure was repeated twice with the plant material

126

residue. Both supernatants were combined and the organic solvent was evaporated

127

under a nitrogen flow. Finally, extracts were reconstituted up to 5 mL with 0.1 % of

128

formic acid in water.

129

A solid-phase extraction (SPE) procedure was carried out to eliminate potential

130

interferences from plant extracts. Oasis mixed-mode anion-exchange cartridges and

131

Oasis MAX 96-well plates (30 mg, 30 µm) from Waters (Milford, USA) were used

132

following a previously reported procedure (Vallverdú-Queralt et al., 2010). Firstly, 1

133

mL of methanol and subsequently 1 mL of sodium acetate (50 mmol/L, pH 7) were

134

loaded into Oasis® MAX cartridges from Waters to equilibrate it; then, 1mL of each

135

extract was diluted with 1 mL of Milli-Q water and acidified with 34 µL of hydrochloric

136

acid (35%) before being loaded into the cartridges separately. These were rinsed with

137

sodium acetate (50 mmol/L, pH 7; 5% methanol). The polyphenols were eluted with

138

1800 µL of methanol (2% formic acid). The eluted fractions were evaporated under

139

nitrogen flow, and the residue was reconstituted with water (0.1 % formic acid) up to

140

250 µL and filtered through a 13 mm, 0.45 µm PTFE filter (Waters) into an insert-

141

amber vial for HPLC analysis. Samples were stored at ‒20 ºC until analysis.

142 143 144

2.2.3. LC-LTQ-Orbitrap-MS and LC-MS/MS analyses

145

For accurate mass measurements, a LTQ Orbitrap Velos mass spectrometer (Thermo

146

Scientific, Waltham, MA) equipped with an ESI source was used operating in negative

147

ion mode. Specific parameters were as follows: spray voltage, 4 kV; sheath gas, 20

148

(arbitrary units); auxiliary gas, 10 (arbitrary units); sweep gas, 2 (arbitrary units); and

149

capillary temperature, 275 °C. Default values were used for most other acquisition

150

parameters (FT Automatic gain control (AGC) target 5·× 10 5 for MS mode and 5·× 104

151

for MSn mode). Plant extracts were first analysed in full MS mode at a resolution of

152

60000 (at m/z 400). Successive analyses were done in MSn mode with the Orbitrap

153

resolution set at 30000 (at m/z 400). The most intense ions detected in full scan

154

spectrum were selected for the data-dependent scan. Parent ions were fragmented by

155

high-energy C-trap dissociation (HCD) with normalised collision energy of 45% and an

156

activation time of 100 ms. The maximum injection time was set to 100 ms with two

157

micro scans for MS mode and to 1000 ms with one micro scan for MSn mode. The mass

158

range was from m/z 100 to 1000.

159

Instrument control and data acquisition were performed with Xcalibur 2.0.7 software

160

(Thermo Fisher Scientific). An external calibration for mass accuracy was carried out

161

the day before the analysis according to the manufacturer’s guidelines.

162

The liquid chromatograph was an Accela system (Thermo Scientific, Hemel

163

Hempstead, UK) equipped with a quaternary pump, a photodiode array detector (PDA)

164

and a thermostated autosampler. A reversed-phase column Atlantis T3 C18 (100 × 2.1

165

mm, 3 µm) from Waters (Milford, MA) maintained at 25 ºC was used. Gradient elution

166

was performed with water/0.1% formic acid (v/v), acetonitrile/0.1% formic acid (v/v) at

167

a constant flow rate of 0.350 mL/min, and injection volume was 5 µL. An increasing

168

linear gradient of solvent B was used. Separation was carried out in 36 min under the

169

following conditions: 0 min, 10 % B; 1 min, 10% B; 15 min, 30% B; 22 min, 50% B; 28

170

min, 100% B; 34 min, 100% B, 36 min, 10% B. The column was equilibrated for 6 min

171

prior to each analysis. These conditions were adapted from a previous study with some

172

modifications (Vallverdú-Queralt, Rinaldi de Alvarenga, Estruch, & Lamuela-Raventos,

173

2013).

