Harvest strategies for optimization of the content of bioactive

Subscriber access provided by University of Sunderland

Bioactive Constituents, Metabolites, and Functions

Harvest strategies for optimization of the content of bioactive alkamides and caffeic acid derivatives in aerial parts and in roots of Echinacea purpurea Maria O. Thomsen, Lars P. Christensen, and Kai Grevsen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03420 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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 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 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.

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 40

Journal of Agricultural and Food Chemistry

1

Harvest strategies for optimization of the content of bioactive

2

alkamides and caffeic acid derivatives in aerial parts and in roots

3

of Echinacea purpurea

4 5

Maria O. Thomsen,† Lars P. Christensen,*,§ and Kai Grevsen*,†

6 7

†Department

8

10, DK-5792 Aarslev, Denmark

9

§Department

10

of Food Science, Faculty of Science and Technology, Aarhus University, Kirstinebjergvej

of Chemistry and Bioscience, Faculty of Engineering and Science, Aalborg University,

Fredrik Bajers Vej 7H, DK-9220 Aalborg Ø, Denmark

11 12 13 14 15 16 17 18 19 20 21

1 ACS Paragon Plus Environment


Page 2 of 40

22 23 24

ABSTRACT

25

Aerial parts and roots of Echinacea purpurea were harvested consecutively in order to find the best

26

strategy for harvest of both types of plant material for an optimal content of bioactive alkamides and

27

caffeic acid derivatives. Four caffeic acid derivatives and 15 alkamides were identified and quantified.

28

The aerial parts were harvested in bud, bloom and wilting stage and the roots were harvested one week,

29

one month and three months after each harvest of aerial parts. The highest yield per area of both

30

alkamides and caffeic acid derivatives is achieved when the aerial parts are harvested late (wilting stage).

31

To obtain an optimal content of alkamides and caffeic acid derivatives it is not recommendable to harvest

32

the aerial parts and the roots in the same year. If the aerial parts must be harvested the roots should be

33

harvested one week after because this will result in the most optimal concentration of bioactive

34

compounds in both products.

35 36

Keywords: Harvest strategies; plant development stage; aerial parts; roots; caffeic acid derivatives;

37

alkamides

38 39 40 41 42 43 44 2 ACS Paragon Plus Environment

Page 3 of 40


45 46 47 48

INTRODUCTION

49

The medicinal plant Echinacea purpurea (L.) Moench (Heliantheae, Asteraceae) also known as purple

50

coneflower is a perennial originating from North America. Native Americans have used the aerial parts

51

and roots of E. purpurea for treatment of a wide variety of diseases and conditions. Today different

52

preparations of E. purpurea are popular herbal medicines in North America and Europe for the prevention

53

or treatment of infectious diseases and enhancement of the immune system.1 The plant is believed to

54

have an immune stimulating effect as well as other pharmacological activities, such as anti-inflammatory

55

and antioxidative effects. The therapeutic compounds of interest in E. purpurea are alkamides, caffeic

56

acid derivatives and polysaccharides.2−6

57

Alkamides is a well-studied group of compounds and comprehensive investigations in E. purpurea

58

plants and preparations have resulted in the identification of more than 18 different alkamides of which

59

several are isomeric pairs differing solely by their E/Z configuration of the conjugated 2,4-diene moiety

60

(Figure 1). Alkamides are known for their anti-inflammatory activity7−10 and have in addition shown both

61

antiviral and antifungal activity as well as antidiabetic effects in vitro.11−14 The lipophilic properties of

62

alkamides makes them highly bioavailable and they are believed to play a significant role in the

63

immunomodulating effect of E. purpurea.15−17 In contrast to alkamides, caffeic acid derivatives have a

64

relatively poor bioavailability.18 Several caffeic acid derivatives, including cichoric acid, have been

65

isolated by hydrophilic extraction of E. purpurea and are known for their antioxidant activity.19 Cichoric

66

acid, which is the major caffeic acid derivative in E. purpurea, has also shown antiviral activity.20

67

Furthermore, in vitro experiments have demonstrated that cichoric acid also has potential antidiabetic 3 ACS Paragon Plus Environment


Page 4 of 40

68

effects being able to prevent insulin resistance and to increase insulin release and glucose uptake,21,22 as

69

well as to enhance immune and anti-inflammatory properties23 and to induce apoptosis in

70

preadipocytes.24

71

Several phytochemical studies of E. purpurea have demonstrated that the content of alkamides

72

varies a lot between cultivars and the different plant parts with the dodeca-2E,4E,8Z,10E/Z-tetraenoic

73

acid isobutylamide isomers (12,13) (Figure 1) being the most abundant in both roots and aerial parts.

74

.5,19,25,26 The content of alkamides is highest in roots with up to 80 % on dry weight base followed by

75

flowerheads but the relative proportions of the tetraene alkamides 12 and 13 appears to be highest in the

76

aerial parts as the roots also contain high levels of C12 diene-diyne alkamides.19,25−27 Cichoric acid on the

77

other hand, shows almost the same contents in roots as in flowers and leaves, leaving only the stems with

78

a lower content.25,26,28,29 The concentration of caffeic acid derivatives (mg g-1 dry weight (DW)) in roots

79

of E. purpurea is highest in the spring but seems to vary very little with the age of the plant;30,31 however,

80

as the yield in plant mass is increasing significantly over time until the roots are around four years, the

81

yield of cichoric acid per plant or per area (kg plant-1 or kg m-2) is expected to be highest in roots with

82

the age of 4 or higher. Echinacea purpurea roots for drug extraction are therefore normally harvested in

83

the autumn of the third or fourth cultivation year.32 Despite this, most scientific investigations on the

84

content of bioactive compounds in roots have been conducted on plants younger than two years,28,33,34

85

probably for practical reasons.

86

Previous investigation on harvest of E. purpurea at different developmental stages have shown that

87

the content of cichoric acid in aerial parts decreases with later harvest from bud stage to wilting stage of

88

the aerial parts,34−36 whereas the content of the dominating alkamides, the two dodeca-2E,4E,8Z,10E/Z-

89

tetraenoic acid isobutylamide isomers (12,13) (Figure 1) in aerial parts tends to increase with harvest of

90

aerial parts in later developmental stages.28,36 Earlier investigations on the content of caffeic acid 4 ACS Paragon Plus Environment

Page 5 of 40


91

derivatives and alkamides in the roots of E. purpurea harvested from summer to autumn have shown that

92

the content of the dominating alkamides 12 and 13 and cichoric acid are decreasing from bloom to seed

93

set and wilting.28,33,34

94

Many of the bioactive compounds in plants are so called secondary metabolites produced by the

95

plants with the purpose of protecting the plant from different biotic and abiotic stresses.37 We therefore

96

hypothesized that harvest of aerial parts will stress the plant and that harvest of aerial parts consequently

97

will have a significant impact on the content of bioactive compounds in the subsequent harvested roots.

98

The purpose of this work was therefore a combination of: 1) To investigate if the developmental stages

99

of aerial parts (flowers) at harvest have an influence on the content of bioactive compounds of aerial

100

parts themselves. 2) To investigate if the content of bioactive compounds in the subsequent harvested

101

roots is affected by the harvest of aerial parts and what would be the most beneficial time to harvest the

102

roots, when the aerial parts have been harvested. We therefore used two seed populations (‘seed

103

companies’) of four-year-old cultivated E. purpurea plants to investigate the most beneficial harvest

104

time, i.e., the highest concentration of bioactive compounds, for both aerial parts and roots, alone and in

105

combination. Two experiments were performed: First, an investigation of the most favorable harvest

106

stage for aerial parts. Secondly, an investigation of whether pre-harvest of aerial parts influences the

107

content of alkamides and caffeic acid derivatives in the subsequent harvested roots. The roots were

108

harvested at fixed time intervals starting from harvest of aerial parts.

