echinacea species. · inden for dyrkningsmetoder blev fire emner undersøgt: art og sort, plantens...

The impact of cultivation techniques and

induced stress on bioactive compounds in

Echinacea species.

Ph.D. Thesis

Maria Obel Thomsen

October 2012

AU AARHUS UNIVERSITY

Department of Food Science

Science and Technology

Aarhus University

Kirstinebjergvej 10

DK-5792 Aarslev

Denmark

ii

Main Supervisor

Senior scientist Kai Grevsen


Aarhus University

Co-supervisor

Professor Lars Porskjær Christensen

Institute of Chemical Engineering, Biotechnology and Environmental

Technology

University of Southern Denmark

Assessment committee

Associate professor Carl Otto Ottesen


Aarhus University

Associate Professor Anna K. Jäger

Department of Drug Design and Pharmacology

University of Copenhagen

Professor Monika Schreiner

The Institute of Vegetable and Ornamental Crops

Leibniz University Hannover

iii

I. Preface

This thesis has been submitted in partial fulfilment of the requirements for the

degree of Doctor of Science at the Department of Food Science, Science and

Technology, Aarhus University. The presented work is part of a three-year re-

search project, entitled “The impact of cultivation techniques and

induced stress on bioactive compounds in Echinacea species”. The project was

partly funded by the The Danish Counsil for Strategic Research (Project:

„Health-promoting bioactive compounds in plants‟ 2101-07-006).

Along the road of this project, a number of people have helped and encouraged

me. I am thankful to my two supervisors for their help and valuable discussions

in relation to this project. Furthermore, Kai Grevsen, whose door was always

open, for his skilled guidance throughout the project and for introducing me to

the world of horticulture, and Lars Porskjær Christensen for always answering

my emails comprehensively and for having valuable advises whenever needed.

I am grateful to everyone in Årslev both former and present colleagues, for

maintaining a pleasant and cheerful environment. I am especially thankful to

the technical staff at the department: Astrid Bergman, Birthe Flyger, Marta

Kristensen, Ruth Nielsen, Jens Barfod and Knud Erik Pedersen, without their

valuable work in the field, explanations to me about hands-on horticulture and

inspiring environment I had probably not been able to realise this project.

Furthermore, I would like to thank Eia Andersen, Martin Jensen and Katrine

Kjær for their thorough and competent proofreading.

Finally, I would like to express my deepest appreciation for my friends and

family who have supported me all the way. Especially my boyfriend Joachim

Vinther, for good discussions, for encourage me and for being present when

needed.

Maria Obel Thomsen, Årslev, October 2012

v

II. Abstract

The genus Echinacea is a perennial originating from North America and Native

Americans have used Echinacea for treatment of a wide variety of conditions.

Today, Echinacea preparations are popular herbal medicines for treatment of

infectious diseases and enhancement of the immune system in Europe and the

United State, and the knowledge and interest in Echinacea is steadily increasing

throughout Australia, South America, North Africa, and Asia. Due to the great

marked of Echinacea, optimisation of the bioactive content is of great interest.

Two main research areas are covered by this thesis. The effect of optimising cul-

tivation techniques, and the effect of exogenous induced stress on the quality of

Echinacea plant material, measured as the enhanced concentration of the bioac-

tive compounds, alkamides and caffeic acid derivatives.

Four areas within the subject cultivation technique were investigated: Species

and varieties, developmental stage of plant, harvest strategy of aerial parts and

roots, and fertilisation in form of nitrogen application. The choice of Echinacea

species, plant part used and seed population had a major influence on the quali-

ty of the raw material for medicinal plant preparations of Echinacea. Harvest at

the right developmental stages of the plant is also very important, and both

plant age and time of year (spring-fall for roots and bud-wilting stage for aerial

parts) as well as harvest of aerial parts before subsequent harvest of roots had a

significant influence on the quality of Echinacea. Moreover, the amount of ap-

plied nitrogen to the soil had a significant influence on the biomass yield and

the concentration of alkamides.

Two kinds of induced stress were investigated in this study: exogenous applica-

tion of four elicitors (Hydrogen peroxide, salicylic acid, and methyl jasmonate

and chitosan oligosaccharide) and direct applied stress in form of a saline shock

with a sodium chloride solution. Exogenous application of different elicitors to

E. purpurea did generally not have a positive effect on the content of alkamides

and cichoric acid derivatives. The exception from this pattern was hydrogen

peroxide which enhanced the content of caffeic acid derivatives significantly in

flowers of E. purpurea. Moreover, induced stress as direct applied stress in the

vi

form of a saline shock two days before harvest did significantly enhance the con-

tent of alkamides and caffeic acid derivatives in flowers on E. purpurea.

In conclusion, while optimisation of cultivation techniques have a major impact

on the quality of raw material of Echinacea for medicinal plant preparations.

Application of induced stresses in form of exogenous application of elicitors

seems to be less affective, although application of hydrogen peroxide and sodi-

um chloride resulted in a significant positive enhancement of the quality of

Echinacea flowers.

vii

III. Resumé

Echinacea (solhat) er flerårige planter fra kurvblomstfamilien som stammer fra

det nordlige Amerika hvor indianerne har benyttet dem i århundreder til be-

handling af en lang række sygdomme. I dag er naturmedicinpræparater med

Echinacea populære i Europa og det nordlige Amerika. Præparaterne benyttes

til behandling af en række infektionssygdomme som for eksempel influenza og

forkølelse, og til styrkelse af kroppens eget immunforsvar. Kendskabet til Echi-

nacea er stærkt stigende i både Australien, Sydamerika, Nordafrika og Asien, og

en øget efterspørgsel gør det interessant at forsøge at øge udbytte af de medicin-

ske stoffer i dyrkede Echinacea planter.

To forskningsområder er dækket af denne afhandling. Effekten af at optimere

dyrkningsmetoder og effekten af at udsætte planten for stress. Effekten måles

som øget kvalitet af plantematerialet, det vil sige som øget produktion af de bio-

aktive stoffer, alkamider og kaffesyrederivater.

Inden for dyrkningsmetoder blev fire emner undersøgt: Art og sort, plantens

udviklingsstadie, høststrategi for overjordiske dele og rødder, og kvælstoftilfør-

sel. Undersøgelserne viste at den valgte art, sort og plantedel har stor indflydel-

se på kvaliteten af det rå plantemateriale. Det er også vigtigt at høste planten på

det rigtige tidspunkt, da både plantens alder og tidspunktet på året har stor be-

tydning, både for de overjordiske dele og for rødderne. Derudover har det vist

sig at høst af de overjordiske dele påvirker kvaliteten af rødderne negativt. Til

sidst har dette studie vist at kvælstoftilførsel signifikant påvirker indholdet af

alkamider positivt i de overjordiske dele.

To former for påført stress blev undersøgt: eksogen påførelse af fire stress udlø-

sere (hydrogen peroxid, salicylsyre, methyl jasmonate og chitosan oligosaccha-

rid) og direkte påført stress i form af et saltchok med en saltopløsning. Stoffer

der udløser stress var eksogen påført til E. purpurea og de havde generelt ingen

positiv effekt på indholdet af alkamider og kaffesyrederivater, bortset fra hydro-

gen peroxid hvor indholdet af kaffesyrederivater steg signifikant i blomster to

dage efter behandling. Derudover var der en markant effekt på indholdet af bå-

viii

de alkamider og kaffesyrederivater i blomsterne, når planten blevet påført di-

rekte stress i form af salt.

Af dette kan man konkludere, at mens optimering af dyrkningsmetoder har stor

betydning for kvaliteten af plante materiale til naturmedicin produktion, så ser

påført stress ud til at have en mindre betydning. Kun påførelse af hydrogen pe-

roxide og salt resulterede i en stigning i indholdet af de bioaktive stoffer i blom-

sterne på Echinacea.

ix

IV. List of publications

Paper I. Maria O. Thomsen, Xavier C. Fretté, Kathrine B. Christensen, Lars P.

Christensen, and Kai Grevsen. Seasonal variations in the concentrations of lipo-

philic compounds and phenolic acids in the roots of Echinacea purpurea and

Echinacea pallida.

Submitted to Journal of Agricultural and Food Chemistry

Paper II. Maria O. Thomsen, Lars P. Christensen, and Kai Grevsen. Harvest

strategies for aerial parts and roots of Echinacea purpurea for high content of

bioactive compounds.

Submitted to Journal of Agricultural and Food Chemistry

Paper III. Maria O. Thomsen, Lars P. Christensen, and Kai Grevsen. Effect of

developmental stage and nitrogen application on the content of bioactive com-

pounds in aerial parts of Echinacea purpurea.

Manuscript for Journal of Herbs, Spices & Medicinal Plants

Paper IV. Maria O. Thomsen, Lars P. Christensen, and Kai Grevsen. Effect of

external stress on the bioactive compound in leaves and flowers of Echinacea

purpurea.

Submitted to journal of Agricultural and Food Chemistry

xi

V. Table of Content

I. Preface iii

II. Abstract v

III. Resume vii

IV. List of publications ix

V. Table of content xi

Chapter 1 Introduction 1

Chapter 2 Echinacea, The plant 5

Chapter 3 Secondary metabolites 13

3.1 Lipophilic compounds 15

3.2 Caftaric acid derivatives 21

3.3 Health beneficial effects 27

3.4 Summary 29

Chapter 4 Cultivation techniques 31

4.1 Species and varieties 32

4.2 Developmental stage of plant 34

4.2.1 Harvest age 34

4.2.2 Aerial Parts 36

4.2.3 Roots 40

4.2.4 Interaction 45

4.2.5 Summary 49

4.3 Nitrogen application 50

xii

Chapter 5 Induced stress 53

5.1 Hydrogen peroxide 55

5.2 Methyl jasmonate 57

5.3 Salicylic acid 59

5.4 Chitosan oligosaccharide 61

5.5 Sodium chloride 64

5.6 Discussion of induced stress 67

Chapter 6 Conclusion 69

Chapter 7 Perspective 71

Bibliography 73

Appendix 87

Appendix A. Paper I

Seasonal variations in the concentrations of lipophilic compounds and

phenolic acids in the roots of Echinacea purpurea and Echinacea pallida.

Appendix B. Paper II

Harvest strategies for aerial parts and roots of Echinacea purpurea for

high content of bioactive compounds.

Appendix C. Paper III

Effect of developmental stage and nitrogen application on the content

of bioactive compounds in aerial parts of Echinacea purpurea.

Appendix D. Paper IV

Effect of external stress on the bioactive compound in leaves and flow-

ers of Echinacea purpurea.

Appendix E.

Plant material and sample treatment

1

Chapter 1

Introduction

Commercial medicinal plant production aims to produce high biomass yields

per hectare with a high content of desired bioactive substances. Nearly all these

bioactive compounds are termed secondary metabolites and are produced in

higher amounts by plants with the purpose of protecting the plant from attack

by insect, herbivores and pathogens, or to survive other biotic or abiotic stresses

[Zhao et al. 2005]. Investigations have confirmed that production of secondary

metabolites responds to environmental stresses [Hadecek 2002] and different

cultivation techniques [Gershenzon 1984; McClure 1979], thus by adjusting the

cultivation techniques or by applying environmental stresses, the secondary me-

tabolite production could be enhanced. Fertilization [Gershenzon 1984;

McClure 1979], harvest stage [Galambosi 2004; McClure 1979], and post-

harvest treatments [Tanko et al. 2005] have shown an effect on the content of

secondary metabolites. Moreover, environmental factors such as low tempera-

ture [Plazek et al. 2011], altitude [Monschein et al. 2010], light [Izquierdo et al.