174

The elemental composition of the detected compounds was based on their accurate mass

175

measurements and isotopic patterns, and then searched for identification in the

176

Dictionary of Natural Products (Chapman & Hall/CRC), the MOTO database

177

(http://appliedbioinformatics.wur.nl/moto) and the Plant Metabolic Network. The

178

interpretation of the observed MS/MS spectra in comparison with those found in the

179

literature was the main tool for tentative identification of polyphenols.

180

Quantification of the previously identified compounds was performed by LC–ESI-

181

MS/MS using an Agilent series 1100 HPLC instrument (Agilent, Waldbronn, Germany)

182

coupled to an API 3000 triple quadrupole mass spectrometer (PE Sciex, Concord,

183

Ontario, Canada) equipped with a Turbo Ionspray source, which was operated in

184

negative-ion mode. Separation was carried out under the same chromatographic

185

conditions used during the identification step. Specific mass spectrometer parameters

186

were as follows: spray voltage, ‒3.5kV; nebuliser gas (N2), 10 (arbitrary units); curtain

187

gas (N2), 12 (arbitrary units); collision gas (N2), 4 (arbitrary units); focusing potential,

188

‒200 V; entrance potential, ‒10 V; drying gas (N2), heated to 400 ºC and introduced to a

189

flow rate of 6000 cm3/min. The declustering potential and collision energy were

190

optimised for each compound in infusion experiments: individual standard solutions (10

191

µg/mL) dissolved in 1:1 (v/v) mobile phase were infused at a constant flow rate of 5

192

µL/min using a model syringe pump (Harvard Apparatus, Holliston, MA). Collision

193

energy and declustering potential are shown in Table 1.

194

For quantification purposes, data was acquired in multiple reaction monitoring (MRM)

195

mode, tracking the transition of parent and product ions specific for each compound.

196

Quantification of polyphenols was performed by the internal standard method. The

197

method of internal standards is used to improve the precision of quantitative analysis.

198

The internal standard was ethyl gallate (400ng/g) and results were expressed as µg/g dry

199

weight (DW).

200 201

2.2.4. Analysis of total polyphenols

202

For the total polyphenols (TP) assay, each sample was analysed three times; 20 µL of

203

the eluted fractions from SPE were mixed with 188 µL of Milli-Q water in a thermo

204

microtitre 96-well plate (nuncTM, Roskilde, Denmark), and 12 µL of Folin-Ciocalteau

205

(F–C) 2N reagent and 30 µL of sodium carbonate (200 g/L) were added, following the

206

procedure described by Vallverdú-Queralt, Medina-Remon, Martinez-Huelamo,

207

Jauregui, Andres-Lacueva and Lamuela-Raventos (2011a). The mixtures were

208

incubated for 1 h at room temperature in the dark. After the reaction period, 50 µL of

209

Milli-Q water were added and the absorbance was measured at 765 nm in a UV/Vis

210

Thermo Multiskan Spectrum spectrophotometer (Vantaa, Finland). Results were

211

expressed as mg of gallic acid equivalents (GAE)/g DW.

212 213

2.2.5. Antioxidant capacity

214

The culinary herb extracts prepared for polyphenol analysis were also analysed for their

215

antioxidant capacity (AC). The AC was measured using an ABTS+ radical

216

decolorisation assay and DPPH assay (Vallverdu-Queralt, Medina-Remon, Casals-Ribes,

217

Amat, & Lamuela-Raventos, 2011b).

218

ABTS+ assay

219

1 mM Trolox (antioxidant standard) was prepared in methanol. Working standards were

220

obtained by diluting 1 mM Trolox with methanol. Solutions of known Trolox

221

concentration were used for calibration. An ABTS+ radical cation was prepared by

222

passing a 5 mM aqueous stock solution of ABTS (in methanol) through manganese

223

dioxide powder. Excess manganese dioxide was filtered through a 13 mm 0.45 µm filter

224

PTFE (Waters). Then, 245 µL of ABTS+ solution were added to 5 µL of Trolox or to

225

herb extracts (0.1% formic acid in water) and the solutions were stirred for 30 s. The

226

homogenate was shaken vigorously and kept in darkness for 1 h. Absorption of the

227

samples was measured on a UV/Vis Thermo Multiskan Spectrum spectrophotometer at

228

734 nm and methanol blanks were run in each assay. Results were expressed as mmol

229

Trolox equivalents (TE)/g DW.