109 110

MATERIALS AND METHODS

111

Plant Material. Echinacea purpurea (L.) Moench plants were propagated from seeds purchased

112

from two seed companies Rieger-Hoffmann Gmbh (Blaufelden-Raboldshausen, Germany) and

113

Pharmasaat Gmbh (Artern, Germany), and the seed populations are hereafter referred to as ‘Rieger5 ACS Paragon Plus Environment


Page 6 of 40

114

Hofmann’ and ‘Pharmasaat’ populations, respectively. The plants were raised in a greenhouse and

115

transplants were established in the field (sandy loam soil at Aarslev, Denmark; coordinates: 55.3° N,

116

10.45° E) in spring. The experimental area was 120 m2 with a plant density of 6 plants/m2 and row

117

distance of 50 cm. The single plots for harvest were 0.85 m2 and consisted of five plants. The crop was

118

fertilized with 50 kg N ha-1, 8 kg P ha-1, and 25 kg K ha-1 every spring and irrigated when necessary. The

119

flowers and 20 cm of the top of the plants were cut off every year (July/August) to simulate harvest of

120

aerial parts and wilted crop remains were removed every spring until the fourth cultivation year, where

121

the experiment took place.

122

Harvest of Aerial Parts: The aerial parts of the two seed populations of E. purpurea were

123

harvested at three different developmental stages (bud, full blooming and wilting (seed set)) (Table 1).

124

Each sample of aerial parts consisted of material from five plants in one plot. The top 20 cm of plants

125

were harvested with stems, leaves and flowers and the material from five plants where pooled together

126

to one sample.

127

Harvest of Roots: There were three subsequent harvest times of root, for every harvest stage of

128

aerial parts: one week after harvest of aerial parts, one month after, and three months after, respectively

129

(Table 1). To compare the content of alkamides and caffeic acid derivatives in roots with a control where

130

aerial parts/flowers were not harvested, two groups of roots (only Pharmasaat) without prior harvest of

131

aerial parts were harvested. The first harvest in this control group was one week after the flowers were

132

in bloom, which coincide with the same time as roots harvested one week after aerial parts were harvested

133

in bloom. The second harvest in the control group was three months after bloom, which coincide with

134

the same time as the roots harvested three months after aerial parts harvested in bloom (see arrows in

135

Figure 3). All samples for root material were dug up by hand (approximately 25 × 25 × 25 cm soil blocks),

136

collected, washed free of soil in a big strainer (mesh 2 × 2 mm), cut into pieces (< 2 cm) and consisted 6 ACS Paragon Plus Environment

Page 7 of 40


137

of root material from five plants. The samples called ‘roots’ consisted of both the fibrous roots and the

138

rhizome part. The harvest of ‘roots’ where done to mimic commercial production. Aerial part and root

139

samples were frozen instantly after harvest at −24 °C.

140

The experimental designs of the field trial was in randomized blocks with treatments: two seed

141

populations, three harvest dates of aerial parts, three harvest dates of roots and six replicates (2 × 3 × 3

142

× 6 = 108 plots) plus the two root harvests in the control group (without aerial part harvest) with only

143

one seed population (Pharmasaat) and six replicates (2 × 1 × 6 = 12 plots).

144

Solvents and Chemicals. Acetonitrile (MeCN), ethanol (EtOH, 96%) and methanol (MeOH),

145

(High-Performance Liquid Chromatography (HPLC) grade for chromatography) were obtained from

146

Fisher Scientific (Roskilde, Denmark). Triflouroacetic acid (TFA) of reagent quality was obtained from

147

Prolabo (Leuven, Belgium). Caftaric acid (> purity 97%), cichoric acid (> purity 95%) and echinacoside

148

(purity > 98%) were obtained from Sigma-Aldrich (Steinheim, Germany) and dodeca-2E,4E,8Z,10E/Z-

149

tetraenoic acid isobutylamides (12,13) (> purity 95%) and chlorogenic acid (> purity 98%) were obtained

150

from Phytolab GmbH & Co. KG (Vestenbergsgreuth, Germany). Milli-Q water was purified locally on

151

own equipment (SG, Ultra Clear Basis, Germany).

152

Extraction and Analysis. Samples of both roots and aerial parts were freeze dried and ground to

153

obtain a particle size of < 500 µm (European Freeze Dry, Kirke Hyllinge, Denmark), then packed airtight

154

(aluminum foil bags) and stored frozen (–20 °C) prior to HPLC-analysis. Extraction, analysis and

155

identification of alkamides and caffeic acid derivatives were performed according to methods previously

156

described.38 In short, 1.0 g of ground plant material was extracted with EtOH/water (70/30), stirred for 2

157

h and filtered. Each sample was analyzed on a Dionex, Ultimate 3000 HPLC system. Separations were

158

performed using a Purospher Star RP-18 column, 5 µm, (250 × 4.6 mm) with a matching pre-column.

159

Alkamides were analyzed by a validated HPLC-photodiode array (PDA) method using a solvent system 7 ACS Paragon Plus Environment


Page 8 of 40

160

of MeOH and water and UV-detection at 210 and 254 nm, while caffeic acid derivatives were analyzed

161

by a validated HPLC-PDA method using a solvent system consisting of MeCN and water containing

162

0.01% TFA and UV-detection at 330 nm. Detailed information on the separation and quantification of

163

alkamides and caffeic acid in extracts by HPLC is described in Thomsen et al.38

164

Identification. Alkamides and phenolic acids in extracts of roots and aerial parts of E. purpurea

165

were identified by liquid chromatography-diode array detection-atmospheric pressure chemical

166

ionization-tandem mass spectrometry (LC-DAD-APCI-MS/MS) resulting in the identification of 15

167

alkamides and 4 caffeic acid derivatives (Figure 1). For details about the LC-MS data (retention time and

168

mass spectra) and the identification of the compounds please see Thomsen et al.38 LC-MS analysis was

169

performed using a LTQ XL (ESI-2D-iontrap, Thermo Scientific) operated in APCI positive mode and

170

hyphenated with an Accela HPLC Pump and a DAD operating from 200 to 600 nm. Settings for the mass

171

spectrometer were 50, 5, and 5 (arbitrary units) for sheath, auxillary, and sweep gas flow rates,

172

respectively, vaporizer temperature 450 °C, discharge current 5 μA, capillary temperature 275 °C,

173

capillary voltage 16 V, tube lens 35 V, and AGC target settings 3 × 104 and 1 × 104 for full MS and

174

MS/MS, respectively. Separations were obtained on a LiChrospher RP18 (5 μm; 250 × 4.6 mm, 100 Å,

175

Phenomenex, Allerød, Denmark). The mobile phase consisted of 0.1% formic acid in water (A) and

176

0.1% formic acid in MeOH (B), and separations were performed using the following linear-programmed

177

solvent gradient: 0 (50% B), 35 (80% B), 45 (80% B), 50 (50% B), and 60 min (50% B). The flow was

178

1 mL min-1, the temperature was 35 °C, and the injection volume was 10 μL.

179

Statistics. Analysis of variance (ANOVA) was performed on each variable using the Statistical

180

Analysis System (SAS Institute, Inc., Cary, NC). The variations [standard errors (SE)], the significances

181

of treatment effects, and interactions (F tests) were calculated and tested using the ANOVA procedure


Page 9 of 40


182

in SAS. If the F tests showed significant treatment effects, least significant difference (LSD) values (p =

183

0.05) were used to separate means of treatments effect in Tables 2 and 3.

184

RESULTS

185

Harvest of Echinacea Plant Material. The air (in 2 m height) and soil (in 10 cm depth)

186

temperature during the five months of experiment are shown in Figure 3. Each measurement point is an

187

average for one day (logged at hourly intervals) from July 1 to December 15. In Figure 3 the harvest time

188

of the three stages of development of aerial parts are also indicated. The aerial parts were harvested with

189

three weeks difference, representing (i) the flowers bud stage, where at least 80 percent of the plants were

190

in bud stage, (ii) the flowers blooming stage, where at least 80 percent were in full bloom, and finally

191

(iii) the wilting stage, where the flowers had started to wilt and develop seeds (Table 1). The roots were

192

harvested one week, one month and three months after each harvest of aerial parts in the three different

193

stages (Figure 3). The first harvest of roots was in July when the soil and air temperature was high and

194

the flowers were about to bloom, while the last harvest was in late November, when the temperature was

195

low, but still above freezing point and the aerial parts were wilted. The exact dates for the nine different

196

harvests of roots can be seen in Table 1. The climate data in Figure 3 for summer and autumn in the

197

harvest year are in reasonable agreement with the ‘normal’ climate (30-year average) at the experimental

198

site. The yield of biomass of both aerial parts and roots is shown in Table S1 (Supporting Information).

