Profiling and Quantification of Phenolics in Stevia rebaudiana Leaves

Subscriber access provided by UNIV LAVAL

Article

Profiling and Quantification of Phenolics in Stevia Rebaudiana Leaves Hande Karaköse, Anja Müller, and Nikolai Kuhnert J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01944 • Publication Date (Web): 03 Sep 2015 Downloaded from http://pubs.acs.org on September 21, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

Journal of Agricultural and Food Chemistry

Profiling and Quantification of Phenolics in Stevia Rebaudiana Leaves

1

2

3

Hande Karaköse, Anja Müller and Nikolai Kuhnert*

4

Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen,

5

Germany

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

*Author to whom correspondence should be addressed Tel: 49 421 200 3120; Fax: 49 421 200

24

3229; E-mail: [email protected] 1 ACS Paragon Plus Environment


25

Abstract

26

Stevia rebaudiana (Bertoni) is a plant from the Asteraceae family with significant economic

27

value due to steviol glycoside sweeteners in its leaves. Chlorogenic acids and flavonoid

28

glycosides of Stevia rebaudiana from seven different botanical varieties, cultivated over two

29

years and harvested three times a year in eight European locations were profiled and quantified in

30

a total of 166 samples. Compounds quantified include chlorogenic acids and flavonoid glycosides

31

and aglycons. All phenolic concentration profiles show a perfect Gaussian distribution. Principal

32

component analyses allow distinction between varieties of different geographical origin and

33

distinction between different plant varieties. While concentrations of all chlorogenic acids

34

showed a positive correlation, no correlation was observed for flavonoid glycosides. Conclusions

35

from these findings with respect to the biosynthesis and functional role of phenolics in Stevia

36

rebaudiana are discussed.

37

38

Keywords: Stevia rebaudiana, statistical evaluation, anova, LC-MS quantification

39

40

41

42

43

44

45

46

47

48

2 ACS Paragon Plus Environment

Page 2 of 39

Page 3 of 39


49

Introduction

50

Polyphenols are ubiquitous plant secondary metabolites encountered in all dietary plants. They

51

have been linked in numerous epidemiological studies and subsequent human intervention studies

52

with a variety of health benefits including prevention of cancer and type 2 diabetes, and

53

improvement of cardiovascular disease.1-4 Dietary plants produce typically a large variety of

54

structurally diverse polyphenols - most plants an average of around twenty distinct compounds.

55

Some exceptions exist such as coffea canephora, which produces in excess of hundred distinct

56

phenolic metabolites.5 Next to a significant number of compounds dietary plants biosynthesize

57

polyphenols in significant quantities, which range from 1% of their dry weight up to 15% of their

58

dry weight in fruits and leaves.6, 7 While quantitative data exist for many polyphenols in small

59

sample sets (between 3 and 10 samples), sparse data is available in the literature for large sample

60

sets with more than 100 samples of a single dietary plant. Accordingly, we have little reliable

61

knowledge on how quantities of polyphenols vary in large sample sets, neither on an individual

62

compound basis nor on a level of the full phenolic metabolic profile of the plant. We have little

63

information about minimum and maximum level of phenolic concentrations, the spread and

64

distribution of phenolic concentrations or correlation of concentrations in plants. Consequently,

65

no knowledge exists how agricultural parameters might be varied to increase plant polyphenol

66

content to improve health benefits or to minimize polyphenol content to avoid excessive

67

bitterness or astringency associated with plants rich in polyphenols. As a result, we lack

68

knowledge on how the biosynthesis of individual compound classes or compounds is regulated

69

within the plant and hence, how compound levels influence one another. Furthermore, the

70

complete polyphenol metabolome might be useful in barcoding samples in order to obtain

71

information on their origin, growth conditions or botanical varieties.



Page 4 of 39

72

In this contribution we present a unique data set on the full polyphenolic metabolite profile of

73

Stevia rebaudiana leaves from a total of 166 samples varying in their geographical origin,

74

botanical varieties, year of harvest, time of harvest and agricultural growth conditions. From the

75

166 samples, six chlorogenic acids (CGA) and six flavonoids were quantified using LC-MS

76

methods and the statistical variation of the compounds quantified analyzed within the full data set

77

and selected data subsets. Statistical analyses of the correlation between the individual secondary

78

metabolites were carried out.