230

DPPH assay

231

The antioxidant capacity was also studied through the evaluation of the free radical-

232

scavenging effect on the DPPH radical. Solutions of known Trolox concentration were

233

used for calibration. Five microlitres of herb extracts (0.1% formic acid in water) or

234

Trolox were mixed with 250 µL of methanolic DPPH (0.025 g L‒1). The homogenate

235

was shaken vigorously and kept in darkness for 30 min. Absorption of the samples was

236

measured on the spectrophotometer at 515 nm. Results were expressed as mmol TE

237

100/g DW.

238 239

2.3. Statistical analysis

240

The significance of the results and statistical differences were analysed using

241

Statgraphics plus v. 5.1 software (StatPoint, Inc., Herndon, VA). Data were analysed by

242

multifactor analysis of variance and a Duncan multiple range test was applied to

243

determine differences between means, with a significance level of p = 0.05.

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Additionally, correlations among variables were evaluated using principal component

245

analysis (PCA) to cluster culinary herbs according to their polyphenol profile. PCA is a

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multivariate statistical technique that allows us to visualise the original arrangement of

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plant herbs in an n-dimensional space, by identifying the directions in which most of the

248

information is retained.

249 250

3. Results and discussion

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3.1. Phenolic profile of culinary herbs and spices

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Culinary herbs and spices are interesting for their content of bioactive compounds that

253

may exert beneficial effects on human health. Table 2 shows a list of 51 phenolic

254

compounds identified by LC–ESI-LTQ-Orbitrap along with their retention times (RT),

255

accurate mass measurements (acc. mass), molecular formula (MF), mDa of error

256

between the mass found and the accurate mass of each polyphenol and the MS/MS

257

fragment ions used for identification. Phenolic compounds were identified by

258

comparing retention times and their masses with those of 24 authentic standards.

259

Identification of the remaining 27 compounds without available standards was based on

260

accurate mass measurements of the [M ‒ H]‒ ion and fragment ions. The fragmentation

261

patterns of the majority of these compounds have been previously identified in other

262

works (Vallverdú-Queralt, Jáuregui, Di Lecce, Andrés-Lacueva, & Lamuela-Raventós, 2011c;

263

Vallverdú-Queralt et al., 2010).

264 265

Caffeic (m/z 179) and caffeic-O-hexoside (m/z 341), protocatechuic (m/z 153),

266

rosmarinic (m/z 359), 3-, 4- and 5-O-caffeoylquinics (m/z 353), coumaroylquinic (m/z

267

337), ferulic-O-hexoside (m/z 355), ferulic (m/z 193), p-coumaric (m/z 163),

268

homovanillic-O-hexoside (m/z 343), gallic (m/z 169), syringic (m/z 197), p- and m-

269

hydroxybenzoic (m/z 137) acids and kaempferol-3-O-glucoside (m/z 447), kaempferol

270

(m/z 285) and quercetin (m/z 301) were detected in all the culinary herbs and spices.

271

Several trimeric proanthocyanidins (m/z 863) and one hexamer (m/z 1727) were also

272

detected in cinnamon and cumin. The proanthocyanidin hexamer identified through its

273

[M ‒ 2H]2‒ ion was confirmed by the 0.5 Da mass differences between the isotopic

274

peaks. The selected resolution of 60000 (at m/z 400) allowed determination of the

275

charge state with high accuracy. The proanthocyanidin hexamer showed doubly-charged

276

ions at m/z 863 corresponding to monoisotopic masses of 1727.3730. The most common

277

classes of proanthocyanidins consist of subunits of catechin, epicatechin, and their gallic

278

acid esters (B-type oligomers). However, the hexamers and trimers found in this study

279

were A-type oligomers (Figure 1), which are structural variations of proanthocyanidin

280

oligomers with the formation of a second interflavanoid bond by C‒O oxidative

281

coupling. Due to the complexity of this conversion, A-type proanthocyanidins are not

282

encountered in nature as frequently as the B-type oligomers (Lazarus, Adamson,

283

Hammerstone, & Schmitz 1999).