199

The biomass yield of aerial parts increased over three fold from early ‘bud’ harvest to late ‘wilting’ stage

200

harvest. The biomass yield of root material on the other hand was very stable in all harvests and only in

201

the late harvests after ‘wilting’, a little difference was observed, with higher yield in the roots harvested

202

one month after harvest of the aerial parts (Table S1).

203

Harvest of Aerial parts. Looking at the concentration of bioactive compounds (alkamides and

204

caffeic acid derivatives) in aerial parts (Figure 4), there were only minor differences between the two 9 ACS Paragon Plus Environment


Page 10 of 40

205

seed populations ‘Pharmasaat’ and ‘Rieger-Hofmann’. Significant differences between the seed

206

populations were only in the content of chlorogenic acid, cichoric acid and total caffeic acid derivatives,

207

where ‘Rieger-Hofmann’ showed a slightly higher concentration. The concentration pattern of caffeic

208

acid derivatives was the same for both seed populations at the different harvest stages (Figure 4). For

209

none of the bioactive compounds the F-tests showed statistical significant interactions between effect of

210

‘Seed population’ and effect of ‘Harvest stage of aerial parts’. The content of the alkamides 12 and 13,

211

together with alkamide 1 and 6 and total alkamides in the aerial parts showed a significant (p < 0.001)

212

increase with harvest from stage 1 to stage 3 in both seed populations, while alkamide 5 showed a

213

significant (p < 0.01) decrease with later harvest (Figure 4). The caffeic acid derivatives caftaric acid,

214

echinacoside, cichoric acid and total caffeic acid derivatives showed a significant (p < 0.001) decrease

215

in content in the period from bud stage to wilting stage (Figure 4). The content of chlorogenic acid was

216

the only caffeic acid derivative that was not affected by the different harvest stages of aerial parts.

217

The yields of the bioactive compounds at the different harvests were calculated as kg ha-1 and there

218

were only minor differences between the two seed populations ‘Pharmasaat’ and ‘Rieger-Hofmann’

219

(Figure 5). The significant differences between the seed populations were in the yield of chlorogenic acid

220

and cichoric acid where ‘Rieger-Hofmann’ showed a higher yield, but otherwise the same pattern as

221

‘Pharmasaat’ with regard to the yield of caffeic acid derivatives at the different harvest stages (Figure 5).

222

In the aerial part material harvested in bud, bloom and wilting stage the yield of nearly all the harvested

223

bioactive compounds (except alkamide 5 and echinacoside) showed a significant (p < 0.001) increase

224

from bud to wilting stage. This result is a combination of concentration of the compounds and harvest of

225

a higher biomass (Figure 5).

226

Harvest of Roots. The average root weight measured at the nine harvest dates of roots in the

227

experiment (Table 1) were not significantly different regarding effects of ‘Seed population’, ‘Harvest 10 ACS Paragon Plus Environment

Page 11 of 40


228

stage of aerial parts’, ‘Harvest time of roots’ or interactions between them (Table 2). The average root

229

weight (fresh weight) of the four year old E. purpurea plants were 360 g (range 181−772 g) and this

230

would mean an average root yield of 21.2 t ha-1 and a dried root yield of about 5.2 t ha-1. The average

231

concentration of bioactive compounds in all harvested roots of the two seed populations of E. purpurea

232

used in the experiment showed some minor differences regarding the alkamides. There was a significant

233

higher content of alkamides 4, 5, 7, 8 and 11 in ‘Rieger-Hofmann’ but a significantly lower concentration

234

of alkamides 6, 12, 13 and 14 compared to ‘Pharmasaat’. This resulted in an average total concentration

235

of alkamides that was not significantly different in the two seed populations, with 3.62 ± 0.17 mg g-1 DW

236

in ‘Pharmasaat’ and 3.41 ± 0.17 mg g-1 DW in ‘Rieger-Hofmann’. The average concentration of the

237

major alkamides 12 and 13 in roots of the two seed populations was as mentioned above significantly

238

higher (p < 0.05; LSD0.05 = 0.27 mg g-1 DW) in ‘Pharmasaat’ (1.84 ± 0.10 mg g-1 DW) than in ‘Rieger-

239

Hofmann’ (1.53 ± 0.11 mg g-1 DW). The average concentration of caffeic acid derivatives in the roots of

240

the two seed populations did not differ, and the total average of caffeic acid derivatives was 2.53 ± 0.12

241

mg g-1 DW for ‘Pharmasaat’ and 2.76 ± 0.14 mg g-1 DW for ‘Rieger-Hofmann’.

242

Table 2 shows the concentration of the bioactive compounds in roots as an effect of the treatments

243

‘Development stages of aerial parts at harvest’ and the succeeding root harvest time (‘Harvest of roots’).

244

Because of the relatively minor differences between the two seed populations, the data in Table 2 are

245

shown as an average over the two seed populations. The results of the ANOVA F-test for treatment

246

effects and interactions of ‘Seed population’ (n = 2), ‘Harvest stage of aerial parts’ (n = 3), succeeding

247

‘Harvest time of roots’ (n = 3) and replicates (n = 6) on the content of single bioactive compounds are

248

shown in the lower part of Table 2. The statistical ANOVA analysis showed that there was no 3-way or

249

2-way interaction involving ‘Seed population’ (ns in Table 2). There were some minor significant 2-way

250

interactions between ‘Harvest stage of aerial parts’ and ‘Harvest time of roots’ on the content of 11 ACS Paragon Plus Environment


Page 12 of 40

251

alkamides 4, 8 and 11 when the data of the two seed populations were pooled (Table 2). There were,

252

however, some more highly significant 2-way interactions between ‘Harvest stage of aerial parts’ and

253

‘Harvest time of roots’ in the content of caffeic acid derivatives (caftaric acid, cichoric acid and total

254

caffeic acid derivatives) (Table 2). Early harvest of aerial parts (in bud stage) resulted in a declining

255

content in subsequent harvested roots from one week to three months (p < 0.001), whereas harvest in

256

bloom resulted in an unaffected content in harvested roots (p > 0.05). Harvest in the wilting stage resulted

257

first in a decrease (roots harvested 1 month after) followed by an increase (roots harvested 3 month after)

258

in the content of caffeic acid derivatives in the subsequent harvested roots (p < 0.001). Figure 6 illustrates

259

the interaction effect of harvest stage of aerial parts on the subsequent harvested roots for the content of

260

caftaric acid, cichoric acid and total caffeic acid derivatives in roots.

261

Harvest of Roots ‘With’ and ‘Without’ Prior Harvest of the Aerial Parts. The effects of not

262

harvesting the aerial parts prior to harvest of roots compared to harvest of aerial parts was tested in the

263

seed population ‘Pharmasaat’. In roots harvested one week after bloom ‘with’ and ‘without’ prior harvest

264

of the aerial parts, there was a significant difference in the content of both alkamides and caffeic acid

265

derivatives (Table 3). Eight of the fifteen identified alkamides (1−3, 5, 6, 10, 11 and 15), caftaric acid

266

and the total content of caffeic acid derivatives all showed lower concentrations when the aerial parts

267

had been harvested prior to harvest of the roots. When the roots were harvested three months after bloom,

268

nine of the fifteen alkamides (1, 3, 5, 7, 8, 11−13 and 15) had a significant lower concentration when the

269

aerial parts had been harvested, including the dominating alkamides (12,13). This also affected the total

270

concentration of alkamides in the roots, which was 2.108 ± 0.551 mg g-1 DW with harvest of aerial parts

271

prior to harvest of roots and 4.300 ± 0.320 mg g-1 DW without harvest of aerial parts prior to harvest of

272

roots, i.e., a doubling of the concentration. The total concentration of caffeic acid derivatives had, on the

273

other hand, a significant higher concentration in the roots, if the aerial parts had been harvested in bloom 12 ACS Paragon Plus Environment

Page 13 of 40


274

three months before with 3.207 ± 0.186 mg g-1 DW compared to 2.269 ± 0.378 mg g-1 DW without

275

harvest of aerial parts (Table 3).