79

We have previously reported on the presence of around 30 different chlorogenic acids (CGAs) in

80

Stevia rebaudiana leaves.8 CGAs are a large family of esters formed between quinic acid and

81

certain trans-hydroxycinnamic acids, most commonly caffeic, p-coumaric, and ferulic acid.6

82

Flavonoids have as well been reported in Stevia rebaudiana leaves and quantified as total

83

flavonoids, however, little detailed structural information on the compounds was reported.9,

84

The current state of the art of Stevia rebaudiana`s phytochemical profile has recently been

85

reviewed by Wülwer-Rieck.11 Similar to chlorogenic acids the presence of flavonoid compounds

86

would add a health benefit to the usage of Stevia rebaudiana leaves in food products, in contrast

87

to purified steviol glycosides currently approved by most legislative authorities. Flavonoids are a

88

class of secondary metabolites that are produced ubiquitously in fruits and vegetables. By

89

definition, flavonoids are compounds with a C6-C3-C6 structure comprising two aromatic rings,

90

one fused as a benzopyran. Flavonoids include several subgroups which vary in the oxidation of

91

the C3 carbon of the C-ring, and in their hydroxylation, methylation and glycosylation, all these

92

classes collectively described by the term flavonoids. Within different subclasses of flavonoids,

93

further differentiation is based on the number, position and nature of substituent groups attached

94

on the rings. Most flavonoids appear in plants as their glycosides with sugars such as glucose,

95

galactose, rhamnose, arabinose, xylose and rutinose conjugated to one or several phenolic OH 4 ACS Paragon Plus Environment

10

Page 5 of 39


96

groups. Flavonoid glycosides have many isomers with the same molecular weight but different

97

aglycone and sugar components at different positions attaching on the aglycone ring.7

98

Stevia rebaudiana belongs to the Asteraceae family of plants, and it is native to Paraguay. The

99

high content of natural sweeteners, the steviol glycosides of the ent-kaurene class of compounds,

100

contained in its leaves makes Stevia rebaudiana of a significant economic value in food industry

101

in many applications as a “zero calorie sweetener”. Purified steviol glycosides have only recently

102

been approved as food additives by European and US legislating authorities, leading to a

103

dramatic increase in its global use and scientific interest in the crop.

104

105

Materials and Methods

106

Chemicals

107

The IUPAC numbering has been used for chlorogenic acids and the chlorogenic acids of 3-

108

caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA), 5-caffeoylquinic acid (chlorogenic

109

acid), 3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), and 4,5-

110

dicaffeoylquinic acid (4,5-diCQA), were purchased from PhytoLab (Vestenbergsgreuth,

111

Germany). Flavonoid glycoside standards were obtained from Sigma Aldrich, HPLC grade

112

acetonitrile, methanol and chloroform was obtained from Carl-Roth GmbH, Karlsruhe.

113

Stevia rebaudiana leaves were obtained from Universität Hohenheim who led the trial cultivation

114

in eight different EU regions.

115

116

Sample Preparation

117

Two grams of Stevia rebaudiana leaves were immersed in liquid nitrogen, ground in a hammer

118

mill, and extracted first with 150 mL of chloroform in a Soxhlet apparatus (Buchi B-811



119

extraction system) for 2 h and then with 150 mL of methanol for another 2 h. Solvents were

120

removed from the methanolic extract in vacuo, and extracts were stored at - 20 oC until required.

121

LC-TOF MS

122

The LC equipment (Agilent 1100 series, Bremen, Germany) comprised a binary pump, an

123

autosampler with a 100 μL loop, and a diode array detector with a light-pipe flow cell (recording

124

at 254 nm and scanning from 200 to 600 nm). This was interfaced with a MicroTOF Focus mass

125

spectrometer (Bruker Daltonics) fitted with an ESI source. The MS parameters were: nebulizer

126

1.6 bar, dry gas 12.0 L/min, dry temperature 220 °C. The MicroTOF was operated in negative ion

127

mode and the mass range was 150 – 1200 m/z. Internal calibration was achieved with 10 mL of

128

0.1 mol/L sodium formate solution injected through a six-port valve prior to each

129

chromatographic run. Calibration was carried out using the enhanced quadratic calibration mode.