284

To our knowledge, three of the polyphenols identified in this work are reported for the

285

first time in these plant extracts. Thus, while apigenin-C-hexoside-C-hexoside (m/z 593)

286

has been previously found in marjoram (Kaiser, Carle, & Kammerer 2013), it has been

287

hitherto unidentified in rosemary and oregano. Sinapic acid-C-hexoside (m/z 385) was

288

also identified for the first time in rosemary and thyme. Lastly, dicaffeoylquinic acid

289

(m/z 515) was detected in rosemary, thyme, oregano, cinnamon and cumin. Hossian et

290

al. (2010) identified dicaffeoylquinic acids in rosemary, thyme, oregano, sage, and basil

291

but, as far as we know, this is the first time they have been reported in cumin and

292

cinnamon. Mass spectra of those compounds identified for the first time are shown in.

293

Figure 2a-c.

294

Consistent differences (p < 0.05) in TP content were observed among the different herbs

295

and spices (Table 3), ranging from 1.12 mg GAE/g DW in bay to 5.82 mg GAE/g DW

296

in cinnamon. A similar pattern was observed in their antioxidant capacities. The ABTS +

297

assay gave results between 0.72 mmol TE/g DW and 4.13 mmol TE/g DW for bay and

298

cinnamon, respectively. The DPPH assay presented 0.30 mmol TE/g DW for bay and

299

2.16 mmol TE/g DW for cumin. The radical-scavenging capacities of oregano,

300

rosemary and thyme extracts have been previously observed in different model systems

301

(Erkan, Ayranci, & Ayranci 2008; Miura, Kikuzaki, & Nakatani 2002; Vichi, Zitterl-

302

Eglseer, Jugl, & Franz 2001).

303 304

3.2. Pattern of similarities among herbs and spices according to the phenolic

305

composition

306

The HPLC-MS/MS performance parameters are reported in Table 4. Recoveries ranged

307

from 85% and 111% and repeatability was less than 8% for all the analytes. Limits of

308

detection (LODs) were between 1.7·× 10‒4 for chlorogenic acid and 8.9 ×·10‒3 for

309

quercetin. The quantification levels of the main polyphenols observed revealed

310

distinctive features among culinary herbs and spices. The results of the quantitative

311

determination of the target polyphenols are summarised in Table 5. The statistically

312

significant differences (p < 0.05) found between the herbs and spices for each

313

polyphenol are highlighted with different superscripts. An HPLC chromatogram of bay,

314

including identification of each peak, is shown in Figure 3.

315

The main phenolic acid in the studied culinary herbs was found to be rosmarinic acid,

316

which varied from 0.39 µg/g DW in bay to 157 µg/g DW in rosemary, being the

317

dominant phenolic compound in oregano, thyme and rosemary. It should be noted that

318

the three species showing similarities - oregano, thyme and rosemary - all belong to the

319

Lamiaceae family. These results are in accordance with another study analysing 26

320

spice extracts (Shan et al., 2005). Rosmarinic acid, along with other compounds also

321

present in rosemary (i.e., carnosol, rosmanol, epi-rosmanol, among others), has shown

322

potent antioxidant activities, and is well correlated with total antioxidant activity

323

(Herrero, Plaza, Cifuentes, & Ibáñez 2010). A similar pattern was observed for p-

324

hydroxybenzoic acid, with the highest levels found in rosemary (15.2 µg/g DW) and the

325

lowest in bay (1.14 µg/g DW).

326

The highest levels of caffeic acid (6.56‒12.6 µg/g DW) were observed in oregano,

327

thyme and rosemary, with lower amounts found in cinnamon, cumin and bay (0.44 to

328

3.06 µg/g DW). As mentioned above, it should be noted that species showing

329

similarities - oregano, thyme and rosemary - belong to the Lamiaceae family. Caffeic

330

acid has been previously identified in oregano and rosemary (Agiomyrgianaki & Dais

331

2012; Herrero et al., 2010). The results for caffeic acid reported by Kivilompolo et al.