2011], and drought [Gershenzon 1984] have for some plants shown an effect on

the content of secondary metabolites.

The medicinal plant, Echinacea is native to North America where Native Ameri-

cans have used these plants for many centuries prior to the European colonisa-

tion. The Native Americans used Echinacea to treat a wide range of diseases, for

instance: cough, toothache, and venereal diseases [Flannery 1999]. Today,

Echinacea is grown nearly all over the world especially in North America and

Europe [Bauer et al. 1988a; Kreft 2005; Laasonen et al. 2002], and the

knowledge and interest in Echinacea has steadily increased throughout Austral-

ia, North Africa, South America and Asia [Yu and Kaarlas 2004]. The medicinal

use of Echinacea is primarily immunomodulatory, for example treatment of

2

upper respiratory infections such as common cold and influenza. There is no

known single compound responsible for the beneficial health effects, but inves-

tigations have shown that lipophilic compounds, phenolic acids, and polysac-

charides all are beneficial, and a complex mixture of all three compound groups

causes significant synergistic effect [Bauer 1999; Dalby-Brown et al. 2005].

Before the industrialisation and urbanisation, medicinal plants were the prima-

ry health care agent [Wills et al. 2000], and throughout the last century more

than 80% of all drug substances were natural products or inspired by natural

compounds [Harley 2008]. Even though the medicinal marked uses more and

more synthetic compounds, Alan Harley (2008) estimated in 2008 that almost

half of all new drugs approved since 1994 were based on natural compounds.

Moreover, the herb sale is steadily increasing. In just five years, the total esti-

mated herb sale in the United States has increased by nearly 20% to a value of

$5.2 bill (~ €4 bill) in 2010 [Blumenthal et al. 2011], and in 17 European coun-

tries the total turnover on dietary supplements in 2005 were around €5 bill with

an increase in some countries such as Poland, the Netherlands and the Czech

Republic of more than 200% in 8 years [European Advisory Services 2006].

Echinacea products are the fourth most selling medicinal herb in Europe

(2005), and the sixth most selling in the United States (2010), with an annual

turnover of nearly €140 mill in Europe alone [Blumenthal et al. 2011; European

Advisory Services 2006].

It has previously been stated that optimisation of cultivation techniques have an

effect on the bioactive content in E. purpurea [Callan et al. 2005; Li 1998; Stu-

art and Wills 2000a]. However, earlier investigations have only measured the

effects on the total content of lipophilic compounds or the dominating caffeic

acid derivative, cichoric acid, even though the compound or mix of compounds

with beneficial effects is still unknown. The investigation reported here goes a

step deeper, and investigates all the major lipophilic compounds and caffeic acid

derivatives (concentration > 0.1 mg/g DW). The aim of the present work was to

investigate and describe methods to enhance production of the content of sever-

al lipophilic compounds and caffeic acid derivatives in Echinacea species and

not only the content of the total lipophilic compounds and the dominating caf-

feic acid derivative. The hypothesis was that cultivation techniques and induced

stress during cultivation could lead to a higher content and/or change the pro-

file of bioactive compounds in Echinacea plants, and hence improve the quality

Chapter 1 - Introduction

3

of raw plant material for medicinal preparations. For both medicinal and horti-

cultural purposes, three different species of Echinacea are widely cultivated: E.

purpurea (L.) Moench, E. pallida var. pallida (Nutt.) and E. angustifolia (DC.)

Hell.. For medicinal preparations and treatments the whole plant of E. pur-

purea is used (root, stem, leaf and flower) while normally only the roots from E.

pallida and E. angustifolia are used. In the present work, the focus is on E. pur-

purea but also roots of E. pallida were investigated.

The objectives of this study were as follows:

To determine the content and profile of secondary metabolites in differ-

ent species and varieties of Echinacea (Chapter 3 and 4.1).

To investigate the effects of harvest of aerial parts and roots at different

developmental stages on the content of secondary metabolites (Chapter

4.2).

To investigate how prior harvest of flowers affects the content of second-

ary metabolites in subsequent harvested roots (Chapter 4.2)

To evaluate the effect of nitrogen fertilization on the secondary metabo-

lites in aerial parts of E. purpurea (Chapter 4.3).

To investigate the effect of applied stress in form of exogenous applied

elicitors or direct stress on the content of secondary metabolites in E.

purpurea (Chapter 5).

The overall purpose of this thesis is to discuss the background and results of the

experiments (on cultivation techniques and applied environmental stresses)

obtained in the present PhD project in relation to established research. There

are overviews of the botanical characteristics of the three main Echinacea spe-

cies in chapter 2, which includes the taxonomical history and plant morphology.

Chapter 3 describes the secondary metabolites of interest (lipophilic compounds

and phenolic acids), and the variation of the secondary metabolites in the three

Echinacea species. Chapter 3 ends with an overview of the knowledge of health

beneficial effects of the secondary metabolites of interest. Chapter 4 is a discus-

sion and a summary of the results of experiments reported in three attached

4

papers (two submitted and one manuscript) (Paper I, II and III), and it is focus-

sing on the objectives described above: The difference between species and vari-

eties, developmental stages in Echinacea roots and aerial parts and nitrogen

fertilization. Charter 5 discusses the last paper (submitted) (Paper IV), regard-

ing induced stress. The appendixes are copies of three submitted papers for

publication in international peer-reviewed journals, one manuscript in prepara-

tion, and a description of the postharvest treatments and analysis of plant sam-

ples. In paper I, the content of secondary metabolites in roots of E. purpurea

and E. pallida during one year of cultivation are examined with the purpose to

find the most beneficial harvest time (Appendix A). The influence of harvest of

aerial part on the quality of subsequent harvested roots is studied and the most

beneficial harvest time for roots from bloom to late fall are investigated in paper

II (Appendix B). The influence of nitrogen fertilization and different develop-

ment stages of aerial parts of E. purpurea are investigated in paper III (Appen-

dix C). Exogenous application of elicitors and direct applied stress are examined

in paper IV (Appendix D). The last appendix (E) is a description of the posthar-

vest treatments and analysis of plant samples used.

5

Chapter 2

Echinacea, The plant

The name Echinacea originates from the Greek word “echino” which means sea

urchin or hedgehog, and when “echino” is used as a prefix it means prickly or

spiny. The name Echinacea is therefore probably referring to the prickly tips of

the paleae in the flowers (Figure 2.1) [Kindscher 1989]. Originally, Echinacea

was published under the name Rudbeckia purpurea in “Species Plantarum” by

Linnaeus in 1753. Later, it was published under the name Brauneria by Necker

(1790) and finally Echinacea by Moench (1794) [Blumenthal and Urbatsch

2006; McGregor 1968]. Up to 1959 both Rudbeckia and Echinacea were widely

used, but at the Ninth International Botanical Congress in Montreal (1959), the

nomenclature Brauneria was voted illegitimate as a generic name and Echina-

cea, as the next oldest name in use, was selected as the proper name [McGregor

1968]. Not only the name of the genus has caused misperception, the number

and name of species and varieties have also been up for much debate. Especially

the two species E. pallida and E. angustifolia have caused much confusion.

Figure 2.1. The prickly tips of the paleae in E. purpurea flowers, giving the name

of the genus.

6

In year 1900, M. Fernald recognised E. pallida and E. angustifolia as two dif-

ferent species as well as several other scientists [Baskin et al. 1993]. However, in

1920 the two species were described as synonyms in “The Homoeopathic Phar-

macopoeia of the United States”, and the differentiation between E. pallida and

E. angustifolia were neglected after that [Bauer and Wagner 1990; Bauer and

Wagner 1991]. In the 1981 edition of “The Homoeopathic Pharmacopoeia of the

United States”, E. pallida and E. angustifolia were still described as synonyms

[Bauer and Wagner 1990]. A. Cronquist described E. pallida and E. angustifolia

as one species, but two varieties in 1955, but R. McGregor (1968) divided them

into two separated species in 1968. The two classification methods was for de-

bate throughout the next couple of decades unto the end of the 1980‟s where R.

Bauer and Co-workers published several articles on how to distinguish different

species of Echinacea by their chemical profile [Bauer et al. 1988a; Bauer et al.

1988b; Bauer et al. 1989a; Bauer and Remiger 1989; Heubl et al. 1988]. E. pal-

lida and E. angustifolia have not been synonyms for the same species since

then. As a consequence of all this confusion, investigations performed before

around 1990 must be viewed with caution considering the species investigated.

The first real attempt to question the taxonomy classification by R. McGregor

(1968) after 1990 came in 2002 from Binns et al. (2002), who recognised only

two subgenus of Echinacea and four species. Again, E. angustifolia and E. pal-

lida were classified as varieties of the same species (Table 2.1). Today both clas-

sifications are questioned, proven and disproven, based on chemical profiles

and amplified fragment length polymorphism [Blumenthal and Urbatsch 2006;

Flagel et al. 2008; Mechanda et al. 2004; Wu et al.2009]. Nevertheless, the

classification by R. McGregor (1968) is still the most used and the one described

in Flora of North America [Urbatsh and Neubig 2006]. Other taxonomical clas-

sifications than the three mentioned here have been proposed in modern time,

but they have not been as debated as Cronquist vs. McGregor and Binns vs.

McGregor.

Echinacea (Heliantheae: Asteraceae) is a herbaceous perennial, native to the

Atlantic drainage area of the United State, but now easily grown many places in

the world. E. purpurea is grown in both temperate, subtropics and tropic zones,

both at sea level and in high altitudes, in-land and near the coast [Paper I; El-

Gengaihi et al. 1998; Lin et al.2011; Loaiza et al. 2005; Stuart and Wills 2000a].

E. purpurea is drought tolerant and can survive a long winter with a

Chapter 2 - Echinacea, The plant

7

Table 2.1. Taxonomic overview of Echinacea by R. McGregor (1968) and Binns et al. (2000)

classified by species and varieties. R. McGregor (1968) recognised nine species and two varieties

within the genus Echinacea, while Binns et al. (2000) only recognised four species and eight

varieties. All species and varieties classified by R. McGregor (1968) are represented in the spe-

cies and varieties of Binns et al. (2000), except for the two varieties of E. angustifolia, which are

synonyms in Binns et al. (2000) classification.

R. McGregor (1968) Binns et al. (2000)

Species Varieties Species Varieties

Echinacea purpurea Echinacea purpurea

Echinacea pallida Echinacea pallida var. pallida

Echinacea simulata var. simulate

Echinacea angustifolia var. angustifolia var. angustifolia

var. strigosa var. angustifolia

Echinacea sanguinea var. sanguinea

Echinacea tennesseensis var. tennesseensis

Echinacea atrorubens Echinacea atrorubens var. atrorubens

Echinacea paradoxa var. paradoxa var. paradoxa

var. neglecta var. neglecta

Echinacea laevigata Echinacea laevigata

soil temperature below 0 °C [Paper I; Gray et al. 2002]. E. pallida and E. an-

gustifolia have often been mixed up as mention above. It is easy to distinguish

the two species biochemically (see chapter 3), but physically, they do look very

similar. Even though it is difficult to distinguish the two species, it is possible

without chemical analysis and microscopes. Three parameters separates the two

species: i) E. angustifolia plants are in general smaller than E. pallida both in

stem length, leaf length (both basal and cauline), ray corolla length, and diame-

ter of the fresh pollen (Table 2.2) [McGregor 1968]. ii) When the species are in

bloom, it can be seen that the fresh pollen is white on E. pallida and yellow on

E. angustifolia [McGregor 1968]. iii) The ray corolla is spreading on E. angusti-

folia while they are dropping on E. pallida so the angle is approximately 45° to

the stem x-axis (Table 2.2) [Binns et al. 2002a]. However, since height is diffi-

cult to validate and flowers are seasonal, E. pallida and E. angustifolia can be

very difficult to distinguish when they are not in bloom. If a microscope is

8

Table 2.2 Visual differences between E. purpurea, E. pallida and E. angustifolia.