130

LC-MSn

131

The LC equipment (Agilent 1100 series, Bremen, Germany) comprised of a binary pump, an

132

autosampler with a 100 μL loop, and a diode array detector with a light-pipe flow cell (recording

133

at 254 nm and scanning from 200 to 600 nm). This was interfaced with an ion-trap mass

134

spectrometer fitted with an ESI source (Bruker Daltonics HCT Ultra, Bremen, Germany)

135

operating in Auto-MSn mode to obtain fragment ions m/z. Tandem mass spectra were acquired in

136

Auto-MSn mode (smart fragmentation) using a ramping of the collision energy. Maximum

137

fragmentation amplitude was set to 1 V, starting at 30% and ending at 200%. MS operating

138

conditions (negative mode) were capillary temperature of 365 °C, a dry gas flow rate of 10

139

L/min, and a nebulizer pressure of 50 psi.

140

HPLC

141

Separation was achieved on a 250 x 3 mm C18 column (Varian Pursuit XRS) with 5 μm particle

142

size. Solvent A was water/formic acid (1000+0.005% v/v), and solvent B was acetonitrile (ACN). 6 ACS Paragon Plus Environment

Page 6 of 39

Page 7 of 39


143

Solvents were delivered at a total flow rate of 0.5 mL/min and the column temperature was set to

144

25 oC. 5 μL of samples were injected in to LC-MS system, unless stated otherwise. The gradient

145

profile was 10 to 80% B in 60 min and a return to 10% B at 65 min and 5 min isocratic to re-

146

equilibrate.

147

Calibration Curve of Standard Compounds

148

Most abundant chlorogenic acid derivatives (3-CQA, 4-CQA, 5-CQA, 3,5-diCQA, 4,5 diCQA)

149

and two flavonoid glycosides (quercetin-3-glycoside and kaempferol-7-glycoside) were chosen

150

for calibration curves.

151

Stock solutions of the standard compounds were prepared in 80% ACN/water. A series of

152

standard solutions was injected (5 μL) into the LC-MS system. The areas of the peaks of each

153

standard from extracted ion chromatograms (EIC) were used to make the respective standard

154

curves.

155

Statistical Analyses

156

Statistical analyses of the data were performed using IBM SPSS 20. The distributions of the

157

variables were tested for normality using the Kolmogorov-Smirnov test. Associations between

158

the variables were investigated using both parametric (Pearson’s correlation) and non-parametric

159

(Spearman’s correlation) techniques. Results were interpreted using the widely accepted 5% level

160

of significance.

161

To test whether there were differences on each chlorogenic acid with respect to its origin or

162

variety, separate one-way ANOVA analyses was employed, followed by two post-hoc tests:

163

Fisher’s Least Significant Difference (LSD) as the least conservative test where equal variances

164

are assumed and Games-Howell test where non-equal variances are assumed for the multiple pair

165

wise comparison tests. All empirical results were interpreted using the widely accepted 5% level

166

of significance (p < 0.05). 7 ACS Paragon Plus Environment


167

A principle component analyses (PCA) based on the LC-MS dataset of stevia phenols was carried

168

out using Profile Analysis 1.1 (Bruker Daltonics) with kernelizing prior to bucketing and

169

normalization to sum of peaks to allow differentiation between different stevia varieties and

170

geographic origins.

171

172

Results and Discussion

173

Within a large agricultural trial, Stevia rebaudiana was cultivated in nine Southern European

174

locations from 2010 to 2011. Up to three times a year leaves were harvested from July to

175

September. In total seven different botanical varieties of Stevia rebaudiana were cultivated.

176

Following harvesting, leaves were dried and analyzed at the latest three months after harvesting.

177

The European Stevia rebaudiana samples were complemented by commercial Stevia rebaudiana

178

samples cultivated in South America and Asia resulting in a total of 166 samples.

179

Compound identification

180

Methanolic Stevia rebaudiana extracts were analyzed by LC-MSn in the negative ion mode using

181

an ESI ion-trap mass spectrometer, allowing assignments of compounds to region-isomeric level,

182

and additionally by high-resolution mass spectrometry using LC-ESI-TOF MS in negative ion

183

mode allowing determination of molecular formulae based on the accurate mass measurements

184

(see Supplementary Information). Molecular formulae were, in general, accepted if an error

185

below 5 ppm was experimentally observed. The Stevia rebaudiana samples under investigation

186

contained up to 29 chlorogenic acid derivatives, published by our group previously.8

187

Furthermore, the samples contained a series of flavonoid glycosides. No new additional

188

compounds were identified in any of the samples. Structures are presented in Figure 1.