276

DISCUSSION

277

Harvest of Aerial Parts. In earlier reports of more botanical nature the content of bioactive

278

compounds in E. purpurea are often measured in specific plants parts, such as flowers, stems, leaves or

279

roots. Our approach is more agricultural production minded and therefore we determine the content of

280

bioactive compounds in what would be the result of a mechanical harvest of the top 20 cm of the plant,

281

i.e., the pooled mass of leaves, stems and flowers. In other investigations of aerial parts of E. purpurea,

282

the harvest height is not stated;35,39 thus it is difficult to compare the absolute concentration of bioactive

283

compounds found in this investigation with others. Nevertheless, the total alkamides in aerial parts

284

harvested in bloom (1.22−1.40 mg g-1 DM, Figure 4) is in the same order of magnitude as the total

285

alkamides in aerial parts harvested in an experiment performed in Australia (0.7−1.3 mg g-1 DM).39

286

The dominating caffeic acid derivative in aerial parts of both E. purpurea lines in the present study

287

was cichoric acid, which is in accordance with previous investigations. From Australia and China the

288

concentration of cichoric acid in aerial parts is reported to be 12.5−13.4 mg g-1 DM and 14.1−15.9 mg

289

g-1 DM, respectively, which is somewhat higher than the concentration of cichoric acid of 4.78−5.57 mg

290

g-1 DM found in the present study (Figure 4).35,39 However, it is known that the concentration of cichoric

291

acid is approximately equal in flowers and leaves and only the stems have a lower concentration.28 Hence,

292

it should be possible to compare concentrations found in flowers, with concentrations in aerial parts. In

293

a Canadian investigation,9 on wild populations of Echinacea they report a concentration of cichoric acid

294

in flowers of 4.17−8.89 mg g-1 and this is more comparable with the results obtained in this study.

295

The next most abundant caffeic acid derivative in the aerial parts of both E. purpurea lines in the

296

present investigation was echinacoside. as shown by LC-DAD-APCI-MS/MS analyses using an 13 ACS Paragon Plus Environment


Page 14 of 40

297

authentic echinacoside standard for comparison.38 Echinacoside, is usually not detected or only found in

298

small amounts in E. purpurea5,35,40−42 hence, this is the first study, which shows that echinacoside also

299

can occur in relative large amounts in aerial parts of E. purpurea. Although, this is not in accordance

300

with previous investigations of aerial parts of E. purpurea,40−42 it is not completely surprising to us,

301

because we have previously shown that echinacoside was the next most abundant caffeic acid derivative

302

in the roots of an E. purpurea line from another Echinacea cultivation experiment in the same

303

experimental field.38 On the other hand, echinacoside was only present in minute amounts in the roots of

304

the two E. purpurea lines in the present investigation (Tables 2 and 3). This indicates that genetic

305

diﬀerences between populations as well as cultivation conditions play a major role in the formation of

306

caffeic acid derivatives in E. purpurea.

307

For aerial parts of E. purpurea our study showed that the content of alkamides 1, 6, 12, 13 and the

308

total alkamides increased when harvesting at the development stages from bud to wilting, while the

309

content of caftaric acid, echinacoside, cichoric acid and total caffeic acid derivatives decreased. These

310

results generally agree with previous studies showing that with later harvest the concentration of total

311

alkamides is increasing and the concentration of cichoric acid is decreasing.28,34−36 Although, there is

312

scientific evidence for beneficial health effects of E. purpurea extracts and/or preparations as described

313

in the introduction, it is still not clear, which specific secondary metabolites that are responsible for these

314

health promoting effects. Thus, it is important to investigate the dynamics of all potential bioactive

315

compounds and not only the total alkamides or the dominating caffeic acid derivatives, in particular

316

cichoric acid. This investigation shows for example that the content of alkamide 5 (6% of total alkamides)

317

respond differently compared to the other alkamides in the aerial parts of E. purpurea meaning that its

318

concentration decreases with later harvest, i.e., from the aerial parts bud stage to the wilting stage.


Page 15 of 40


319

If the purpose is to have the highest concentration of the alkamides (except alkamide 5), aerial parts

320

should be harvested in a late developmental stage after flowering. Late harvest will also give the highest

321

amount of alkamides harvested per area because the biomass of aerial parts is increasing three fold from

322

early bud harvest (142 g m-2 DM) to late wilting stage harvest (460 g m-2 DM) (Table S1). If higher

323

concentrations of caffeic acid derivatives or alkamide 5 are wanted, the aerial parts should be harvested

324

in an early developmental stage when flower buds are forming. The fully developed flowers and the

325

bigger leaf mass within the top 20 cm of the aerial parts in the late developmental stages gives the highest

326

yield of biomass. Consequently, the highest yield per area of caffeic acid derivatives is in the late

327

developmental stage (wilting) even though the concentration at this stage is lower. Thus, the highest yield

328

per area of both alkamides and caffeic acid derivatives are achieved when the aerial parts are harvested

329

in the late developmental stage (Figure 5 and Table S1).

330

Harvest of Roots. The content of the dominating alkamides 12 and 13 in roots of E. purpurea

331

grown in Denmark are comparable with the results from several other investigations.25,39,43,44 Even

332

though there is a significant difference between the two seed population with regard to their content of

333

single alkamides, the average concentration of the major alkamides 12 and 13 (1.84 ± 0.10 mg g-1 DW

334

and 1.53 ± 0.11 mg g-1 DW for ‘Pharmasaat’ and ‘Rieger-Hofmann’, respectively) are comparable with

335

other investigations from The United States, New Zealand and Finland, who report contents ranging from

336

1.12 to 2.03 mg g-1 DW.25,39,40,43,44 The average concentration of cichoric acid in roots of the two seed

337

populations grown in Denmark were 2.46 ± 0.12 mg g-1 DW for ‘Pharmasaat’ and 2.67 ± 0.13 mg g-1

338

DW for ‘Rieger-Hofmann’. Cichoric acid was the most abundant caffeic acid derivative in both E.

339

pupurea lines, followed by caftaric acid, chlorogenic acid, and echinacoside (Tables 2 and 3), which is

340

in accordance with other studies.5,19,40−42,43,45 However, the concentration of cichoric acid in the roots of

341

both investigated E. purpurea lines is rather low compared with other groups’ findings, who reported 15 ACS Paragon Plus Environment


Page 16 of 40

342

concentrations ranging from 4.81 up to 30.60 mg g-1 DW.5,8,28,29,39,43,46 We cannot explain why we find

343

such a low content of cichoric acid compared to other studies as we can exclude degradation of cichoric

344

acid in the freeze-dried samples, since we also measured the content of cichoric acid in fresh root material

345

directly after harvest in the field, which gave the same low content as in the freeze-dried samples (data

346

not shown). Furthermore, comparison of the content of caftaric acid, chlorogenic acid and echinacoside

347

in the fresh roots samples with freeze-dried samples clearly indicated that no enzymatic degradation of

348

caffeic acid derivatives occurred during freeze-drying and storage until analysis (data not shown). These

349

observations are in accordance with Brown et al.,47 who did not find any significant degradation of

350

cichoric acid and other caffeic acid derivatives during sample preparation of E. purpurea raw materials

351

(roots and aerial parts) and HPLC analysis. Thus, the considerable variation in the concentration of

352

cichoric acid in E. purpurea indicates that genetic diﬀerences between populations, climate, and

353

cultivation conditions play a major role, and thus may explain the relatively low content of this caffeic

354

acid derivative in this investigation compared to other studies. This is also supported by the fact that the

355

concentration of alkamide 12 and 13, total alkamides, cichoric acid and total caffeic acid derivatives are

356

all in agreement with values from another Echinacea experiment conducted in the same experimental

357

field.38

358

The results of our experiment showed that harvest of aerial parts at different developmental stages

359

results in a significant effect on the content of cichoric acid in the succeeding harvested roots (Figure 6).