E. purpurea E. pallida E. angustifolia

ROOT

Shape Fibrousa Fusiform taproot. With thick lateralsa

Taproot. With sparse thin lateralsa

Colour Reddish brownc Pale brownc Pale brownc

Cell size (µm) 50 x 30c 40 x 80c 45 x 30c

STEM

Length (cm) 60-180b 40-90b 10-50b

Branched yesb Rarelyb Occasionallyb

LEAF

Basal

Shape Ovate to ovate-lanceolateb

Oblong lanceolate to long ellipticalb

Oblong lanceolate to ellipticalb

Length (mm) 155-220a 130-200 a 80-120 a

Width (mm) 50-100 a 10-50 a 10-50 a

Cauline

Shape Lanceolate-ovate a Lanceolate-ovate to elliptocal a

Length (mm) 70-200b 100-250b 40-150b

Width (mm) 15-80b 10-25b 5-38b

FLOWER HEAD

Ray corolla Reflexed parallel to stem axis a

Spreading 45° to stem axis a

Spreading a

Colour Purplishb Purplish, pink or whiteb White, pink or purplishb

length (mm) 35-50 a 40-90b 20-38b

Width (mm) 10-13 a 5-8b 5-8b

Phyllary

length (mm) 10-15a 8-18b 6-11b

Width (mm) 2-4a 2-4b 2-3b

Series 4a 3-4b 3-4b

Cypsela

Length (mm) 4-4.5b 3.7-5b 4-5b

Pappus A low crown of equal teethb

A toothed crownb A toothed crownb

Fresh pollen

Colour Yellow/lemonb Whiteb Yellowb

Diameter (µm) 19-21b 24-28.5b 19-26b

CHROMOSOME Nr. 11b 22b 11b

a Binns et al.(2002a), b R. McGregor (1968), c Bauer and Wagner (1990).


9

available, the two species can be separated by the size of the root cells or by the

number of chromosomes, since E. pallida is a tetraploid, while all other Echina-

cea species are diploids (Table 2.2) [McGregor 1968]. We have ourselves in the

work here experienced that there still are problem with distinction of E. pallida

and E. angustifolia. We ordered and bought E. angustifolia seeds from a seed

company, however, in the first year with flowers; the plant looked very much

like E. pallida and the chemical profile confirmed that suspicion. This mistake

by the seed company is the reason why E. Angustifolia is not part of this study.

E. purpurea is very easy to distinguish from E. pallida and E. angustifolia (ta-

ble 2.2). The basal leaves have another shape, as they are ovate to ovate-

lanceolate, while the basal leaves on E. pallida and E. angustifolia are oblong

lanceolate to elliptical (Figure 2.2) [McGregor 1968]. The ray corolla is wider

and reflexed parallel to stem axis and the stems are often branched, while it is

rarely or only occasionally branched in E. pallida and E. angustifolia (Table 2.2)

[McGregor 1968]. Besides that, roots of E. purpurea are fibrous and reddish

brown, while E. pallida and E. angustifolia have a pale brown taproot (Figure

2.2).

All taxa of Echinacea are self-sterile [McGregor 1968] which makes outcrossing

and a variety of hybrids common. Since R. McGregor (1968) easily could make

over 5000 successfully hybrids in his crossing programme, gene flow is very

common and considerable natural hybridisation occurs in natural populations

and even cultivated population can be disturbed [McKeown 1999]. The genus

Echinacea is therefore either a very young genus with rapid speciation or a ge-

nus where the genetic barriers have been incompletely formed [Flagel et al.

2008]. An example of a variety of hybrid origin is E. angustifolia DC var. strigo-

sa. R. McGregor (1968) and Wu et al. (2009) both argue that it must have been

hybridised in the past. R. McGregor‟s (1968) conclusions are made on observa-

tions of morphological variations, while Wu et al. (2009) concludes on the basis

of the metabolic profile. They suppose that E. angustifolia DC var. strigosa is a

hybrid from E. angustifolia var. angustifolia and either E. atrorubens or E.

paradoxa var. neglecta. R. McGregor (1968) recognises E. atrorubens and E.

paradoxa var. neglecta as two species while Binns et al. (2002) classifies them

as two varieties of one species.

10

Figure 2.2 Flower, leaf and root of E. purpurea (left) and E. pallida (right)


11

In summary, the names and numbers of the genus, species and varieties have

been debated since Echinacea has become known outside the Native North

Americans population, starting with the name of the genus. It was decided in

1959 that the proper name was Echinacea and in the end of the 1980‟s R. Bauer

and co-workers proved that E. angustifolia and E. pallida definitely were differ-

ent species (for detail see chapter 3) [Bauer et al. 1988a; Bauer et al. 1988b;

Bauer et al. 1989a; Bauer and Remiger 1989; Heubl et al. 1988]. Visually, it is

nearly impossible to distinguish E. angustifolia and E. pallida. E. purpurea on

the other hand is easy to distinguish from the other two.

13

Chapter 3

Secondary metabolites

Despite, great variation within all living organisms, the synthesis of fats, pro-

teins, carbohydrates and nucleic acid in cells are generally the same and are es-

sential for development, growth and reproduction. These processes are identical

in nearly all living matter and are hence the definition of primary metabolism

[Seigler 1977]. Secondary metabolites on the contrary, are specific to the organ-

isms or groups of organisms; as such, one specific secondary metabolite is only

found in some organism or groups of organism, and not in all lining matter.

During the last 20 to 30 years secondary metabolites have been recognised to

fulfil many important plant functions, including protection against attack of

pests or the hazardous environment; protection against UV-radiation; attraction

of pollinators and seed-dispersing animals; participation in allelopathy; me-

tabolism and other plant-animal interactions [Wills et al. 2000]. With the

greater knowledge of the important functions of secondary metabolites, the

terminology “secondary” has been debated [Bentley 1999]. Despite, names such

as “Specific” or “Special” metabolite have been suggested [Bennett and Bentley

1989], “secondary” has not been replaced yet. Most of the bioactive compounds

in medicinal plants are termed secondary metabolites [Wills et al. 2000].

Herbal medicine preparations of Echinacea species have shown a number of

beneficial effects such as non-specific immunomodulatory properties and early

treatment of upper respiratory infections [Barrett et al. 2003]. Several groups of

bioactive compounds have been identified in Echinacea species, including lipo-

philic constituents such as alkamides and essential oils, and hydrophilic con-

stituents such as caffeic acid derivatives, glycoproteins and polysaccharides

[Barnes et al. 2005; Bauer 1999]; however, the most investigated compounds in

E. purpurea and the compounds believed to be responsible for the im-

14

monumodulating properties are derivatives of caffeic acid and lipophilic com-

pounds [Pietta et al. 2004]. Even though, lipophilic compound, caffeic acid de-

rivatives and polysaccharides all have shown beneficial activities, such as anti-

inflammatory affect [Clifford et al. 2002; Lalone et al. 2007; Speroni et al.

2002], the complex mixture of all three can lead to significant synergistic effects

[Dalby-Brown et al. 2005; Lalone et al. 2007]. The beneficial effects of Echina-

cea preparations are therefore believed to be a synergistic effect of a mixture of

many compounds and research within this area is still required in order to fully

understand their mode of action.

In this thesis, focus is on the lipophilic compounds (primarily alkamides) and

derivatives of caffeic acid. Both groups will be described in the following sec-

tions. The aim of this chapter is to give an overview of the existing knowledge in

combination with our observations on lipophilic compound and caffeic acid de-

rivatives in Echinacea, and an account for the health beneficial effects of these

compounds.

Chapter 3 - Secondary metabolites

15

3.1 Lipophilic Compounds

The lipophilic compounds in Echinacea species consist of both ketoal-

kenes/alkynes and alkamides. Ketoalkenes/alkynes are aliphatic unsaturated

carbon chains combined with the functional group, ketone (e.g. Figure 3.1,

Tetradeca-8Z-ene-11,13-diyn-2-one, compound 8). Ketoalkenes/alkynes are by-

products from the breakdown of fatty acids. Alkamides are compounds with an

amide bond that link an amine in form of either isobutylamide or 2-

methylbutylamide with an unsaturated aliphatic fatty acid (e.g. Figure 3.1, Un-

deca-2E,4Z-diene-8,10-diynoic acid isobutylamide, compound 1). The lipophilic

compounds in Echinacea species are generally aliphatic compounds with a

chain length of C11 to C16 (Figure 3.1). The knowledge about the biosynthesis of

alkamides is still in its infancy. It is known that alkamides are produced by liga-

tion of an alkyl or aryl amine with a polyalkenyl or acetylenic fatty acid. Howev-

er, the enzymes catalysing the processes have yet to be identified [Minto and

Blacklock 2008].

The content and type of lipophilic compound vary between the three main spe-

cies of Echinacea and it is possible alone in the variation of lipophilic com-

pounds in roots to distinguish the three species (Table 3.1). E. pallida roots con-

tains mainly ketoalkenes/alkynes, while E. purpurea and E. angustifolia roots

mainly contain alkamides. The two dodeca-2E,4E,8Z,10E/Z-tetraenoic acid iso-

butylamide isomers (alkamide 13, 14, Figure 3.1) are the main lipophilic com-

pounds in both E. purpurea and E. angustifolia, they are, however, absent or in

very low concentration in E. pallida. The alkamides in E. angustifolia are

Table 3.1 Scheme to distinguish between three species of Echinacea according to

the presence or absence of lipophilic compounds [Paper I; Bauer 1999].

E.

purpurea E.

pallida E.

angustifolia

Ketoalkynes/alkenes +

Alkamides, mainly 2,4-diene + +

Alkamides, mainly 2-monoene +

Alkamides 13, 14 + +


16

H NH

O

1: Undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide

A,B

H NH

O

3: Undeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide

A

NH

O

5: Dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide

A,B

NH

O

7: Dodeca-2E,4E,10E-triene-8-ynoic acid isobutylamide

A

O

NH

9: Dodeca-2E,4E-diene-8,10-diynoic acid 2-methylbutylamide

A

O

NH

11: Dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamideA

NH

O

13: Dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide

A,B

O

15: Pentadeca-8Z-ene-11,13-diyn -2-oneC

O

17: Pentadeca-8Z,13Z-dien-11-yn-2-one

C

NH

O

19: Dodeca-2E,4E-dienoic acid isobutylamide

A

O

21: Pentadeca-8Z,11E,13Z-trien-2-one

C

H NH

O

2:Undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide

A,C

NH

O

4: Dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide

A,C

H

O

NH

6: Trideca-2E,7Z-diene-10,12-diynoic acid isobutylamideA;B

H

O

8: Tetradeca-8Z-ene-11,13-diyn-2-oneC

O

NH

10: Dodeca-2Z,4E-diene-8,10-diynoic acid 2-methylbutyl-amideA

O

NH

12: Dodeca-2E,4E,10E-triene-8-ynoic acid 2-methylbutyl-amide

A

NH

O

14: Dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide

A,B

H NH

O

16: Pentadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide

C

NH

O

18: Dodeca-2E,4E,8Z-trienoic acid isobutylamide

A

O

20: Pentadeca-8Z,11Z,13E-trien-2-oneC

Figure 3.1 Chemical structure and systematic names of the 21 lipophilic compounds identified

in paper I. ALipophilic compounds found in E. purpurea roots. BLipophilic compounds found in

aerial parts of E. purpurea. CLipophilic compounds found in E. pallida roots [Paper I].