189

All analytes showed baseline separation with exception of the pair 3,4- and 4,5 dicaffeoylquinic

190

acid. Peak assignments of CGAs have been made on the basis of structure diagnostic hierarchical 8 ACS Paragon Plus Environment

Page 8 of 39

Page 9 of 39


191

keys previously developed,12-15 supported by means of their parent-ion high-resolution mass, UV

192

spectra, and retention times relative to 5-CQA using validated methods in our laboratory.5 The

193

base peak chromatogram of Stevia rebaudiana extract is shown in Figure 2. Within the

194

chromatogram, CGAs and flavonoid glycosides elute between 8 and 25 minutes, whereas steviol

195

glycosides elute at later retention times between 28 and 40 minutes.

196

Four peaks were detected at m/z 353.1 and assigned using the hierarchial keys previously

197

developed as well-known 3-CQA, 5-CQA, and 4-CQA and cis-5CQA.12 Three dicaffeoylquinic

198

acid isomers were identified by their parent ion m/z 515.2 and were assigned as 3,5-diCQA, 3,4-

199

diCQA, and 4,5-diCQA using the hierarchial key.13 Two further peaks present as minor

200

components showed fragmentation patterns, similar to that of 4,5-diCQA, which are identified as

201

cis-isomers of 4,5-diCQA.16 Tri-caffeoylquinic acids and caffeoylshikimates were present as

202

minor compounds and their regiochemistry assigned using published tandem MS methods.14, 15

203

A total of fifteen peaks in the chromatogram correspond to flavonoid glycosides with their

204

characteristic fragmentation patterns in tandem MS showing neutral losses of sugar moieties of

205

162 Da (- C6H10O5) followed by characteristic fragment spectra of the aglycones quercetin (m/z

206

300), kaempferol (m/z 285), luteolin (m/z 285) and apigenin (m/z 269) in MS3 (see Table 1)

207

(Figure 3). For example, three peaks were located with an m/z value of 447.1 (Figure 4)

208

showing, after a neutral loss of 162 Da a base peak at m/z 285 in MS2. Further fragmentation in

209

MS3 with the ion at 285.1 as a precursor ion revealed two fragment ions characteristic for

210

kaempferol and one fragment ion characteristic for luteolin. Additional hydrolysis experiments

211

followed by LC-MS analyses confirmed the presence of aglycones of kaempferol, quercetin,

212

luteolin and apigenin by comparison of retention times, high resolution MS data and tandem MS

213

data compared to reference substances in Stevia rebaudiana leaves. Therefore, we tentatively



Page 10 of 39

214

assigned the flavonoid glycosides as hexose conjugates of kaempferol, quercetin, luteolin and

215

apigenin.

216

Comparison of the fifteen flavonoid glycosides present in Stevia rebaudiana leaves with

217

reference standards, either commercial or obtained through preparative HPLC purification in

218

previous work, showed that all compounds, with the exception of rutin and naringenin, were not

219

identical to the reference compounds available (all eight glucosides reported in dietary plants and

220

two galactosides) as judged by their retention times and fragment spectra. We suggest that the

221

flavonoids present in Stevia rebaudiana are presumably based on fructose, galactose or mannose.

222

For other plants of the Asteraceae family it was shown by Harrison et al17 that poly-fructans are

223

present and by the group of Goffner18 that additionally, galactomannans form the most abundant

224

polymeric carbohydrate structures in this plant family.18 Hence it can be speculated that these C6-

225

sugars are present in Stevia rebaudiana leaf flavonoids, for which no reference materials are

226

available.

227

Quantification of Chlorogenic acids and Flavonoids

228

Quantitation was carried out using eight point calibration curves. Chlorogenic acids were

229

quantified by LC-UV at 320 nm and by LC-MS using extracted ion chromatograms of the

230

pseudomolecular ions at m/z 353.1 and 515.2 in negative ion mode using an ESI-TOF MS

231

spectrometer. Additionally, flavonoids were quantified by LC-UV using kaempferol-7-glucoside

232

and quercetin-3-glucoside as reference standards, resulting in relative values rather than absolute

233

values for flavonoids. All values obtained are quoted in g/100g dried leaf material.