360

Moreover, there is a significant interaction (Table 2) between the effects of harvest of aerial parts at

361

different developmental stages and the effect of harvest time of roots one week, one month and three

362

month after harvest of aerial parts. This is true for the content of both cichoric acid, caftaric acid, total

363

caffeic acid derivatives and the alkamides 4, 8 and 11 (Table 2), but not for the dominating alkamides 12

364

and 13. Hence, if the farmer decides to harvest both aerial parts and roots in the same cultivation year, 16 ACS Paragon Plus Environment

Page 17 of 40


365

the content of cichoric acid, caftaric acid, total caffeic acid derivatives, and alkamide 4, 8 and 11 in roots

366

can be affected in different ways. The yield of biomass of roots on the other hand is not affected or nearly

367

not affected by the prior harvest of aerial parts at different developmental stages. Only late root material

368

after harvest of aerial parts in the wilting stage has a little effect on the dry matter yield of root material

369

(Table S1). Root biomass yield harvested one month after (384 g m-2 DM) is a little higher than biomass

370

harvested one week after (307 g m-2 DM) and three month after (331 g m-2 DM).

371

Only very few investigations have looked at the effect of harvest of aerial parts in combination

372

with the content of bioactive compounds in the succeeding harvested roots. The question is, however,

373

important for medicinal plant producers because it allows a double harvest of plant material in one year

374

on the same crop. In a North American study, Callan et al.34 determined that the content of cichoric acid

375

in roots was not affected by harvest of flowers or aerial parts, when they investigated roots harvested two

376

month after harvest of the aboveground parts. However, the present investigation shows that harvest of

377

the aerial parts with subsequent harvest of roots have a significant effect on several alkamides and caffeic

378

acid derivatives, when they are harvested one week or three months after the aerial parts blooming stage.

379

This is important, since the concentration of total alkamides in roots harvested one week and three months

380

after bloom are lower, when the aerial parts have been harvested (Table 3), and more than half of the

381

alkamides present in the roots show significantly lower concentrations. Moreover, the concentration of

382

caffeic acid derivatives are also lower in the roots where the aerial parts have been harvested, if the roots

383

are harvested one week after bloom. Harvest of roots one week after harvest of the aerial parts, which

384

are harvested in bloom, is therefore not recommendable. If the roots are harvested three months after

385

bloom, the content of caftaric acid, cichoric acid and caffeic acid derivatives are all higher in the roots

386

with prior harvested aerial parts. This means that the highest concentration of caffeic acid derivatives is

387

achieved by harvest of roots one week after bloom without prior harvest of aerial parts, while the highest 17 ACS Paragon Plus Environment


Page 18 of 40

388

concentration of alkamides is achieved by harvest of roots three months after bloom, without prior

389

harvest of aerial parts.

390

In conclusion, the highest concentration of alkamides (except alkamide 5) in aerial parts is achieved

391

when the aerial parts are harvested in the wilting stage, while the highest concentration of caffeic acid

392

derivatives is achieved when the aerial parts are harvested in the bud stage. However, the highest yield

393

per area of both alkamides and caffeic acid derivatives is achieved when the aerial parts are harvested

394

late (wilting stage). It is not entirely recommendable to harvest the aerial parts the same year the roots

395

are meant to be harvested, since the content of bioactive compounds in the subsequent harvested roots is

396

lower when the aerial parts are harvested. However, if the aerial parts are to be harvested, it is

397

recommendable to harvest the roots one week after, as this will result in the most optimal concentration

398

of bioactive compounds.

399 400

AUTHOR INFORMATION

401

Corresponding Authors

402

*(L.P.C.) Phone: +45 2778 7494. E-mail: [email protected]

403

*(K.G.) Phone: +45 8715 8342. E-mail: [email protected]

404 405

ORCID

406

Kai Grevsen: https://orcid.org/0000-0002-6102-8723

407

Lars Porskjær Christensen: https://orcid.org/0000-0002-5035-9201

408 409

Funding


Page 19 of 40


410

We greatly acknowledge the financial support from The Danish Council for Strategic Research (Project

411

“Health promoting effects of bioactive compounds in plants” 2101-07-006).

412 413 414

Notes

415

The authors declare no competing financial interest.

416 417

Supporting Information

418

Supporting Information Available: Table S1. The influences of harvest stage of aerial parts on the yield

419

of biomass and dry matter of aerial parts and roots harvested one week, one month and three month after

420

harvest of aerial parts. This material is available free of charge via the Internet at http://pubs.acs.org.

421 422

REFERENCES

423

(1)

Barrett, B. Medicinal properties of Echinacea: A critical review. Phytomedicine 2003, 10, 66−86.

424

(2)

Bauer,

R.

Chemistry,

analysis

and

immunological

investigations

of

Echinacea

425

phytopharmaceuticals. In: Immunomodulatory Agents from Plants. Wagner, H.; Birkhäuser: Basel,

426

Switzerland, 1999; 8, 41−88.

427

(3)

Goel, V.; Chang, C.; Slama, J. V.; Barton, R.; Bauer, R.; Gahler, R.; Basu, T. K. Alkylamides of

428

Echinacea

429

Immunopharmacol. 2002, 2, 381−387.

430

(4)

purpurea

stimulate

alveolar

macrophage

function

in

normal

rats.

Int.

Goel ,V.; Lovlin, R.; Barton, R.; Lyon, M. R.; Bauer, R.; Lee, T. D.; Basu, T. K. Efficacy of a

431

standardized Echinacea preparation (EchinilinTM) for the treatment of the common cold: a

432

randomized, double-blind, placebo-controlled trial. J. Clin. Pharm. Ther. 2004, 29, 75−83. 19 ACS Paragon Plus Environment


433

(5)

Page 20 of 40

Binns, S. E.; Livesey, J. F.; Arnason, J. T.; Baum, B. R. Phytochemical variation in Echinacea

434

from roots and flowerheads of wild and cultivated populations. J. Agric. Food Chem. 2002, 50,

435

3673−3687.

436

(6)

437 438

Manayi, A.; Vazirian, M.; Saeidnia, S. Echinacea purpurea: Pharmacology, phytochemistry and analysis methods. Pharmacogn. Rev. 2015, 9, 63−72.

(7)

LaLone, C. A.; Hammar, K. D. P.; Wu, L.; Bae, J.; Leyva, N.; Liu, Y.; Solco, A. K.; Kraus, G. A.;

439

Murphy, P. A.; Wurtele, E. S.; Kim, O. K.; Seo, K. L.; Widrlechner, M. P.; Birt, D. F. Echinacea

440

species and alkamides inhibit prostaglandin E2 production in RAW264.7 mouse macrophage cells.

441

J. Agric. Food Chem. 2007, 55, 7314−7322.

442

(8)

Hou, C. C.; Chen, C. H.; Yang, N. S.; Chen, Y. P.; Lo, C. P. Wang, S. Y.; Tien, Y. J.; Tsai, P. W.;

443

Shyur, L. F. Comparative metabolomics approach coupled with cell- and gene-based assays for

444

species classification and anti-inflammatory bioactivity validation of Echinacea plants. J. Nutr.

445

Biochem. 2010, 21, 1045−1059.

446

(9)

Gulledge, T. V.; Collette, N. M.; Mackey, E.; Johnstone, S. E.; Moazami, Y.; Todd, D. A.; Moeser,

447

A. J.; Pierce, J. G.; Cech, N. B.; Laster, S. M. Mast cell degranulation and calcium influx are

448

inhibited by an Echinacea purpurea extract and the alkylamide dodeca-2E,4E-dienoic acid

449

isobutylamide. J. Ethnopharmacol. 2018, 212, 166−174.

450

(10) Oláh, A.; Szabó-Papp, J.; Soeberdt, M.; Knie, U.; Dähnhardt-Pfeiffer, S.; Abels, C.; Bíró, T.

451

Echinacea purpurea-derived alkylamides exhibit potent anti-inflammatory effects and alleviate

452

clinical symptoms of atopic eczema. J. Dermatol. Sci. 2017, 88, 67−77.

453 454

(11) Binns, S. E.; Purgina, B.; Bergeron, C.; Smith, M. L.; Ball, L.; Baum, B. R.; Arnason, J. T. Lightmediated antifungal activity of Echinacea extracts. Planta Med. 2009, 66, 241−244.


Page 21 of 40


455

(12) Christensen, K. B.; Petersen, R. K.; Petersen, S.; Kristiansen, K.; Christensen, L. P. Activation of

456

PPARγ by metabolites from the flowers of purple coneflower (Echinacea purpurea). J. Nat. Prod.

457

2009, 72, 933–937.

458

(13) Kotowska, D.; El-Houri, R. B.; Borkowski, K.; Petersen, R. K.; Fretté, X. C.; Wolber, G.; Grevsen,

459

K.; Christensen, K. B.; Christensen, L. P.; Kristiansen, K. Isomeric C12-alkamides from the roots

460

of Echinacea purpurea improve basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes.