17

mainly 2-monoene moiety (Figure 3.1, compound 8), while the alkamides in E.

pallida and E. purpurea mainly are 2,4-diene moiety (Figure 3.1, compound 1).

Even from a quick overview of the HPLC chromatograms of root extracts it is

easy to distinguish the three species of Echinacea, as the pattern is very differ-

ent. The difference in appearance of lipophilic compounds between E. pallida

and E. purpurea is illustrated in the HPLC chromatograms in figure 3.2.

Comprehensive investigations in Echinacea plants and products have resulted

in characterization of more than 25 alkamides, of which several are isomeric

pairs differing solely by the double-bond configuration [Barnes et al. 2005]. As

a double-bond inhibits the free rotation around the bond axis, two different

compounds called isomeric pairs are possible. Compound 9 and 11 are an ex-

ample of an isomeric pair (Figure 3.3). Other E/Z-isomeric alkamide pairs in

figure 3.1 are compound 1-2; 4-5; 9-11; 13-14 and 20-21. Even though E/Z-

isomers are nearly identical, it is important to distinguish the compounds, since

E/Z-isomers often have different physical and chemical properties. Z-isomers

have for example generally a higher boiling point and a lower melting point than

E-isomers and enzymes can often distinguish E/Z-isomers .

NH

O

NH

O

9

11

Figure 3.3. The two E/Z-isomeric compounds 9 and 11. The only difference

between the two compounds is the E/Z-configuration.

The content of the dominating isomeric alkamide pair (alkamide 13, 14) in roots

of E. purpurea grown in Denmark can be compared with the results of others

(Table 3.2). It can be noticed from table 3.2 that the concentration of alkamide

13, 14 are in the same order of magnitude, whether it is grown in Europe, North

America or New Zealand. Our values of grown E. purpurea are a little lower

than the values obtained in Canada and New Zealand and a little higher than the

values reported from The United States. Overall, the concentration of alkamides

13, 14 are very similar in roots of E. purpurea across the world.


18

Figure 3.2 HPLC-chromatograms of roots of E purpurea and E. pallida at a wave-

length of 210 and 254 nm [Paper I; Appendix E].


19

Table 3.2 Content of the domination alkamide (alkamide 13, 14) in roots of E. pur-

purea, sorted by investigating country.

Country mg Alkamide 13, 14 / g DW References

Denmark 1.45 Paper I

Denmark 1.68 Paper II

Germany 0.04-0.39 Bauer 1999

Finland 1.73 Laasonen et al. 2002

Canada 2.79/3.12a Binns et al. 2002b

USA 1.66/1.94b Gray et al. 2002

USA 0.806 Schieffer and Kohn 2002

USA 1.12 Qu et al. 2005

New Zealand 1.7/5.7c Perry et al. 1997

New Zealand 2.03 Perry et al. 2000

a Young seedlings/Wild harvested. b Season 1/season 2. c Root/rhizome.

The composition of the different lipophilic compounds in roots of E. purpurea is

also rather similar from growth site to growth site. It is agreed that alkamide 2,

4 and 13, 14 are the major lipophilic compounds and additionally compound 5

and 11 in some growth sites [Paper I; Binns et al. 2002b; Perry et al. 1997; Stu-

art and Wills 2000a]. It should be noticed that even though compounds 13, 14

are the dominating alkamides and the one all directs the attention towards, it is

not even counting for 50% of the total amount of lipophilic compounds in roots

of E. purpurea [Paper I; Binns et al. 2002b; Perry et al. 1997; Stuart and Wills

2000a] and in New Zealand and North America compounds 13, 14 are in some

investigations only counting for approximately 25% of the total content of alka-

mides [Binns 2002b; Perry et al. 1997].

The diversity in alkamides in E. purpurea is much higher in roots than in aerial

parts. As can be seen in table 3.1, we identified 15 alkamides in roots, but only 5

alkamides in the aerial parts of E. purpurea. Alkamides 13, 14 are generally the

only compounds investigated in aerial parts [Paper IV]. Even though, our inves-

tigations indicate that alkamide 6 is in an equal or higher concentration (aerial

parts) [Paper II and IV] than alkamide 13, 14. Alkamide 6 has opposite to al-

kamide 13, 14 and most other alkamides found in E. purpurea a UV-maximum


20

at 210 nm and not at 254 nm (Figure 3.2), where most analysis are performed.

Hence, if the alkamide concentrations are measured at 254 and without a stand-

ard for all the alkamides, the high concentration of alkamide 6 is never found.

For instance, Wills and Stuart (1999) do not find alkamide 6 in aerial parts

grown in Australia, they are only analysing at 254 nm where alkamide 6 is diffi-

cult to discover. Investigations on effects of cultivation techniques should in-

clude the content of other lipophilic compounds than compound 13, 14, since

the compounds or the mixture of compounds that can be assigned for the bene-

ficial health effects of E. purpurea are still unknown. Future investigations

should at least measure the effect of treatments on compound 5 and 6 in aerial

parts and compound 2 and 4 and maybe compound 1, 5 and 11 in roots.

In summary, it is very difficult to distinguish E. pallida and E. angustifolia vis-

ually (Chapter 2). However, the chemical profile of lipophilic compounds in

roots of the three species makes it easy to distinguish E. purpurea, E. pallida

and E. angustifolia from each other, since E. pallida mainly contains ketoal-

kenes/alkynes, E. angustifolia mainly contains 2-monoene alkamides and E.

purpurea primarily contains 2,4-diene alkamides. Most investigations on

treatment effects (both cultivation techniques and applied stress) are only inves-

tigating the content of the dominating isomeric alkamide pair, alkamide 13, 14.

Even though the beneficial compounds or mix of compounds are unknown and

compound 2 and 4 in roots and compound 5 and 6 in aerial parts also are major

alkamides in terms of a high content. Compound 6 is even the dominating al-

kamides in aerial parts of Danish grown E. purpurea. As it will be reported later

(Chapter 4.2), the alkamides are not always reacting similar on different cultiva-

tion techniques, a positive effect on the content of alkamide 13, 14 might there-

fore not be the most desired effect for a high beneficial health effect. Analysis of

other major alkamides should therefore be included in investigations of cultiva-

tion techniques and applied stress.


21

3.2 Caffeic Acid Derivatives

Phenolic acids are a large group of aromatic secondary plant metabolites, that

when describing plant metabolites, consist of a phenol part with a distinct group

of organic acids, generally formic, acetic or propanoic acid [Robbin 2003]. De-

rivatives of the last one are more commonly known as phenylpropanoids and

are a group of compounds derived from cinnamic acid, which itself is synthe-

sised from the amino acid phenylalanine (Figure 3.4) [Dixon and Pavia 1995]. In

Echinacea, all the phenolic acids of interest are derivatives of the phenylpro-

panoid, caffeic acid [Bauer 2000] (Figure 3.4). The major derivatives of caffeic

acid in Echinacea are produced from a reaction with tartaric acid (caftaric acid

and cichoric acid), quinic acid (chlorogenic acid and cynarin) or glycoside

(echinacoside) (Figure 3.4).

It has been stated that echinacoside is present in E. pallida and E. angustifolia,

but absent in E. purpurea and that echinacoside therefore can be used to distin-

guish E. purpurea from the other two [Bauer 2000; Binns et al. 2002b]. How-

ever, in our plant population and in other populations of E. purpurea in Den-

mark [Mølgaard et al. 2003], China [Liu et al. 2007] and Taiwan [Chen et al.

2008] the roots contain echinacoside. Even though Binns et al. (2002b) states

that E. purpurea lack echinacoside, they, themselves find echinacoside in both

wild harvested and in wild-transplanted E. purpurea, only the young seedlings

of E. purpurea lack echinacoside. How to distinguish the three species by caffeic

acid derivatives varies from area to area, as can be seen by table 3.3 where in-

vestigations on roots from Germany, New Zealand, Denmark and two from

Canada are listed [Paper I; Binns et al. 2002b; Perry et al. 2001; Pietta et al.

1998; Sabra et al. 2012]. It is agreed that E. purpurea contains cichoric acid and

caftaric acid. E. pallida contains echinacoside, but not cynarine and E. angusti-

folia contains echinacoside, chlorogenic acid and cynarine in all five areas.

Hence, it can be concluded that E. angustifolia can be distinguished from E.

pallida by its content of cynarine (Table 3.3). There is a tendency for E. pur-

purea to contain cichoric acid and lack echinacoside and cynarine. However,

both compounds have been found in different grown populations (Table 3.3).

Thus, it must be concluded, that the distribution of caffeic acid derivatives can-

not on its own be used to distinguish between E. purpurea, E. pallida and E.

angustifolia.


22

COOH

NH2

COOH COOH

OH

COOH

OH

OH

phenylalanine cinnamate p-coumarate

caffeic acid

HOOC

OHO

O

OH

OH

OH OH

Quinic acid Chlorogenic acid

O

OOH

OH

COOH

OHOH

O

O

OH

OH

Cynarin

O

O

OH

OH

OH

OOH

OH

O

OH

OH

O

OOH O

O

OO

OH

OH

OH

Tartaric acid

Cichoric acid

Caftaric acid

Glycoside

O

OOO

O

OH

OH

OH

OHO

OH

OHOH

O

OHOH

OH

OH

OH

Echinacoside

PAL

Cinnamate-4-hydroxylase

Figure 3.4 Chemical structure and name of the five derivatives of caffeic acid in

the three Echinacea species and their biosynthetic pathway.


23

Table 3.3 Scheme showing the distinguishing between the three species of

Echinacea according to the presence or absence of caffeic acid derivatives in

roots.

E. purpurea E. pallida E. angustifolia

Echinacoside A+ O + Δ X ◊ O + Δ X ◊

Cichoric acid + Δ X ◊ O + X O + ◊

Chlorogenic acid + Δ X O + Δ ◊ O + Δ X ◊

Cynarine X + Δ X ◊

Caftaric acid + Δ X ◊ O + X ◊ O +

+ Canada [Binns et al. 2002b], Δ Germany, [Pietta et al. 1998], X Canada [ Sa-

bro et al.2012] ◊ New Zealand [Perry et al. 2001] and O Danmark [Paper I].

aEchinacoside were in wild harvested E. purpurea roots, but not in young seed-

lings.

The content of cichoric acid is low in roots of our E. purpurea grown in Den-

mark compared with the content reported from other investigations (Table 3.4).