234

Chlorogenic acid standard solutions were analyzed by LC-MS using the same chromatographic

235

method as used for Stevia rebaudiana leaf extracts. Calibration curves for mono-caffeoyl

236

derivatives, 3-CQA, 4-CQA and 5-CQA and for dicaffeoyl derivatives 3,4-diCQA, 3,5-diCQA

237

and 4,5 diCQA, were obtained using extracted ion chromatograms (see Table 2). Pearson 10 ACS Paragon Plus Environment

Page 11 of 39


238

correlation coefficients of the calibration curves using LC-MS quantifications are given in Table

239

2. All calibration curves were linear within the quantification range. Values obtained from LC-

240

UV and LC-MS quantification gave the same absolute concentration values with an error of less

241

than +/- 5% showing validation of the method. All quantitative CGA values for all 166 samples

242

are given in the Supplementary Information. The quantitative values obtained for all chlorogenic

243

acid derivatives were found to be a factor of three-to-five higher than values previously reported

244

by our group.8 On occasions, the data showed a ten-fold increase of CGA concentrations. It must

245

be concluded that the material previously investigated, obtained from commercial sources was of

246

unknown age and origin. Fresh leaves of Stevia rebaudiana contain significant quantities of

247

CGAs, much higher than previously determined. In terms of absolute quantities, Stevia

248

rebaudiana shows, following green coffee beans and mate leaves the third highest concentration

249

of CGAs amongst all dietary materials.

250

The four flavonoid aglycones quercetin, kaempferol, luteolin and apigenin were quantified using

251

a LC-UV method monitoring absorption at 280 nm for selected Stevia rebaudiana samples

252

following hydrolysis of the sugar moiety in dilute acid. It is well established that the molar

253

extinction coefficient of flavonoid glycosides varies only to a small degree if structural variation

254

of the sugar moiety occurs.19,

255

Supplementary Information. All flavonoid glycosides were quantified using a LC-MS method

256

with quercetin-3-glucoside and kaempferol-7-glucoside as reference compounds, complementing

257

the aglycone structure of the flavonoid glycosides.

258

Analyses of data

259

With the quantitation carried out we have obtained a unique data set of the phenolic metabolome

260

of Stevia rebaudiana. This data set requires statistical evaluation allowing interesting

261

observations and conclusions. We decided to address the following questions in this contribution.

20

Quantitative values for the four glycones are given in the



262



Page 12 of 39

What is the statistical distribution of quantitative data? Little data is available in the literature that allows for a definite answer to this question.

263

264



How do phenolic quantities vary with variation of origin, harvest and plant variety?

265



Does the data set allow distinction between varieties or origin in a predictive manner by using quantitative phenol concentrations?

266

267



Do quantities of a set of distinct secondary metabolites correlate with one another?

268



Further questions addressing the influence of the phenolic metabolome on sensory

269

properties or the influence of agricultural parameters on the phenolic metabolome are

270

outside the scope of this contribution and will be commented on at a later stage.

271

Statistical Spread of Data

272

The distribution of the dataset is an essential step for examination of data in statistical analyses.

273

The most frequent distribution of data is the Gaussian or normal distribution. A normal

274

distribution can be easily characterized by observing its symmetrical bell shaped curve on a

275

histogram (see Supp. Info., Figure S1). Skewness and Kurtosis values below one show also that

276

the data is normally distributed. The Kolmogorov-Smirnov (KS) test was used for the analyses of

277

data distribution. The significance value above 0.05 means the data is normally distributed.

278

Each mono- and di-CQAs as well as total mono- and di-CQAs quantities obtained from 166

279

Stevia rebaudiana samples showed normal distribution. The KS test result, mean values, standard

280

deviations, skewness and kurtosis of the curve for each CGA is represented in Table 3. A

281

histogram of 5-CQA is presented as an example in supplementary information. The observation

282

of an almost perfect Gaussian distribution in all quantitative data came as a surprise. Normally,

283

phenolic metabolites are assumed to act as plant defense compounds produced by the plant in

284

various stress situations, in particular, stress induced by pathogens. Hence, a bimodial shaped

285

distribution of CGA concentrations could be expected with two maxima, whereby the higher 12 ACS Paragon Plus Environment

Page 13 of 39


286

concentration maximum corresponds to compound production under stress. The experimental

287

data clearly show this is not the case. It can be speculated that none of the 166 Stevia rebaudiana

288

samples were under stress. A more reasonable scenario would suggest that CGA quantities right

289

of the maximum of the Gaussian curve shifted to higher concentrations constitute a situation of a

290

plant under stress, and hence, CGA concentrations are only gradually increased by the plant.