332

are in line with our study, with less than 50 µg/g DW found in rosemary and oregano,

333

although they describe higher levels in thyme (129 µg/g DW) (Kivilompolo &

334

Hyotylainen 2007; Park 2011). Papageorgiou et al. reported higher levels of caffeic

335

acid in rosemary (300 to 1500 µg/g DW) than in our study, but with undetectable

336

amounts in bay (Papageorgiou, Mallouchos, & Komaitis 2008). Differences in phenolic

337

acid levels from those in the literature can be attributed to genotypic and environmental

338

differences within species, choice of plant parts tested, when samples were taken and

339

determination methods.

340

Syringic acid showed the same pattern as the aforementioned phenolic acids, with

341

rosemary containing the highest amount (3.46 µg/g DW), followed by oregano (1.26

342

µg/g DW), while bay and cumin showed the lowest levels (0.40‒0.47 µg/g DW).

343

Kivilompolo et al. found syringic acid below 50 µg/g DW in thyme, with undetectable

344

levels in the other herb extracts analysed (Kivilompolo & Hyotylainen 2007; Park

345

2011). In contrast, Hossain et al. reported the presence of syringic acid in thyme,

346

rosemary, oregano and bay (Hossain, Rai, Brunton, Martin-Diana & Barry-Ryan, 2010).

347

Levels of chlorogenic acid were similar in all culinary herbs and spices, with the

348

exception of cumin, which contained 4.18 µg/g DW. Results in line with our study are

349

reported in the literature (Hossain et al., 2010).

350

Levels of protocatechuic acid were highest in cinnamon (10.2 µg/g DW) followed by

351

oregano (9.94 µg/g DW) and rosemary (8.42 µg/g DW), with the lowest in bay and

352

thyme (2.05‒2.55 µg/g DW). Our results for protocatechuic acid are higher than those

353

reported by Papageorgiou et al., who found levels between 3.20 and 4.50 µg/g DW for

354

rosemary, 0.10 and 2 µg/g DW for oregano, and undetectable amounts in bay

355

(Papageorgiou et al., 2008). In another study investigating the phenolic content of 26

356

common spice extracts from 12 botanical families, protocatechuic acid was not detected

357

in any of the species studied in our work, instead being found in sweet basil, dill, star

358

anise and coriander (Shan et al., 2005).

359

In contrast with the aforementioned phenolic acids, levels of p-coumaric acid were

360

highest in bay (9.64 µg/g DW), followed by rosemary (5.57 µg/g DW) and oregano

361

(4.90 µg/g DW), with the lowest levels observed in cumin (0.74 µg/g DW). Coumaric

362

acid is one of the main compounds found in all herbs and spices, together with

363

chlorogenic and p-hydroxybenzoic acids (Lv et al. 2012; Miron, Plaza, Bahrim, Ibáñez,

364

& Herrero 2011; Shan et al., 2005). Lastly, ferulic acid levels were highest in bay and

365

oregano (2.12‒2.15 µg/g DW) and lowest in cinnamon (0.33 µg/g DW), in accordance

366

with other studies detecting these compounds (Baatour et al. 2012; Shan et al., 2005).

367

Shan et al. reported between 0.85 and 7.55 µg/g DW ferulic acid in rosemary, and

368

between 1.30 and 4.90 µg/g DW in oregano, with none detected in bay.

369

Catechin and epicatechin were quantified in cumin and cinnamon, being under the

370

detection limits in the other studied herbs. Catechin levels ranged from 14.1 µg/g DW to

371

16.1 µg/g DW in cumin and cinnamon, respectively. Similarly, epicatechin levels were

372

6.43 µg/g DW for cumin and 7.25 µg/g DW for cinnamon. Catechin and epicatechin

373

have been previously identified by other authors (Shan et al., 2005).

374

Quercetin has been previously detected in rosemary, oregano, sage, bay and thyme

375

(Hossain et al., 2010). In our study, quercetin levels ranged between 0.32 µg/g DW in

376

oregano and 7.50 µg/g DW in cumin, in accordance with another study that reported

377

between 0.20 and 6 µg/g DW quercetin in rosemary, 0.20 and 2.30 µg/g DW in

378

oregano, and none in bay (Papageorgiou et al., 2008).