The low values are found even in fresh material, extracted immediately after

harvest; and degradation of cichoric acid during storage and processing of sam-

ples can therefore be excluded. Our values of 2.65 - 2.89 mg/g (Table 3.4) [Pa-

per I and II] are comparable with values from Finland, Canada and some parts

of The United States (Table 3.4), with values in the range from 4.99 – 9.4 mg/g

(Table 3.4) [Binns et al. 2002b; Gray et al. 2002; Hu and Kitts 2000; Laasonen

et al. 2002; Qu et al. 2005], however; even compared to this our values are ra-

ther low. The content of cichoric acid is in general very different from investiga-

tion to investigation, varying from 2.65 - 30.6 mg/g in roots. Even from investi-

gations in Denmark with the same climate (Table 3.4), a very large difference in

content of cichoric acid can be observed. There can be many reasons for the var-

ying concentrations of cichoric acid. i) It has been reported that extraction

methods can have a major impact on the measured concentration [Stuart and

Wills 2000b]. ii) Investigations have shown that cultivation techniques such as

harvest time of year have a significant impact on the concentration of cichoric

acid; however, harvest time, the age of the plant or how the plant was cultivated

is often not reported [Paper I]. iii) Growth location has a major impact. This can

be seen by the values reported by Stuart and Wills (2000) from Australia (Table

3.4) where the concentration of cichoric acid in E. purpurea grown on tableland


24

Table 3.4 Content of the domination caffeic acid derivative, cichoric acid, found in

roots of E. purpurea, sorted by investigating country.

Country mg cichoric acid / g DW Reference

Denmark 2.89 Paper I


Denmark 24.3/35.7a Mølgaard et al. 2003

Germany 6-21 Bauer 1999

Finland 9.4 Laasonen et al. 2002

Canada 8.06/5.88b Binns et al. 2002b

Canada 4.9 Hu and Kitts 2000

USA 18.1/16.1c Callan el al. 2005

USA 18.97 Pellati et al. 2005

USA 4.99/8.69d Gray et al. 2002

USA 15.2 Schieffer and Kohn 2002

USA 8.95 Qu et al. 2005

China 15.9 Liu et al. 2007

Australia 18.9/30.6e Stuart and Wills 2000

Australia 12.5/13.8f Wills and Stuart 1999

New Zealand 22.7/16.8g Perry et al. 2001

a Root/Root-stock. b Young seedlings/Wild harvested. c Fine roots/thick roots. d Season

1/season 2. e Tableland/coastal site. f South/north Australia. g January (summer)/April

(fall).

(humid elevated inland) is 30% lower than the concentration in E. purpurea

grown on a coastal site even though the same seed population was used. iv) It

has been reported that there are significant differences in concentration of

cichoric acid between different seed populations (paper II and III, Binns 2002).

v) It has been reported that cichoric acid is easy degradable [Bergeron et al.

2002; Nüsslein et al. 2000]. A decrease in for example drying temperature from

70 to 40°C enhanced the concentration of cichoric acid with more than 50% in

samples of flowers of E. purpurea [Stuart and Wills 2003] and at a storage at

20°C and a humidity of 80%, the content of cichoric acid in dried roots of E.

purpurea is declining with more than 60 % from storage day 45 to storage day

180 [Lin et al. 2011]. Hence, different extraction methods; cultivation tech-

niques; growth location and climate; plant age; seed population and drying and

storage conditions can cause relatively large variation in the concentration of


25

cichoric acid. Moreover, nearly all the investigations lined in table 3.4 uses dif-

ferent analytical techniques/separation methods, that might also explain the

varying values.

In our investigation, we have tried to optimize the contents of all the bioactive

compounds in one analysis, which mean that we have made a compromise,

where both the content of alkamides and caffeic acid derivatives are high. How-

ever, the compromise cannot explain the very low value compared to another

Danish investigation (Table 3.4) [Mølgaard et al. 2003]. The climate is very sim-

ilar, as Denmark is a very small country. Considerations regarding handling and

storage have been into our mind through the whole process, and the low con-

centrations are even found in fresh material [Appendix E].

Not only does the content of cichoric acid vary largely in investigations through-

out the world, the same pattern can be observed with the content of other caffeic

acid derivatives. We found concentrations of chlorogenic acid below 0.1 mg/g

DW, which is similar to concentrations of chlorogenic acid found in an New Zea-

land population [Paper I and II, and Perry et al. 2001]; however, the concentra-

tion was 0.18 mg/g DW in two locations in The United States [Gray et al. 2002;

Schieffer and Kohn 2002] and 3.8 mg/g DW in a Chinese grown population [Liu

et al. 2007]. The concentration of caftaric acid was below 0.1 mg/g DW in one of

our investigations on E. purpurea roots [Paper II], between 0.1 and 1 mg/g DW

in population grown in the other of our investigations, in one location in The

United States and New Zealand [Paper I, Gray et al. 2002, Perry et al. 2001]

and the concentration of caftaric acid was 3.47 and 7.5 mg/g DW in experiments

in another location in the United State and in China [Liu et al. 2007; Schieffer

and Kohn 2002]. Moreover, the content of cichoric acid in flowers and aerial

parts of E. purpurea is varying largely throughout the world (Table 3.5). As in

the roots, the content of cichoric acid in aerial parts in Denmark is quite low and

the levels vary across the world from 2.26-94.81 mg/g in flowers and 1.52-20.2

in the aerial parts (table 3.5). According to our review on the investigations rep-

resented in table 3.4 and 3.5, it has not been possible to find a pattern in the

diverse content of caffeic acid derivatives. Neither climate zones; extraction

methods; cultivation techniques; nor handling and storage can by themselves

explain the big variation. Analytical techniques/separation methods varies from

one investigation to another and that might explain some of the variations.

However, the variation must be a combination of several factors.


26

Table 3.5 Content of the dominating caffeic acid derivative, cichoric acid, in flowers

and aerial parts of E. purpurea, sorted by investigating country.

Country mg cichoric acid / g DW Reference

Flowers

Denmark 2.26 Paper IV

Slovakia 35.5 Mistríková and Vaverková 2009

Slovenia 10.76 Kreft 2005

USA 46.7 Letchamo et al. 1999

USA 54 Callan et al. 2005

USA 10.9 Qu et al. 2005

Canada 8.89 Binns et al. 2002b


Taiwan 94.81 Chen et al. 2008

Australia 32.3/31.5a Stuart and Wills 2000

Aerial parts


Denmark 1.52 Paper III


New Zealand 20.2 Perry et al. 2001

Australia 12.5/13.4b Wills and Stuart 1999

a Tableland/coastal site. b South/north Australia.

In summary, it can be disproven that echinacoside is absent in roots of E. pur-

purea and that cichoric acid is absent in roots of E. pallida and that these two

compounds can be used as marker compounds to distinguish E. purpurea and

E. pallida. It is very difficult or near impossible to use the chemical profile of

caffeic acid derivatives to distinguish the three Echinacea species, since the pro-

files are very different from one investigation to another. The reported content

of the caffeic acid derivatives is also very different from each investigation.


27

3.3 Health beneficial effect

The different Echinacea species have been used extensively by the Indians in

North America long before the modern American man arrived [Bauer and Wag-

ner 1991] and the species have been used for a variety of problems both external

(against wounds, burns and insects bites) and internal (against toothache,

headache, stomach cramps, coughs, chills, measles and gonorrhoea) [Bauer and

Wagner 1991; Kindscher 1989]. The modern Echinacea adventure started in

1885, when H.C.F. Meyer introduced the pharmaceutical firm - Lloyd Brothers -

for his “Meyer‟s Blood Purifier” with E. angustifolia as the secret ingredient,

with a claim of beneficial properties [Flannery 1999]. After discovering this

wonder plant, several physicians started to experiment with the genus and many

reported of successful treatments [Bauer and Wagner 1991]. Today, Echinacea

remedies are primarily used as an immunomodulating agent, predominantly for

treatment of upper respiratory infections such as flu and cold. Several in vitro

investigations have shown that Echinacea preparations have immune stimulat-

ing effects [Bauer et al. 1989b; Burger et al. 1998; Steinmüller et al. 1993], ex-

tracts of E. purpurea have shown antibacterial, antifungal and antiviral activi-

ties [Binns et al. 1999; Binns et al. 2002c; Sisti et al. 2008; Vimalanathan et al.

2005], and displays an antioxidative activity [Dalby-Brown et al. 2005; Hu and

Kitts 2000; Matthias et al. 2007; Sloley et al. 2001; Thygensen et al. 2007; Tsai

et al. 2012] and anti-inflammatory effect [Chen et al. 2005; Raso et al. 2002].

No single compound has up till now been assigned for the beneficial health ef-

fects in Echinacea, but scientific research has shown that alkamides, phenolic

acids and polysaccharides [Bauer 1999] all are beneficial and a complex mixture

of all three causes significant synergistic effects [Barnes et al. 2005; Dalby-

Brown et al. 2005].

Alkamides have been shown to induce anti-inflammatory responses by inhibit-

ing cyclooxygenase-2 (COX-2) activity [Hinz et al. 2007; Müller-Jakic et al.

1993; Woelkart et al. 2006] and in macrophages by inhibiting prostaglandin E2

production [Lalone et al. 2007]. Moreover, alkamides have shown both antiviral

[Binns et al. 2002c; Vimalanathan et al. 2005] and anti-oxidative activity [Dal-

by-Brown et al. 2005]. The caffeic acid derivatives found in Echinacea species

are known for their anti-oxidative activities [Dalby-Brown et al. 2005; Pellati et

al. 2004; Thygensen et al. 2007] and several of the compounds have shown po-

tential health beneficial effects in vitro. Cichoric acid has shown several antiviral

activities. It is a potential anti-HIV compound since it is active against the en-

3.3 Health beneficial effect

28

zyme, integrase (IN) by inhibition of 3‟-processing and strand transfer, two

steps of the integration process of the IN catalysis [Bailly and Cotelle 2005] and

it has been found active against herpes simplex virus [Binns et al. 2002c].

Chlorogenic acid has shown antimicrobial activities [Lou et al. 2011; Luo et al.

2011]. A docking study on chlorogenic acid reports that chlorogenic acid is a po-

tential inhibitor of H5N1 influenza A virus neuraminidase [Luo et al. 2011] and

an investigation on gram positive and gram negative bacteria have shown that

chlorogenic acid has an antibacterial activity in vitro [Lou et al. 2011]. Echina-

coside has shown both anti-inflammatory and wound healing properties in vitro

and in vivo [Speroni et al. 2002].

That Echinacea extracts have an immunostimulating effect in vivo has been

demonstrated in several investigations [Brinkeborn et al. 1999; Goel et al. 2004;

Lindenmuth and Lindenmuth 2000], although several other investigations have

shown that extracts of Echinacea are without effect [Barrett et al. 2002; Mel-

chart et al. 1998; Yale and Liu 2004]. The contradictory results regarding clini-

cal investigations might be due to difference in quality of the used plant material

and extraction methods and thus large differences in bioactive compound pro-

file of the crude extracts tested [Linde et al. 2009]. The composition of alka-

mides, caffeic acid derivatives and polysaccharides is nearly never clarified,

sometimes it is not even stated which plant part is used [Barrett et al. 2003]. An

investigation of 19 different commercial products containing Echinacea species

purchased in shops in Denmark demonstrated that the content of cichoric acid,

alkamide 2 and alkamide 13, 14 were varying significantly from product to

product [Mølgaard et al. 2003]. One of the products, which is approved as herb-

al medicine by the Danish Food and Drug Administration did not even contain

these compounds. For investigations of the immunostimulating effect, quality

should be more in focus, so more reliable investigation could be obtained.