291

Sample Variation

292

Variation between varieties

293

Seven defined botanical varieties of Stevia rebaudiana were cultivated and their phenolic profile

294

was determined. Average values determined for the seven botanical varieties are presented in a

295

radar plot in Figure 5. From the data, it can be seen that the average concentration of all

296

monocaffeoylquinic acids remains rather constant over all seven varieties (2.123 - 2.686 g/100g),

297

whereas a larger spread of data is observed for dicaffeoylquinic acids (1.484 – 2.432 g/100g).

298

Varieties 5, 6, 7 and 3 show on average increased levels of chlorogenic acids compared to

299

varieties 2 (Figure 5).

300

Variation between origins

301

Stevia rebaudiana cultivated in nine different locations within the EU and additionally samples

302

from outside the EU (see Supplementary Information) were available for comparison. According

303

to the literature, polyphenol concentrations are due to their physiological function as UV

304

protection agents, which are a direct function of growth altitude and climatic conditions,

305

particularly in sunshine hours. Accordingly, variations of chlorogenic acid concentrations

306

between different origins should be expected. Indeed, the data reveal significant variations in

307

average CGA concentrations varying from 3.090 -1.637 g/100g for total monocaffeoylquinic

308

acids and 2.890 - 1.144 g/100g for dicaffeoylquinic acids (see Supplementary Information for

309

average values of all origins). EU cultivated Stevia rebaudiana shows concentrations of CGAs 13 ACS Paragon Plus Environment


Page 14 of 39

310

sandwiched between extreme values at both ends observed in samples from outside the EU (e.g.

311

highest for origin G samples with an average value of 2.890 g/100g dicaffeoylquinic acids and

312

trial location L and E samples with a lowest average value of 1.448 g/100g and 1.144 g/100g

313

respectively). A radar plot shown in Figure 6 was used to display variations between different

314

origins.

315

The data allow insight into variations between minimum values and maximum values of

316

phenolics. Our data show that between minimum and maximum values encountered in plants

317

from an individual harvest, an average factor of five to eight, depending on the compound in

318

question is observed, indicating a large statistical spread of phenol concentrations..

319

Principal component analyses (PCA)

320

To allow differentiation between different Stevia rebaudiana varieties and geographic origins a

321

principal component analyses (PCA) based on the LC-MS dataset of Stevia rebaudiana phenols

322

was carried out. PCA analyses were carried out with an aim to distinguish EU cultivated samples

323

from non-EU cultivated samples. For this purpose 20 EU and 20 non-EU samples were subjected

324

to a PCA analyses. Score and loading plots are shown in Figure 7. From the score plot it can be

325

seen that the samples fall in three groupings. Group A from South American samples can clearly

326

be distinguished from all other samples based on their high diCQA content (from loading plot).

327

A second group B contains exclusively European samples from the Uconor cooperative. The final

328

group C contains both EU and non-EU samples e.g. from Turkey, Ukraine and India, which

329

group together. The sample distinction information from the loading plot suggests that a

330

combination of rebaudioside A concentrations and diCQA concentrations allows distinction here.

331

A second PCA analyses was carried out to compare variations between different botanical

332

varieties. For this purpose data from four different botanical varieties were chosen and a PCA

333

analyses on the full LC-MS data set carried out. Score plot of PC1 versus PC2 shows little 14 ACS Paragon Plus Environment

Page 15 of 39


334

differentiation between sample varieties (Figure S2). However, when looking at higher order

335

principle components, e.g., PC2 versus PC4, three of the varieties group in the score plot with

336

two varieties grouping together in the same area of the plot. The loading plot reveals that the

337

compounds responsible for the variations include dicaffeoylquinic acids along with rutin.

338

Statistical evaluation of data

339

From the obtained data, a series of statistical analyses was carried out. For each variety, origin or

340

harvest, average values and standard deviations were determined. Additionally, the statistical

341

pattern and type of statistical distribution of the data was analyzed for each subgroup. The

342

correlation studies were performed by Pearson's correlation, with the significance value of p

Profiling and Quantification of Phenolics in Stevia rebaudiana Leaves

Recommend Documents