379

A PCA was carried out to discriminate among culinary herbs and spices (Figure 4)

380

according to their phenolic profile. The two principal components (PC1 and PC2)

381

obtained for each herb or spice accounted for 79.89 % of the variability of the original

382

data. The closer the location of variable Y (= loading) to the axis origin, the lower its

383

contribution to the class distinction among herbs and spices. Thus, plant metabolites

384

such as protocatechuic and chlorogenic acid showed a low discriminating power (Figure

385

4), while large loadings for variables such as catechin, epicatechin, rosmarinic and

386

caffeic acids were highly discriminatory. It can be clearly observed that catechin,

387

epicatechin and quercetin are highly correlated with cumin and cinnamon. In contrast,

388

bay, thyme and oregano, which are situated in the middle and bottom of the plot, are

389

related to low levels of these metabolites and higher levels of p-coumaric and ferulic

390

acids. On the other hand, rosemary, which is situated in the upper-right hand side of the

391

plot, is highly correlated with rosmarinic, caffeic, syringic and p-hydroxybenzoic acids.

392

393

In summary, high-resolution mass spectrometry provided a powerful tool for the

394

identification of polyphenolic diversity in culinary herbs and spices of the families

395

Lamiaceae (rosemary, thyme and oregano), Apiaceae (cumin) and Lauraceae (cinnamon

396

and bay), even in the absence of standards. Quantification levels of phenolic compounds

397

revealed distinguishing features among these plant families. Our results show that these

398

culinary ingredients are rich in phenolic constituents and demonstrate good antioxidant

399

capacities, and the use of them in cooking and food processing may have beneficial

400

effects for human health.

401

402

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511 512

Protocatechuic acid p-Hydroxybenzoic acid Chlorogenic acid Catechin Caffeic acid Syringic acid Epicatechin p-Coumaric acid Ferulic acid Rosmarinic acid Quercetin

DP -40 -40 -40 -50 -40 -40 -50 -40 -50 -40 -50

CE513 -20 -20514 -20 -25515 -20 516 -20 -25517 -20 -20518 -20 -30519

520 521 522

DP: declustering potential; CE: collision energy

Table 1: Optimized parameters for MRM conditions

Table 2: List of compounds identified in culinary herbs and spices

Compound

RT (min) [M ‒ H]‒ MS/MS ions

Acc Mass

Mda MF

detected in

1

Gallic acid*

1.43

169

125 (100)

169.0142

0.8

C7H6O5

R,T,O,Ci,Cu,B

2

Vanillic acid-O-hexoside

1.50

329

329 (10), 167 (100)

329.0877

0.7

C14H18O9

R,T,O,B

3

Syringic acid*

1.70

197

182 (40), 167 (40),

197.0455

0.5

C9H10O5

R,T,O,Ci,Cu,B

4

Caffeic acid-O-hexoside 1

2.10

341

179(100), 135 (10)

341.0877

0.7

C15H18O9

R,T,O,Ci,Cu,B

5

Neochlorogenic acid (3-Ocaffeoylquinic acid )

2.13

353

191 (100), 179 (40), 135 (20)

353.0877

0.8

C16H18O9

R,T,O,Ci,Cu,B

6

Protocatechuic acid*

2.36

153

153 (40), 109 (90)

153.0193

0.4

C7H6O4

R,T,O,Ci,Cu,B

7

Caffeic acid-O-hexoside 2

2.82

341

179(100), 135 (10)

341.0877

0.9

C15H18O9

R,T,O,Ci,Cu,B

8

Homovanillic acid-O-hexoside 1 3.14

343

181 (100), 137 (10)

343.1034

0.7

C15H20O9

R,T,Ci,B

9

3-O-p-Coumaroylqunic acid

3.29

337

191 (10), 163 (100)

337.0930

1.5

C16H18O8

Cu

10 Caffeic acid-O-hexoside 3

3.30

341

179(100)

341.0877

0.7

C15H18O9

R,T,O,Ci,Cu,B

11 p-Hydroxybenzoic acid*

3.52

137

93 (100)

137.0244

0.4

C7H6O3

R,T,O,Ci,Cu,B

Chlorogenic acid (5-O12 caffeoylquinic acid )*

3.58

353

191 (100)

353.0877

0.9

C16H18O9

R,T,O,Ci,Cu,B

13 Catechin*

3.69

289

245 (100)