461

Planta Med. 2014, 80, 17121720.

462

(14) Choi, K. M.; Kim, W.; Hong, J. T.; Yoo, H. S. Dodeca-2(E),4(E)-dienoic acid isobutylamide

463

enhances glucose uptake in 3T3-L1 cells via activation of Akt signaling. Mol. Cell Biochem. 2017,

464

426, 9−15.

465

(15) Woelkart, K.; Koidl, C.; Grisold, A.; Gangemi, J. D.; Turner, R. B.; Marth, E.; Bauer, R.

466

Bioavailability and pharmacokinetics of alkamides from the roots of Echinacea angustifolia in

467

humans. J. Clin. Pharmacol. 2005, 45, 683689.

468 469 470 471

(16) Woelkart, K.; Bauer, R. The role of alkamides as an active principle of Echinacea. Planta Med. 2007, 73, 615−623. (17) Ardjomand-Woelkart, K.; Bauer, R. Review and assessment of medicinal safety data of orally used Echinacea preparations. Planta Med. 2016, 82, 17−31.

472

(18) Chicca, A.; Pellati, F.; Adinolfi, B.; Matthias, A.; Massarelli, I.; Benvenuti, S.; Martinotti, E.;

473

Bianucci, A. M.; Bone, K.; Lehmann, R.; Nieri, P. Cytotoxic activity of polyacetylenes and

474

polyenes isolated from the roots of Echinacea pallida. Br. J. Pharmacol. 2008, 153, 879885.

475 476

(19) Pellati, F.; Benvenuti, S.; Magro, L.; Melagari, M.; Soragni, F. Analysis of phenolic compounds and radical scavenging activity of Echinacea spp. J. Pharm. Biomed. Anal. 2004, 35, 289301.



477 478

Page 22 of 40

(20) Vimalathan, S.; Kang, L.; Amiguet, V. T.; Livesey, J.; Arnason, T.; Hudson, J. Echinacea purpurea aerial parts contains multiple antiviral compounds. Pharm. Biol. 2005, 43, 740−745.

479

(21) Tousch, D.; Lajoix, A. D.; Hosy, E.; Azay-Milhau, J.; Ferrare, K.; Jahannault, C.; Cros, G.; Petit,

480

P. Chicoric acid, a new compound able to enhance insulin release and glucose uptake. Biochem.

481

Biophys. Res. Commun. 2008, 377, 131−135.

482

(22) Zhu, D.; Wang, Y.; Du, Q.; Liu, Z.; Liu, X. Cichoric acid reverses insulin resistance and suppresses

483

inflammatory responses in the glucosamine-induced HepG2 cells. J. Agric. Food Chem. 2015, 63,

484

10903−10913.

485 486

(23) Matthias, A.; Banbury, L.; Bone, K. M.; Leach, D. N.; Lehmann, R. P. Echinacea alkylamides modulate induced immune responses in T-cells. Fitoterapia 2008, 79, 53−58.

487

(24) Xiao, H.; Wang, J.; Yuan, L.; Xiao, C.; Wang, Y.; Liu X. Chicoric acid induces apoptosis in 3T3-

488

L1 preadipocytes through ROS-mediated PI3K/Akt and MAPK signaling pathways. J. Agric. Food

489

Chem. 2013, 61, 1509−1520.

490 491 492 493 494

(25) Qu, L.; Chen, Y.; Wang, X.; Scalzo, R. Patterns of variation in alkamides and cichoric acid in roots and aboveground parts of Echinacea purpurea (L.) Moench. HortScience 2005, 40, 1239−1242. (26) Chen, C. L.; Zhang, S. C.; Sung, J. M. Caffeoyl phenols and alkamides of cultivated Echinacea purpurea and Echinacea atrorubens var. paradoxa. Pharm. Biol. 2009, 47, 835−840. (27) Perry, N. B.; van Klink, J.W.; Burgess, E. J.; Parmenter, G.A.Alkamide

levels

in

Echinacea

495

purpurea: a rapid analytical method revealing differences among roots, rhizomes, stems, leaves

496

and flowers. Planta Med. 1997, 63, 58−62.

497 498

(28) Stuart, D. L.; Wills, R. B. H. Alkylamide and cichoric acid levels in Echinacea purpurea tissues during plant growth. J. Agric. Food Chem. 2000, 7, 91−101.


Page 23 of 40


499

(29) Mølgaard, P.; Johnsen, S.; Christensen, P.; Cornett, C. HPLC method validated for the

500

simultaneous analysis of cichoric acid and alkamides in Echinacea purpurea plants and product. J.

501

Agric. Food Chem. 2003, 51, 6922−6933.

502

(30) Seemannová, Z.; Mistríková, I.; Vaverková, Š. Effects of growing methods and plant age on the

503

yield, and on the content of flavonoids and phenolic acids in Echinacea purpurea (L.) Moench.

504

Plant Soil Environ. 2006, 52, 449−453.

505

(31) Hajimehdipoor, H.; Shekarchi, M.; Khanavi, M.; Roostaie, A.The effects of plant age and

506

harvesting time on chicoric and caftaric acids content of E. purpurea (L.) Moench. Iranian J.

507

Pharm. Sci. 2012, 8, 203−208.

508

(32) Marquard, R.; Kroth, E. Purpur-Sonnenhut (Echinacea purpurea (L.) Moench), schmalblättriger

509

sonnenhut (E. angustifolia DC) und blasser sonnenhut (E. pallida Nutt.). In Anbau und

510

qualitätsanforderungen ausgewählter arzneipflanzen, Buchedition Agrimedia GmbH: Bergen,

511

Germany, 2001; pp. 261−274.

512

(33) El-Gengaihi, S. E.; Shalaby, A. S.; Agina, E. A.; Hendawy, S. F. Alkylamides of Echinacea

513

purpurea L. as influenced by plant ontogony and fertilization. J. Herbs Spices Med. Plants 1998,

514

5, 35−41.

515

(34) Callan, N. W.; Yokelson, T.; Wall-MacLane, S.; Westcott, M. P.; Miller, J. B.; Ponder, G. Seasonal

516

trends and plant density effects on cichoric acid in Echinacea purpurea (L.) Moench. J. Herbs,

517

Spices Med. Plants 2005, 11, 35–46.

518 519 520 521

(35) Liu, Y.; Zeng, J.; Chen, B.; Yao, S. Investigation of phenolic constituents in Echinacea purpurea grown in China. Planta Med. 2007, 73, 1600−1605. (36) Mistríková, I.; Vaverková, S. Morphology and anatomy of Echinacea purpurea, E. angustifolia, E. pallida and Parthenium integrifolium. Biologia 2007, 62, 2−5. 23 ACS Paragon Plus Environment


522 523

Page 24 of 40

(37) Zhao, J.; Davis, L. C.; Verpoorte, R. Elicitor signal transduction to production of plant secondary metabolites. Biotechnol. Adv. 2005, 23, 283−333.

524

(38) Thomsen, M. O.; Frette, X. C.; Christensen, K. B.; Christensen, L. P.; Grevsen, K. Seasonal

525

variations in the concentration of lipophilic compounds and phenolic acids in the roots of

526

Echinacea purpurea and Echinacea pallida. J. Agric. Food Chem. 2012, 60, 12131−12141.

527

(39) Wills, R. B. H.; Stuart, D. L. Alkylamide and cichoric acid levels in Echinacea purpurea grown in

528

Australia. Food Chem. 1999, 67, 385−388.

529

(40) Perry, N.B.; Wills, R. B. H.; Stuart, D. L. Factors affecting Echinacea quality: agronomy and

530

processing. In: Miller, S.C., Ed. Echinacea. The genus Echinacea. Boca Raton, Florida, CRC Press,

531

2004; pp. 111−126.

532

(41) Perry, N. B.; Burgess, E. J.; Glennie, V. L. Echinacea standardization: analytical methods for

533

phenolic compounds and typical levels in medicinal species. J. Agric. Food Chem. 2001, 49,

534

1702−1706.