In summary, Echinacea is a popular traditional medicine used for centuries and

it is still very popular today. Preparations of Echinacea have been reported to

have numerous health beneficial effects both the crude extracts and the individ-

ual compounds. However, a single compound group cannot explain the full im-

munostimulating effect and the effects are believed to be a synergistic effect be-

tween several compound groups. Clinical investigations on Echinacea species

reports contradictory results regarding the beneficial effects; however the quali-

ty of the plants material and the distribution of secondary metabolites are often

unclear, thus the investigations are difficult to compare. The immunostimulat-


29

ing effects of Echinacea are still a mystery and much more research should be

put into this area, before it is fully understood.

3.4 Summary

Bioactive compounds are often secondary metabolites. Despite, several groups

of compound are found in Echinacea species, caffeic acid derivatives and lipo-

philic compounds are the most investigated and the ones believed to be respon-

sible for the beneficial effects.

While E. pallida is dominated by ketoalkenes/alkynes, E. angustifolia is prima-

ry containing 2-monoene moiety alkamides and E. purpurea 2,4-diene moiety

alkamides (Table 3.1) The chemical profile of the lipophilic compounds makes it

therefore easy to distinguish the three species. Alkamides 13, 14 are the domi-

nating alkamides in roots of E. purpurea and the content seems to be similar in

all E. purpurea plants grown throughout the world (Table 3.2). Despite, alka-

mides 13, 14 are the only alkamides investigated in aerial parts of E. purpurea;

they are not the dominating alkamides. In aerial parts, alkamide 6 is the domi-

nating alkamide, and alkamide 6 has a UV-maximum at 210 nm and not at 254

nm as most other alkamides in E. purpurea (Figure 3.2). It is therefore very

easy to overlook, that alkamide 6 is the dominating alkamide in aerial parts of

E. purpurea. The roots of E. purpurea have a more diverse content of alkamides

than the aerial parts, and in this work, we have identified 15 alkamides in roots

and only 5 in aerial parts.

It is not as easy to distinguish the three Echinacea species based on their chemi-

cal profiles of caffeic acid derivatives. E. angustifolia can be recognised by its

content of cynarine, which is absent or in low concentrations in E. purpurea

and E. pallida. However, the presence or absence of all other caffeic acid deriva-

tives are varying from growth site to growth site, especially echinacoside and

cichoric acid (Table 3.3). Echinacoside and cichoric acid can therefore not be

used to distinguish E. purpurea and E. pallida as earlier claimed. Moreover the

content of the caffeic acid derivatives are likewise varying from experiment to

experiment

31

Chapter 4

Cultivation techniques

Approximately 85% of the traditional medicine sold is collected from natural

populations and Echinacea populations has been reported as threat by overhar-

vesting [Chen et al. 2008; Cordell and Colvard 2012]. This is problematic now

and in the future. First of all, colleting from natural populations is not sustaina-

ble, and many plant species are disappearing as consequence of over harvesting

[Cordell and Colvard 2012]. Second, the quality of the material is not always

optimal. Different genotypes; growth environment, and harvest and postharvest

treatment may lead to large differences in content of bioactive compounds. A

direct focus on harvest and postharvest techniques is therefore of great interest

regarding sustainability and good quality of the traditional medicine prepara-

tions. This chapter will examine the possibility of optimising different cultiva-

tion techniques. The first section will focus on the importance of selecting the

right species or variety. Second, it will be investigated how developmental stage

of Echinacea affects the content of the bioactive compounds: At what age is the

Echinacea plant most beneficial to harvest? Should the aerial parts be harvested

in bud, in bloom or during the wilting stage? When should the roots be harvest?

In addition, does harvest of aerial parts influence the quality of later harvested

of roots? The third and last section addresses nitrogen application – do high

quantities or lack of nitrogen application influences the content of bioactive

compounds in Echinacea?

4.1 Species and varieties

32

4.1 Species and varieties

From a medicinal point of view, there are three species of interest: E. purpurea,

E. pallida and E. angustifolia. There has been some confusion about E. pallida

and E. angustifolia in the past as mentioned in chapter 2 (Echinacea, the plant).

Now a-days it is easy to distinguish these species, as their chemical profiles of

lipophilic compounds are very dissimilar (Chapter 3 – Secondary metabolites).

All three species are widely used for medicinal preparations and the species

primarily used varies from one country to another. In Denmark, the food sup-

plements, herbal medicines and herbal remedies primarily contains extract of E.

purpurea, the same in Germany, whereas, most preparations from Italy are

made on E. pallida and the principal species in France is E. angustifolia [Ga-

lambosi 2004; Mølgaard et al. 2003]. Since the chemical profiles are so differ-

ent between the three species, the beneficial effects are probably a result of dif-

ferent compounds. The beneficial compounds or mix of compounds is still un-

known, but they are not the same in all three species.

The chemical profile is not the only difference between the three species. The

two species E. pallida and E. purpurea also have different ideal harvest time of

roots as shown in our investigation [Paper I]. The differences were most visible

for the content of the lipophilic compounds, where the alkamides in E. pur-

purea were divided into two groups, one group had the significantly highest

content of alkamides in early spring, when the aerial parts were about to sprout,

and the other group showed the significant highest content of alkamides in

summer, when the flowers were in bloom and some had just started wilting. In

E. pallida there were only one lipophilic compound with a significant highest

content and that was in early spring. However, all the other compounds in E.

pallida had the same tendency. So, for E. pallida, the most beneficial harvest

time of roots according to content of lipophilic compounds was in early spring,

where it in E. purpurea was dependent on the compounds of interest [Paper I].

Not only species are significantly different chemically. Different seed popula-

tions of E. purpurea throughout the world have shown different content and

composition of caffeic acids derivatives, as could be seen in chapter 3.2 „Caffeic

acid derivatives‟. Table 3.3 shows that the presence of echinacoside, cichoric

acid and caftaric acid vary within the varieties from one investigation to another

and table 3.4 and 3.5 showed that the content of cichoric acid in both roots and

aerial parts of E. purpurea varied significant between different cultivation are-

Chapter 4 - Cultivation techniques

33

as. There are even several differences in the two seed populations used in our

investigations (Pharmasaat and Rieger Hoffmann) grown under the same

growth condition in Denmark. i) There was a significant difference (p < 0.001)

between the two seed populations exposed to three different application of ni-

trogen in the content of alkamide 13, 14 in the aerial parts (see chapter 4.3 Ni-

trogen application)[paper III]. ii) The content of chlorogenic acid, cichoric acid

and total caffeic acid derivatives in the aerial parts were significantly (p < 0.01)

higher in the Rieger Hoffmann seed population, than in the Pharmasaat seed

population, even though they were exposed to the same environment [Paper II].

iii) The average content of the dominating alkamide 13, 14 in roots also differs

between the two seed populations where the content in Pharmasaat was higher

than the content in Rieger Hoffmann [Paper II]. Even within a field with the

same species and the same seed population there has been observed highly vari-

able morphological, agronomic and biochemical traits, both in our investiga-

tions and in investigations from Taiwan and Slovenia [Chen et al. 2008; Kreft

2005]. E. purpurea is self-sterile, very easy to cross and produces several hy-

brids [McGregor 1968]. It is therefore not surprising that there is a large varia-

bility within a single field. Consequently, S. Kreft (2005) recommends that at

least five plants are used to evaluate the quality of a harvest.

In summary, Echinacea is not just Echinacea, even though some Danish herbal

remedies do not clarify the type of plant material further. The chemical profile

in the three species of therapeutic interest is so different, that they are easy to

distinguish and this difference means they most likely have different mode of

action. It is therefore important to grow the species, which have the compound

with the right health beneficial effect. Unfortunately, the compounds or mix of

compounds with the beneficial effects are not identified yet. In addition, differ-

ent seed populations within the species, E. purpurea are of significant im-

portance, since the quality of the plants is varying greatly. In order to cultivate

high quality of Echinacea, several seed populations must be investigated before

the right seed population is grown in large scale.

4.2 Developmental stage of plant

34


E. purpurea is a perennial and dormant during the winter. In the beginning of

the spring, aerial parts start to sprout. The plant is growing to a height of ap-

proximately one meter during the spring and it is flowering in mid-summer

(Denmark: 55.3° N, 10.45° E). During fall, the flowers wilt, seeds are spread,

and then the aerial parts are wilting before the plant goes into dormancy again

[McGregor 1968]. During a year the appearance of E. purpurea is changing sig-

nificantly, the same is the content of bioactive compound in the aerial parts and

roots [Tanko et al. 2005]. E. purpurea is a perennial which means that during

the first couple of years the root is the main growing part, after three to four

years, root growth rate is close to zero [Marquard and Kroth 2001]. This might

also affects the content of the bioactive compounds. Since the content of bioac-

tive compounds potentially is changing with age and season, it is important to

determine the optimum time for harvest.

This section will examine the importance of harvest time of both aerial parts

and roots, starting by investigate the best age of the perennial, E. purpurea to

harvest both aerial parts and roots. The next two subsections describes the most

beneficial time of the year to harvest (during a season), first focusses on the aer-

ial parts, then secondly on the roots. Last, we investigated, if harvest of aerial

parts influences the bioactive compound in the subsequence harvested roots.

4.2.1 Harvest age

It is generally agreed that the biomass yield of roots is increasing with the age of

the plant and reaches a desirable size in the third to fourth growth year [Li

1998; Gray et al. 2002; Seemannová et al. 2006; Seider-lozykowska and

Dabrowska 2003]. Further, we and others have demonstrated that the biomass

yield of the aerial parts and flowers, likewise increase from the first to the fourth

growth year after sowing (Figure 4.1) [Seemannova et al. 2006; Paper II and

III]. It should be noted that E. purpurea plants normally not are flowering the

first year [Li 1998]. A large investigation from Slovenia has shown that the bio-

mass yield of aerial parts decrease again after four years growth. Harvest of the

aerial parts in the fifth or sixth growth year is reducing the biomass yield by


35

Figure 4.1 Biomass yield of aerial parts and flowers is increasing with the age of E.

purpurea. Error bars are left out in order to make the pattern clear. [Seemannová

et al. 2006; own data from paper II and III]

20% and 30%, respectively [Kreft 2005]. Moreover, the roots growth rate is

nearly zero after the third to fourth growth year and later harvests are not in-

creasing the root yield further [Marquard and Kroth 2001]. It is therefore most

beneficial to harvest the aerial parts yearly until the third or fourth growth year

where the roots with advantage can be harvested, based on the biomass yield.

A high biomass yield is not the only goal in commercial medicinal plant produc-

tion, the quality and the content of the bioactive compounds is an equally im-

portant factor. It has been reported that there is no variation in the concentra-

tion of phenolic acids in roots and flowers of E. purpurea or in the concentra-

tion of cichoric acid and caftaric acid in the aerial parts during the first six years

of growth [Kreft 2005; Seemannová et al. 2006]; however, the root concentra-

tion of cichoric acid and caftaric acid increases during the first two years [Gray

et al. 2002]. Since there is either no variation or an increased concentration of

the caffeic acid derivatives in both roots and aerial parts, the yield of caffeic acid

derivatives will increase as the biomass yield increases with age. The recom-

mended harvest for a high yield of caffeic acid derivatives is therefore following

the recommendation for a high biomass yield. Few have investigated the effect

of age on the lipophilic compounds; however, a North American investigation

has reported that the concentration of alkamides is decreasing from the first

growth year to the second in roots of E. purpurea [Gray et al. 2002].