289.0718

1.4

C15H14O6

Ci,Cu

14 Coumaric acid-O-hexoside 1

3.70

325

163 (100), 119 (20)

325.0928

0.8

C15H18O8

R,T,O,Ci,B

15 m-Hydroxybenzoic acid*

3.89

137

93 (100)

137.0244

0.5

C7H6O3

R,T,O,Ci,Cu,B

Cryptochlorogenic acid (4-O16 caffeoylquinic acid)

3.91

353

191(50), 173 (100), 135 (20)

353.0877

0.4

C16H18O9

R,T,O,Ci,Cu,B

17 Homovanillic acid

4.07

181

137 (100)

181.0506

0.2

C9H10O4

T,O,B

18 Proanthocyanidin trimer 1

4.65

863

711 (60), 575 (100), 287 (10)

863.1829

1.5

C45H36O18 Ci,Cu

19 Caffeic acid*

4.84

179

135 (100)

179.0349

0.3

C9H8O4

20 Proanthocyanidin trimer 2

5.02

863

711 (60), 575 (100), 287 (10)

863.1829

1.3

C45H36O18 Ci,Cu

21 Epicatechin*

5.32

289

245 (100)

289.0718

1.3

C15H14O6

22 Apigenin-C-hexoside-C-hexoside 5.36

593

503 (30), 473 (100), 383 (20), 353 (40),

593.1511

0.7

C27H30O15 R,O

23 4-O-p-Coumaroylqunic acid

5.67

337

191 (20), 173 (100), 163 (30)

337.0930

0.1

C16H18O8

R,T,O,Ci,Cu,B

24 Ferulic acid-O-hexoside

5.78

355

193 (100)

355.1034

0.9

C16H20O9

R,T,Ci

25 Coumaric acid-O-hexoside

6.03

325

163 (100), 119 (20)

325.0928

1.2

C15H18O8

B

26 Sinapic acid-C-hexoside

6.95

385

325 (50), 295 (100), 265 (70), 223 (25)

385.1139

0.6

C17H22O10 R,T

27 Vanillic acid

7.03

167

167 (50), 152 (20), 108 (50)

167.0350

0.4

C8H8O4

R,T,O,Ci,Cu,B

Ci,Cu

T,B

28 Proanthocyanidin trimer 3

7.48

863

711 (60), 575 (100), 287 (10)

863.1829

1.8

C45H36O18 Ci,Cu

29 Kaempferol-O-dihexoside

7.70

609

447 (60), 285 (100)

609.1460

1.2

C27H30O16 O

30 Coumaric acid*

7.90

163

119 (100)

163.0400

0.3

C9H8O3

31 Rosmarinic acid-O-hexoside

8.11

521

359 (100)

521.1300

0.2

C24H26O13 R,O

32 Ferulic acid*

8.22

193

193 (5), 178 (40), 149 (10), 134 (80)

193.0506

0.3

C10H10O4

1727.3730 [M-2H]2- 2.6

C90H72O36 Ci,Cu

R,T,O,Ci,Cu,B

R,T,O,Ci,Cu,B

33 Proanthocyanidin hexamer

8.30

863

1006 (20), 863 (100), 755 (50), 575 (40), 287 (10)

34 Rutin*

8.68

609

301 (100)

609.1460

1.4

C27H30O16 R,T,Ci,Cu,B

35 Kaempferol-3-O-rutinoside*

8.79

593

285 (100)

593.1511

0.5

C27H30O15 R,T,O,Cu,B

36 Quercetin-3-O-glucoside*

9.08

463

301 (100)

463.0881

1.3

C21H20O12 R,T,O,Ci,Cu

37 Kaempferol-3-O-glucoside*

9.15

447

285 (100)

447.0932

0.5

C21H20O11 R,T,O,Ci,Cu,B

38 Dicaffeoylquinic acid 1

10.03 515

353 (100), 173 (10), 179 (8)

515.1194

0.5

C25H24O12 R,T,O,Ci,Cu

39 Naringenin-C-hexoside*

10.81 433

373(50), 343(50), 303 (20)

433.1140

0.4

C21H22O10 R,T,Ci

40 Hesperidin*

10.91 609

301 (100)