535

(42) Brown, P. N.; Chan, M.; Betz, J. M. Optimization and single-laboratory validation study of a high-

536

performance liquid chromatography (HPLC) method for the determination of phenolic Echinacea

537

constituents. Anal. Bioanal. Chem. 2010, 397, 1883−1892.

538

(43) Laasonen, M.; Wennberg, T.; Harmia-Pulkkinen, T.; Vuorala, H. Simultaneous analysis of

539

alkamides and caffeic acid derivatives for the identification of Echinacea purpurea, Echinacea

540

angustifolia, Echinacea pallida and Parthenium integrifolium roots. Planta Med. 2002, 68,

541

572−574.

542

(44) Gray, D. E.; Pallardy, S. G.; Garrett, H. E.; Rottinghaus, G. E. Acute drought stress and plant age

543

effects on alkamides and phenolic acid content in purple coneflower roots. Planta Med. 2003, 69,

544

50−55. 24 ACS Paragon Plus Environment

Page 25 of 40


545

(45) Lin S.; Sung J.; Chen, C. Effect of drying and storage conditions on caffeic acid derivatives and

546

total phenolics of Echinacea purpurea grown in Taiwan. Food Chem. 2011, 125, 226−231.

547

(46) Stuart, D.; Wills, R. Effect of drying temperature on alkamide and cichoric acid concentrations of

548

Echinacea purpurea. J. Agric. Food Chem. 2003, 51, 1608−1610.

549

(47) Brown, P. N.; Chan, M.; Paley, L.; Betz, J. M. Determination of major phenolic compounds in

550

Echinacea spp. raw materials and finished products by high-performance liquid chromatography

551

with ultraviolet detection: single-laboratory validation matrix extension. J. AOAC Int. 2011, 94,

552

1400−1410.



553

Page 26 of 40

Figure legends

554 555

Figure 1. Chemical structures of 15 alkamides in Echinacea purpurea identified and quantified in this

556

study according to Thomsen et al.38 Alkamides 12 and 13 are the most dominating alkamides in E.

557

purpurea. These two alkamides are isomers and difficult to separate on HPLC-UV and are therefore

558

treated as a mixture. The ratio between these two isomers in E. purpurea is approximately 1:1.

559 560

Figure 2. Chemical structures of caffeic acid derivatives found in Echinacea purpurea. Cichoric acid is

561

the dominating caffeic acid derivative.

562 563

Figure 3. Temperatures in °C from July to December in the harvest year (Aarslev, Denmark, coordinates:

564

55.3° N, 10.5° E). Temperatures are measured as a daily average (logged at hourly intervals) by Danish

565

Meteorological institute (DMI). Root sampling dates are indicated on the graph and were one week, one

566

month and three months after harvest of aerial parts in the three developmental stages: bud ( ), bloom

567

() and wilting (). Arrows ↓ marks sampling days of roots without prior harvest of aerial parts.

568 569

Figure 4. The concentrations of compounds in aerial parts of E. purpurea harvested in bud, bloom, and

570

wilting stage (Table 1). The concentration of major alkamides 1, 5, 6, 12, 13 and total alkamides (TA)

571

(above) and caftaric acid (CA), chlorogenic acid (CH), cichoric acid (CI), echinacoside (EC) and the

572

total caffeic acid derivatives (TC) (below). Results for the seed population ‘Pharmasaat’ (left) and for

573

‘Rieger-Hofmann’ (right). Bars are SE (n = 6).

574


Page 27 of 40


575

Figure 5. The resulting yields per area of bioactive compounds in aerial parts of E. purpurea harvested

576

in bud, bloom, and wilting stage (Table 1). The yields of major alkamide 1, 5, 6, 12 ,13, and total

577

alkamides (TA) (above) and caftaric acid (CA), chlorogenic acid (CH), cichoric acid (CI), echinacoside

578

(EC), and the total caffeic acid derivatives (TC) (below). Results for the seed population ‘Pharmasaat’

579

(left) and for ‘Rieger-Hofmann’ (right). Bars are SE (n = 6).

580 581

Figure 6. The content of caftaric acid, cichoric acid and total caffeic acid derivatives in roots (mg g-1

582

DW) harvested one week, one month and three months after the aerial parts were harvested in the three

583

developmental stages: bud, bloom, and wilting (Table 1). The results are given as average of the two

584

seed populations ‘Pharmasaat’ and ‘Rieger-Hofmann’. Bars are SE (n = 12).



Page 28 of 40

Table 1. Experimental design of harvest of aerial parts and subsequent harvest of roots in two Echinacea purpurea seed populations: ‘Pharmasaat’ and ‘Rieger Hofmann’

Developmental stage aerial part

Bud

bloom

Wilting

Harvest date aerial parts

July 12rd

August 2rd

August 25th

Description

Harvest of root after harvest of aerial parts

Harvest date roots

1 week

July 19th

1 month

August 9th

3 months

October 14th

1 week

August 11th

1 month

August 31th

3 months

November 4th

1 week

August 30th

1 month

September 22rd

3 months

November 24th

At least 80 % of the plants were in bud

At least 80 % in full bloom

The flowers had started to wilt


Page 29 of 40


Table 2. Content of bioactive compounds in roots. The influences of harvest stage of aerial parts on the content of alkamides and caffeic acid derivatives in roots harvested one week, one month and three month after harvest of aerial parts. Values are average concentration of the two seed populations ‘Pharmasaat’ and ‘Rieger-Hofmann (mg g-1 DW) followed by SE in brackets (n = 12). The results of the statistical ANOVA F-test of treatment effects and interactions are shown below. Devlp. Stagea

Harvest of roots

1

2

3

4

5

6

7

8

10

11

1 week

0.194

0.502

0.071

0.367

0.124

0.079

0.058

0.235

0.021

(0.056)

(0.057)

(0.009)

(0.031)

(0.011)

(0.010)

(0.008)

(0.016)

(0.002)

1 month

0.166

0.466

0.057

0.347

0.147

0.077

0.054

0.210

0.021

(0.028)

(0.054)

(0.007)

(0.041)

(0.020)

(0.009)

(0.007)

(0.023)

(0.003)

3 months

0.199

0.536

0.068

0.421

0.177

0.067

0.053

0.267

0.025

(0.056)

(0.040)

(0.007)

(0.040)

(0.026)

(0.006)

(0.007)

(0.024)

(0.004)

1 week

0.151

0.420

0.061

0.289

0.116

0.068

0.043

0.185

0.020

(0.017)

(0.057)

(0.007)

(0.031)

(0.015)

(0.010)

(0.005)

(0.021)

(0.003)

1 month

0.241

0.524

0.078

0.442

0.167

0.081

0.066

0.261

0.023

(0.019)

(0.048)

(0.006)

(0.041)

(0.012)

(0.014)

(0.008)

(0.015)

(0.002)

3 months

0.228

0.550

0.088

0.407

0.151

0.076

0.062

0.264

0.024

(0.024)

(0.045)

(0.010)

(0.029)

(0.013)

(0.070)

(0.006)

(0.017)

(0.002)

1 week

0.185

0.474

0.069

0.341

0.137

0.072

0.048

0.218

0.022

(0.016)

(0.030)

(0.005)

(0.017)

(0.014)

(0.009)

(0.004)

(0.013)

(0.003)

1 month

0.174

0.488

0.062

0.374

0.146

0.070

0.049

0.231

0.021

(0.012)

(0.044)

(0.006)

(0.025)

(0.014)

(0.010)

(0.004)

(0.020)

(0.003)

3 months

0.191

0.512

0.072

0.360

0.121

0.070

0.051

0.226

0.019

(0.015)

(0.056)

(0.006)

(0.029)

(0.014)

(0.005)

(0.004)

(0.020)

(0.002)

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

Seed pop. x harvest of roots

ns

ns

ns

ns

ns

ns

Harvest of aerial x harvest of roots

ns

ns

ns

*

ns

Harvest of aerialh

ns

ns

ns

ns

Harvest of rootsi

ns

ns

ns

Seed pop.j

ns

ns

ns

Bud

Bloom

Wilting

12, 13

14

15

0.039

1.75

0.119

(0.004)

(0.26)

(0.017)

0.032

1.35

0.105

(0.004)

(0.19)

(0.015)

0.037

1.57

0.115

(0.004)

(0.18)

(0.016)

0.030

1.51

0.109

(0.004)