Plant age at harvest (Years after transplant)

0 1 2 3

Bio

ma

ss y

ield

(g

/m2)

0

500

1000

1500

2000

2500

Slovakia

Denmark Pharmasaat

Denmark Rieger Hoffmann

Flowers

Aerial parts(top 20 cm)


36

In summary, it is recommended to harvest the aerial parts every year until the

third or fourth growth year, thereafter the roots should be harvested too. This

harvest strategy is securing the highest biomass yield of both roots and aerial

parts and the highest yield of caffeic acid derivatives.

4.2.2 Arial parts

Most investigations on the content of alkamides and caffeic acid derivatives in

aerial parts are investigating the different plant parts separately: stem, leaf and

flower. In practice, it is much easier to harvest the aerial parts mechanically (ex-

ample the top 20 cm), and it has been reported that the content of cichoric acid

is near equal in flowers and leaves and that the content of alkamides is only 50%

lowers in leaves compared to flowers [Stuart and Wills 2000a]. Therefore, har-

vest of upper leaves concurrent with the flowers is only beneficial due to the

higher gain of alkamides and cichoric acid

The most beneficial time to harvest the flowers of E. purpurea has been investi-

gated in several studies (Figure 4.2 and 4.3) [Paper II and III; Letchamo et al.

1999; Mistríková and Vaverková 2006; Stuart and Wills 2000a]. The flowers are

often harvested in pre-flowering/bud, flowering, mature/release of pollen and

overblown/senescent stages. The definition of the different stages is often not

precisely described and can therefore be difficult to compare. Hence, as can be

seen from the two figures (Figure 4.2 and 4.3) the pattern is quite similar at the

different locations. Generally, the content of alkamides is slightly increasing

from pre-flowering to the flowering stage followed by a considerable increase to

the flowers mature stage. From the mature stage to the senescent stage, the con-

tent is stable or slightly increasing or decreasing. The difference in the last stage

from one investigation to another can be due to different definitions of senes-

cent. Even though we in our investigation examined aerial parts and not only

flowers, the pattern is the same. The content of cichoric acid varies with the op-

posite pattern, the highest content is in the flowers pre-flowering stage and the

lowest content is in fall when the flowers are in a senescent stage. Liu et al.

(2007) has investigated the content of cichoric acid in the aerial parts of one

year old plants during a whole growth year and reports that the content of

cichoric acid is increasing in the aerial parts from seedling stage to the flowering

stage followed by decrease until the withering stage, this corresponds well with

the investigations shown in figure 4.3, where the content of cichoric acid is de-

creasing in all the investigations.


37

Figure 4.2 Content of total alkamide in six

experiments on aerial parts of E. purpurea

due to different developmental stages of

flowers (A-D) or aerial parts (E-F). A: The

United States [Letchamo et al. 1999], B: Slo-

vakia [Mistríková and Vaverková 2009], C:

Australia - coastal [Stuart and Wills 2000a],

D: Australia - tableland [Stuart and Wills

2000a], E: Denmark, seed population –

Pharmasaat [Paper II], F: Denmark, seed

population - Rieger Hoffmann [Paper II].

Error bars are left out in order to show the

pattern clearly.

Figure 4.3 Content of cichoric acid in eight

experiments on E. purpurea due to different

developmental stages of flowers (A-D) or

aerial parts (E-H). A: The United State

[Letchamo et al. 1999], B: Slovakia [Mis-

tríková and Vaverková 2009], C: Australia -

coastal, D: Australia, tableland [Stuart and

Wills 2o00a], E: Denmark, Pharmasaat, 2

years old [Paper III], F: Denmark, Rieger

Hoffmann, 2 years old [Paper III], G: Den-

mark, Pharmasaat, 3 years old [Paper II], H:

Denmark, Rieger Hoffmann, 3 years old

[Paper II]. Error bars are left out in order to

show the pattern clearly.

0.0

0.2

0.4

0.6

0.8

1.0

To

tal

alk

am

ide

con

ten

t (m

g/g

DW

)

0.0

0.5

1.0

1.5

2.0

Preflo

werin

g

Flo

werin

g

Ma

ture

Sen

escent

0.0

0.6

1.2

1.8

2.4

3.0

Developmental stage of plant

Preflo

werin

g

Flo

werin

g

Ma

ture

Sen

escent

A B

C D

E FC

ich

oir

c a

cid

co

nte

nt

(mg

/g D

W)

0.0

0.3

0.6

0.9

1.2

1.5

Preflo

werin

g

Flo

werin

g

Ma

ture

Sen

escent

3.0

3.7

4.4

5.1

5.8

0

10

20

30

40

50

0

8

16

24

32


Preflo

werin

g

Flo

werin

g

Ma

ture

Sen

escent

A B

C

E

G

D

F

H


38

Our investigations show among other results that even though the other major

caffeic acid derivative follows the pattern of cichoric acid on different harvest

stages, the alkamides do not follow a uniform pattern (Figure 4.4). The content

of alkamides 1, 6, 13 and 14 are all increasing significantly with later harvest

stages, while the content of alkamide 5 is decreasing significantly (p < 0.05).

Knowledge about the content of the secondary metabolite is not enough, since a

flower in bud stage has a much lower weight and by that lower yield (mg/plant)

of secondary metabolites than a fully developed flower. It has been reported that

the weight of a single flower increases with more than a factor of four from the

early bud stage to the senescent stage [Callan et al. 2005]. So, even though the

content of cichoric acid in the flower heads is decreasing with the flowers devel-

opmental stages, the yield (mg/plant) of cichoric acid is increasing, as can be

seen in figure 4.5. All the caffeic acid derivatives are reacting similar to cichoric

acid according to our investigation [Paper II]. A high yield of alkamides and

Figure 4.4 Content of alkamides and caffeic acid derivatives in aerial parts of

E. purpurea grown in Denmark and harvested at different developmental stages

[Paper II]. Bars are SE (n = 6).

Developmental stage

Bud Bloom Wilting

Co

nte

nt

in a

eria

l p

art

s (m

g/g

DW

)

0.0

0.5

1.0

4.0

6.0

8.0

Bud Bloom Wilting

'Pharmasaat'

0.000.050.100.150.20

0.501.001.502.002.503.00

'Rieger Hoffmann'

1

5

6

13,14

Total alkamides

Caftaric acid

Chlorogenic acid

Cichoric acid

Echinacoside

Total caffeic acid derivatives


39

Figure 4.5 Yield of cichoric acid in four experiments on E. purpurea in Denmark

with harvest of different developmental stages of aerial parts. The yield of 2 years

(above) and 3 years (below) old plants. Results for the seed population „Phar-

masaat‟ (left) and the results for „Rieger-Hoffmann‟ (right). Error bars are left out

in order to make the pattern clear, they can be found in paper II and III.

caffeic acid derivatives is therefore achieved with a harvest when the aerial

parts/flowers are in the mature stage.

In summary, all investigations on the most beneficial harvest stage of aerial

parts are only investigating the content of the total alkamides and the major caf-

feic acid derivative, cichoric acid, even though the beneficial compounds are

unknown. Our investigation has shown that alkamide 5 is reacting dissimilar to

the other major alkamides in the aerial parts of E. purpurea.

If the purpose with aerial part harvest is a high content of the bioactive com-

pounds, the aerial part should be harvested in the mature flower stage for a high

content of alkamides (except alkamide 5) or harvested in the preflowering stage

for a high content of caffeic acid derivatives and alkamide 5. If on the other

hand a high yield per area is desired, the mature flower stage is the most benefi-

Preflo

werin

g

Flo

werin

g

Ma

ture

Cic

ho

ric

aci

d y

ield

(m

g/p

lan

t)

0

200

400


Preflo

werin

g

Flo

werin

g

Ma

ture

'Pharmasaat'

0

200

400

600

'Rieger Hoffmann'

2 year old plants

3 year old plants


40

cial stage to harvest aerial parts for both a high yield of alkamides and caffeic

acid derivatives.

4.2.3 Roots

The diversity of the bioactive compounds in the roots is much more complex

than in the aerial parts of E. purpurea, especially regarding the lipophilic alka-

mides. We have identified and quantified 15 different alkamides in the roots and

only 5 alkamides in the aerial parts of E. purpurea (Figure 3.1). Even though the

isomeric alkamide pair 13, 14 are major compounds in the roots and the aerial

parts, alkamide 1, 2, 4, 5, 9 and 18 are also major compounds in the roots,

while the major compound in the aerial parts - alkamide 6 – only are at trace

levels in roots (Chapter 3.1). Cichoric acid is the dominating caffeic acid deriva-

tive in both aerial parts and roots, but while caftaric acid, chlorogenic acid and

echinacoside are represented at trace levels in roots, they are in a 10 times high-

er content in the aerial parts [Paper II]. There are therefore great differences in

medicinal preparations made on roots compared to preparations made on aerial

parts.

A German growers guide recommends a fall harvest of E. purpurea roots [Mar-

quard and Kroth 2001] and T. Li (1998) states that roots of E. purpurea normal-

ly are harvested in fall when the first frost have occurred. Nevertheless, is har-

vest of roots in fall really the most beneficial time? The most beneficial harvest

time for E. purpurea roots has been investigated throughout the aerial parts

flowering season and the results are contradictory. The content of total alka-

mides is increasing from the seedling to the fruiting stage in Egypt (Figure 4.6)

[El-gengaihi et al. 1998], while it is decreasing from the pre-flowering stage to

the mature stage according to two growth sites in Australia [Stuart and Wills

2000a]. Both investigations harvested roots in their first growth year. An expla-

nation on the contradictory reaction to different harvest dates could be the dis-

similar content of the single alkamides (Table 4.1). Our investigation reveals

that the different alkamides in the roots are reacting dissimilar to different har-

vest times [Paper I], alkamide 1, 2, 3 and 7 have all the significantly (p < 0.05)

highest content when the flowers were in bloom, while alkamide 4, 5 and 9 had

the significantly (p < 0.05) highest content in early spring when the first signs of

new aerial shoots were just visible (Figure 4.7). As can be noted by table 4.1, the

alkamides identified in the Australian and Egyptian investigation are nearly


41

Figure 4.6 Content of total alkamide in roots harvested in Australia and Egypt

from the aerial parts seedling stage to senescent of one year old plant [El-gengaihi

et al. 1998, Stuart and Wills 2000a]. Error bars are not stated in the two articles.

Table 4.1 Relative level (Percent of total concentration) of

alkamides in roots of E. purpurea.

Compound Denmark1 Australia2 Egypt3

1 6 6 3

2 16 18 3

3 2 2 Trace

4 12 13 1

5 5 5 -

6 2 Trace

7 2

9 7

11 1 6 Trace

12 1

13, 14 42 45 70

18 3 1

19 2 1

Others4 6 1 Paper I. 2 Stuart and Wills 2000a. 3El-gengaihi et al. 1998

Harvest of roots according to developmental stage of aerial parts

Seed

ling

preflo

wer

Flo

werin

g

Ma

ture

Sen

escent

To

tal

alk

am

ide

con

ten

t (m

g/g

DW

)

0

2

4

6

8

10

12

14

Australia - Coastal

Australia - Tableland

Egypt


42

Figure 4.7 Content of seven alkamides (1, 2, 3, 4, 5, 7 and 9) according to different harvest

dates throughout one year in E. purpurea. Alkamide 1, 2, 3 and 7 had the highest content when

the flowers were in bloom and alkamide 4, 5 and 9 had the highest content in early spring when

the first signs of new aerial shoots were just visible [Paper I]. Bars are SE (n = 6).

identical. However, while the roots from Egypt primarily contain alkamide

13, 14 and the other alkamides are in low or at trace levels, there is a high con-

tent of the other alkamides in the Australian roots. The effect on the content of

total alkamides is mostly influenced by alkamide 13, 14 reaction to different

harvest times in the Egyptian investigation, whereas the Australian investiga-

tion have less than 50% alkamide 13, 14 and a high content of both alkamide 1,

2, 4, 5 and 11 in their total content. The most beneficial harvest time concerning

a high content of total alkamides, seems therefore to be dependent of the distri-

bution of alkamides in the roots. It is therefore relevant to know the most bene-

ficial harvest time for all the alkamides. Note from figure 4.7, that the content of

all the shown alkamides are low in fall, the normally recommended harvest time

of roots!