609.1825

0.9

C28H34015 R,T,O

41 Apigenin-7-O-glucoside*

10.93 431

269 (100)

431.0983

1.1

C21H20O10 R,T,O

42 Rosmarinic acid*

12.05 359

197 (30), 161 (100)

359.0772

1.1

C18H16O8

R,T,O,Ci,Cu,B

43 Naringenin-O-hexuronide

13.99 447

271 (100),175 (10)

447.0932

1.2

C21H20O11 Cu

44 Kaempferol*

15.24 285

285 (40), 151 (100)

285.0405

0.9

C15H10O6

R,T,O,Ci,Cu,B

45 Quercetin*

15.33 301

301 (10), 151 (100)

301.0353

0.5

C15H10O7

R,T,O,Ci,Cu,B

46 Naringenin*

17.40 271

271 (15), 151 (100)

271.0611

1.1

C15H12O5

B

47 Apigenin*

17.55 269

269 (10), 151 (100)

269.0455

0.6

C15H10O5

R,T,O,Ci

48 Hesperetin*

17.91 301

286 (30), 151 (100)

301.0718

1.2

C16H14O6

R,T,O,Cu,B

49 Rosmanol

19.00 345

301 (100)

345.1707

0.8

C20H26O5

R,O

50 Carnosol

21.90 329

329 (10), 285 (100)

329.1758

0.6

C20H26O4

R,O

51 Carnosic acid

23.09 331

331 (70), 287 (100)

331.1915

0.8

C20H28O4

R,T,O,Ci

R: Rosemary; T:Thyme; O: Oregano; Cu: Cumin; Ci: cinnamon; B: bay *Comparison with standard

Table 3. Total polyphenols (mg GAE/g DW) and antioxidant capacity of culinary herbs and spices through ABTS+ and DPPH assays (mmol TE/g DW) expressed as mean ± SD. Different letters in the columns represent statistically significant differences (p < 0.05).

Herbs/ Spices Rosemary

TP

ABTS+

DPPH

5.02 ± 0.43a

2.39 ± 0.17a

1.98 ± 0.17a

Thyme

3.36 ± 0.14b

1.38 ± 0.13b

1.15 ± 0.06b

Oregano

2.23 ± 0.18c

1.34 ± 0.13b

0.78 ± 0.07c

Cumin

4.98 ± 0.31a

3.26 ± 0.29c

2.16 ± 0.06d

Cinnamon

5.82 ± 0.44d

4.13 ± 0.43d

1.88 ± 0.10e

Bay

1.12 ± 0.08e

0.72 ± 0.07e

0.30 ± 0.02f

GAE: gallic acid equivalents; TE: Trolox equivalents; SD: standard deviation.

Table 4. Performance parameters of the HPLC-MS/MS methodology

Protocatechuic acid p-Hydroxybenzoic acid Chlorogenic acid Catechin Caffeic acid Syringic acid Epicatechin p-Coumaric acid Ferulic acid Rosmarinic acid Quercetin a

LODa Recoveries Repeatibility (µg/g DW) (%) (RSD%)b 2.4·× 10-4 95.30 ± 2.22 5.69 -4 6.8·× 10 90.12 ± 1.56 4.21 1.7·× 10-4 91.23 ± 2.44 7.66 1.2·× 10-3 89.20 ± 1.98 6.37 -3 2.6·× 10 98.50 ± 1.33 4.43 2.7·× 10-3 92.43± 1.42 5.15 -3 1.7·× 10 89.44 ± 2.09 7.76 1.3·× 10-3 94.56 ± 2.33 4.94 -4 2.8·× 10 94.20 ± 2.59 4.85 2.0·× 10-3 111.30 ± 3.22 5.33 -3 8.9·× 10 85.81 ± 3.03 3.29

LOD: limit of detection

b

RSD: relative standard deviation

Table 5: Quantification of individual polyphenols (mean ± SD) of culinary herbs expressed as µg/g DW. Different letters in the columns represent statistically significant differences (p < 0.05).

Caffeic acid

Catechin

Chlorogenic acid

Epicatechin

Ferulic acid

p-Coumaric acid

p-Hydroxybenzoic acid

Protocatechuic acid

Rosmarinic acid

Rosemary

12.58 ± 0.44a