(0.23)

(0.014)

0.040

2.02

0.152

(0.003)

(0.27)

(0.019)

0.043

2.12

0.153

(0.003)

(0.27)

(0.018)

0.034

1.57

0.118

(0.003)

(0.17)

(0.013)

0.035

1.65

0.113

(0.003)

(0.23)

(0.015)

0.033

1.62

0.113

(0.003)

(0.15)

(0.011)

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

ns

*

ns

ns

ns

*

*

ns

***

**

**

ATb

CAc

CHd

CIe

ECf

0.038

3.33

(0.004)

(0.26)

nq

0.041

3.60

0.087

(0.004)

(0.42)

(0.010)

0.037

3.03

0.082

0.036

3.04

(0.005)

(0.38)

(0.008)

(0.005)

(0.17)

0.042

3.57

0.070

0.050

2.15

(0.006)

(0.33)

(0.009)

(0.004)

(0.17)

0.037

3.03

0.057

0.038

2.19

(0.004)

(0.39)

(0.008)

(0.004)

(0.24)

0.054

4.10

0.075

0.035

2.78

(0.009)

(0.43)

(0.008)

(0.003)

(0.24)

0.049

4.22

0.081

0.047

2.33

(0.005)

(0.35)

(0.011)

(0.004)

(0.29)

0.041

3.31

0.094

0.035

3.18

(0.004)

(0.25)

(0.010)

(0.003)

(0.29)

0.039

3.45

0.056

0.043

1.81

(0.004)

(0.34)

(0.006)

(0.004)

(0.18)

0.037

3.33

0.107

0.054

2.27

(0.003)

(0.27)

(0.017)

(0.006)

(0.20)

ns

ns

ns

ns

ns

ns

-

ns

ns

ns

ns

ns

ns

ns

ns

-

ns

ns

ns

ns

ns

ns

ns

ns

ns

-

ns

ns

*

ns

ns

ns

ns

***

ns

***

-

***

ns

ns

ns

ns

ns

ns

ns

ns

ns

**

-

ns

ns

*

ns

ns

ns

ns

ns

ns

ns

***

***

-

***

*

***

ns

***

*

*

ns

ns

ns

ns

ns

-

ns

nq nq nq nq nq nq nq nq

PTg 3.43 (0.28)

2.80 (0.18)

2.22 (0.20)

2.31 (0.26)

2.92 (0.34)

2.47 (0.29)

3.32 (0.29)

1.93 (0.18)

2.42 (0.21)

Treatment effects Seed pop. x harvest of aerial x harvest of roots Seed pop. x harvest of aerial

ANOVA F-test

aDevelopmental

stages of aerial parts at harvest. bTotal content of alkamides. cCaftaric acid. dChlorogenic acid. eCichoric acid. fEchinacoside content could not be quantified (nq) due to lack of baseline separation. gTotal content of caffeic acid derivatives. hSignificance of harvest of aerial parts at three developmental stages on the content in roots. iSignificance of difference between roots harvested one week, one month and three month after harvest of aerial parts. jSignificance of differences between the two seed populations. ns not significant , *, **, *** , p < 0.05, p < 0.01 and p < 0.001, respectively.



Page 30 of 40

Table 3. The influence of harvest of aerial parts on the concentration of alkamides and caffeic acid derivatives in roots of Echinacea purpurea (‘Pharmasaat’). The values are concentration (mg g-1 DW) followed by SE (n = 6) in brackets. If the statistical ANOVA F-test showed treatment effects, least significant difference (LSD) values (p = 0.05) are listed. Roots harvested one week after bloom with harvest of aerial parts

without harvest of aerial parts

1

0.139

0.398

(0.023)

(0.085)

2

0.412

0.775

(0.086)

(0.147)

3

0.053

0.131

(0.008)

(0.024)

4

0.270

0.389

(0.052)

(0.057)

5

0.098

0.173

(0.018)

(0.026)

6

0.072

0.116

(0.017)

(0.013)

7

0.041 (0.008)

0.072

8

0.161

0.241

(0.031)

(0.034)

10

0.017

0.041

(0.003)

(0.009)

11

0.025

0.050

(0.005)

(0.009)

12,13

1.599

1.893

(0.268)

(0.328)

14

0.112

0.174

(0.019)

(0.078)

15

0.038

0.081

(0.007)

(0.020)

AT

3.027

4.251

(0.513)

(0.446)

CA

0.063

0.138

(0.013)

(0.007)

CH

-

CI

Roots harvested three months after bloom with harvest of aerial parts

without harvest of aerial parts

0.178

0.119

0.225

(0.041)

(0.018)

0.342

0.453

0.557

(0.074)

(0.069)

0.051

0.046

0.089

(0.017)

(0.008)

ns

0.327

0.390

(0.049)

(0.043)

0.054

0.094

0.145

(0.021)

(0.016)

0.044

0.061

0.083

(0.010)

(0.010)

ns

0.034

0.056

(0.009)

(0.007)

ns

0.175

0.257

(0.032)

(0.024)

0.017

0.017

0.023

(0.003)

(0.004)

0.021

0.022

0.040

(0.005)

(0.005)

ns

0.693

2.230

(0.325)

(0.186)

ns

0.042

0.160

(0.033)

(0.014)

0.039

0.033

0.476

(0.009)

(0.009)

ns

2.108

4.300

(0.551)

(0.320)

0.030

0.123

0.073

(0.007)

(0.009)

-

-

0.046

0.055

(0.006)

(0.008)

2.309

3.021

2.149

(0.205)

ns

2.426

(0.379)

(0.177)

(0.329)

EC

-

-

-

-

-

PT

2.444

3.970

3.207

2.269

(0.414)

(0.225)

(0.186)

(0.378)

Alkamides

(0.014)

LSD00.5

LSD0.05 0.080 ns 0.032 ns 0.049 ns 0.018 0.077 ns 0.012 0.693 0.058 ns 1.194

Caffeic acid derivatives

0.976


0.048 ns ns 0.867

Page 31 of 40


Figure 1




Page 32 of 40

Page 33 of 40




Figure 2

OH O

HO HO

OH

O

O HO

O

O

O

O

OH

HO OH

O

HO HO

OH

Echinacoside

O HO

O

O

OH

OH

O

HO

OH

O

HO

O

Cichoric acid

O

OH HO

OH

OH O

O

OH O

Caftaric acid O HO

O

HO

O HO

OH OH

OH

Chlorogenic acid


Page 34 of 40

Page 35 of 40


Figure 3

30

Mean temperature °C

25 20 15 10 5

Soil (10 cm depth) Air (2 m height) Bud Bloom Wilting Roots without prior harvest of aerial parts

0 -5 -10

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Days



Figure 4 'PharmaPlant'

'Rieger Hoffmann'

Caffeic acid derivative content in aerial parts (µg g-1 DW)

Alkamide content in aerial parts (µg g-1 DW)

3000 1 5 6 12,13 TA

2500 2000 1500 1000 500 0 8000 6000

CA CH CI EC TC

4000 1000 500 0

Bud

Bloom

Wilting

Developmental stage

Bud

Bloom

Wilting

Developmental stage


Page 36 of 40

Page 37 of 40


Figure 5

'Rieger Hoffmann'

'PharmaPlant'

Alkamide -1 yield in aerial parts (kg ha )

12000 10000

1 5 6 12,13 TA

8000 6000 4000 2000 0

Caffeic acid derivative -1 yield in aerial parts (kg ha )

20000 15000

CA CH CI EC TC

10000 5000 1200 800 400 0

Bud

Bloom

Wilting

Developmental stage

Bud

Bloom

Wilting

Developmental stage



Figure 6

0,15 Caftaric acid

0,05 Cichoric acid a

X Data

b

c

3 months

0,00 4 3 2 1 0 4 3 2 1 0

1 month

Caffeic acid derivative -1 content in roots (mg g DW)

0,10

Total

Bloom

1 week

3 months

1 month

1 week

3 months

1 month

1 week

Bud

Wilting

Harvest of aerial parts and subsequent harvest of roots


Page 38 of 40

Page 39 of 40


TOC GRAFIC



1693x952mm (120 x 120 DPI)

ACS Paragon Plus Environment

Page 40 of 40

Harvest strategies for optimization of the content of bioactive

Recommend Documents