The most beneficial harvest time for the content of total alkamides is not the

only result that is varying from one investigation to another; the same is the

content of cichoric acid. The content of cichoric acid is nearly stable in a North

American investigation, with only a minor non-significant decrease in nine har-

vests from early summer over the plants flowering time to late fall [Callan et al.

2005] (Figure 4.8). Whereas, the content of cichoric acid is decreasing signifi-

cantly from early spring to the flowers are in bloom in a Chinese investigation,

followed by a small increase during the aerial parts wilting [Liu et al. 2007], and

No

v

Ja

n

Ma

r M

ay

J

ul

Sep

N

ov

Alk

am

ides

co

nte

nt

(mg

/g D

W)

0.0

0.2

0.4

0.6

0.8

1.0

No

v

Ja

n

Ma

r M

ay

J

ul

Sep

N

ov

Harvest date

No

v

Ja

n

Ma

r M

ay

J

ul

Sep

N

ov

No

v

Ja

n

Ma

r M

ay

J

ul

Sep

N

ov

No

v

Ja

n

Ma

r M

ay

J

ul

Sep

N

ov

No

v

Ja

n

Ma

r M

ay

J

ul

Sep

N

ov

No

v

Ja

n

Ma

r M

ay

J

ul

Sep

N

ov

7 91 2 3 4 5


43

Figure 4.8 Normalised content of cichoric acid in roots of E. purpurea grown in China, The

United States, Denmark, and Australia according to different harvest dates and stages. The

normalisation (%) is based on the highest value (100) in each investigation. Error bars are left

out in order to make the pattern clear. Error bars from our investigation can be seen in paper I.

[Paper I; Callan et al. 2005; Liu et al. 2007; Stuart and Wills 2000a]

in an Australian investigation, the pattern is completely different, since the con-

tent of cichoric acid is decreasing significantly from the aerial parts flowering

stage to the senescent stage (Figure 4.8) [Stuart and Wills 2000a]. Our investi-

gation in Denmark during a whole cultivation year revealed that the content of

cichoric acid was increasing from early spring to late spring (the aerial parts

growing phase) followed by a significant decrease until fall (the whole flowering

phase from bud to wilted flower). The content of cichoric acid show approxi-

mately same pattern due to different developmental stage in The United States

(no significant decrease) [Callan et al. 2005], two areas in East Australia [Stuart

and Wills 2000] and in Denmark [Paper I] where the content is decreasing from

pre-flower /flowering to the wilting stage. The different pattern in China [Liu et

al. 2007] might be a result of a different climate, since this investigation is made

in a subtropical environment and all the other investigations are made in a tem-

perate climate. The climate might have an impact on the pattern

Harvest stage

Preflo

werin

gF

low

ering

Ma

ture

Sen

escent

Australia - Coastal

Australia - Tableland

Harvest date

Dec

Ja

n

Feb

M

ar

Ap

r M

ay

Ju

n

Ju

l A

ug

S

ep

Oct

No

v

Dec N

orm

ali

sed

co

nte

nt

of

cich

ori

c a

cid

0

20

40

60

80

100

China

The United states

Denmark


44

Figure 4.9 Content of the caffeic acid derivatives in roots of E. purpurea accord-

ing to different harvest dates throughout one year in E. purpurea. * marks the sig-

nificantly (p < 0.05) highest content. Bars are SE (n = 7).

in content of cichoric acid during a whole year. In Denmark, the content of all

the caffeic acid derivatives are highest in spring (Figure 4.9). Caftaric acid,

cichoric acid and the total content of caffeic acid derivatives have the signifi-

cantly (p < 0.05) highest content in late spring (May) whereas echinacoside

shows a significantly (p < 0.05) highest content in early spring (Marts). All the

caffeic acid derivatives in roots of E. purpurea show a low content in fall, the

time of the year where it earlier has been recommended to harvest the roots [Li

1998; Marquard and Kroth 2001].

In summary, most beneficial harvest of roots from E. purpurea is dependent of

the compound of interest. A high content of caffeic acid derivatives and half of

the alkamides is achieved with a harvest of roots in spring; moreover, a spring

harvest gives opportunity to use the field for new cash crop the same year. A

high content of the other half of the alkamides is achieved with a harvest of

roots in summertime and harvest in summertime gives the farmer an oppor-

tunity to harvest the aerial parts as well. A harvest in fall cannot be recommend-

ed since the content of all the bioactive compounds are low at that time.

Harvest date

No

v

Dec

Ja

n

Feb

M

ar

Ap

r M

ay

J

un

J

ul

Au

g

Sep

O

ct N

ov

D

ec

Co

nte

nt

(mg

/g D

W)

0

1

2

3

4

5

6

Caftaric acid

Echinacoside

Cichoric acid

Total caffeic acidderivatives

*

*

*

*


45

4.2.4 Interaction between harvest of aerial parts and roots

The aerial parts can be harvested the first three years and it is recommended

harvesting them in the flowers wilting stage in order to secure a high yield of

alkamides and caffeic acid derivatives in E. purpurea. The roots have the high-

est biomass in the third or fourth year and that is securing a high yield of caffeic

acid derivatives. When to harvest the roots depends of the compounds of inter-

est as outlined above. Half of the alkamides and the caffeic acid derivatives have

the highest content in spring, while the other half of the alkamides have the

highest content in mid-summer. However, many farmes chose to harvest the

aerial parts in summertime followed by harvest of roots in fall when the first

frost have arrived (Li 1998). A higher content of secondary metabolites are

normally produced in plants as a reaction to biotic or abiotic stress [Zhao et al.

2005]. So, is the bioactive content in roots affected by harvest of the aerial

parts?

An investigation from The United States reports that harvest of flowers or 50%

of the aerial parts have no influence on the content of cichoric acid in roots, but

there was a slight decrease in the biomass of the two year old root [Callan et al.

2005]. A grower‟s guide from Germany recommends harvest of aerial parts be-

fore harvest of roots [Marquard and Kroth 2001] and a North American book

about Echinacea states that harvest of aerial parts does not affects the quality of

the subsequent harvested roots. However, this seems not to be true in Danish

grown E. purpurea. Figure 4.10 shows the content of the major alkamides (1, 2,

4, 5, 9 and 13, 14) and the major caffeic acid derivatives (caftaric acid and

cichoric acid) in roots of E. purpurea harvested one week and three months af-

ter the aerial parts blooming stage, with and without prior harvest of aerial

parts. The content of alkamides is lower in the harvested roots with prior har-

vest of aerial parts both in roots harvested one week and three months later

(Figure 4.10). The content of caffeic acid derivatives is also lower in the roots

harvested one week after harvest of the aerial parts, but higher in the roots har-

vested three months later. It must be concluded that the bioactive content in

roots are affected by prior harvest of aerial parts. However, if the aerial parts

were to be harvested the same year as the roots, what would then be the best

time to harvest the aerial parts in order to get the highest content of alkamides

and caffeic acid derivatives in roots? The majority of the alkamides in roots are

not affected by different harvest time of the aerial parts or different harvested


46

Figure 4.10 The content of the major alkamides and caffeic acid derivatives in

roots of E. purpurea harvested one week and three months after the aerial parts

blooming stage. Bars (SE, n = 7) marked with * indicate that the content is signifi-

cant differences (p < 0.05) between roots with harvest of aerial parts and roots

without prior harvest of aerial parts. TA: Total content of alkamides, CA: Caftaric

acid, CI: Cichoric acid, TC: Total content of caffeic acid derivatives. [Paper II].

Harvest of roots one week after harvest of aerial parts

Alkamides and caffeic acid derivatives

1 2 4 5 9 13,14 TA CA CI TC

Co

nte

nt

(mg

/g D

W)

0

1

2

3

4

5

6With harvest of aerial parts

Without harvest of aerial parts

Harvest of roots three months after harvest of aerial parts

Alkamides and caffeic acid derivatives

1 2 4 5 9 13,14 TA CA CI TC

Co

nte

nt

(mg

/g D

W)

0

1

2

3

4

5

6With harvest of aerial parts

Without harvest of aerial parts


47

times of the roots. The only alkamides slightly affected is alkamide 4, and the

minor alkamides, alkamide 8 and 11. This is not the case for the content of caf-

feic acid derivatives. There is an interaction between harvest stage of aerial parts

and subsequent harvest time of roots for caftaric acid, cichoric acid and the total

content of caffeic acid derivatives, as can be observed in figure 4.11. If the aerial

parts are harvested in bud stage (for example in order to achieve a high content

of caffeic acid derivatives) the contents of caftaric acid, cichoric acid and total

caffeic acid derivatives are declining in the subsequently harvested roots from

one week to three months after harvest of the aerial parts (p < 0.001). If the aer-

ial parts are harvested in bloom (for example in order to achieve an acceptable

content of both alkamides and caffeic acid derivatives) the content of caffeic ac-

id derivatives are not affected by the different harvest times of roots. Aerial part

harvest in the wilting stage (for example in order to achieve a high yield of both

alkamides and caffeic acid derivatives) results in first a decrease followed by an

increase in the content of caffeic acid derivatives in the subsequent harvested

roots (p < 0.001). Overall, no matter when the aerial parts are harvested, a high

content of caffeic acid derivatives in roots can be achieved when the roots are

harvested one week after the aerial parts are harvested.

In summary, harvest of aerial parts affects the content of bioactive compounds

in roots harvested one week and three month after harvests of aerial parts. If

aerial parts must be harvested the same year as the roots, it is recommended to

harvest the roots in the summer time, one week after harvest of the aerial parts.

Alternatively, aerial parts and roots could be harvested at the same time, in that

way harvest of aerial parts will not affect the content of bioactive compound and

a high bioactive content can be achieved in both roots and aerial parts.


48

Figure 4.11 Interaction between harvest of aerial parts in bud, bloom and wilting

stage with subsequent harvest of roots one week, one month and three months af-

ter harvest of the aerial parts for caftaric acid, cichoric acid and total caffeic acid

derivatives. Bars are SE (n = 12).

Harvest of aerial parts and subsequent harvest of roots

Ca

ffei

c a

cid

der

iva

tiv

eco

nte

nt

in r

oo

ts (

mg

/g D

W)

Harvest of aerial parts

Harvest of roots after harvest of aerial parts

0.000.030.060.090.120.15

Caftaric acid

01234

Cichoric acid

Bud1 w

eek

1 mo

nth

3 m

on

ths

01234

Bloom

1 week

1 mo

nth

3 m

on

ths

Wilting

1 week

1 mo

nth

3 m

on

ths

Total caffeic acidderivative


49

4.2.5 Summary

Aerial parts: The content of cichoric acid is equal in flowers and leaves, and the

content of total alkamides is only 50% lower in the leaves compared to flowers

[Stuart and Wills 2000a]. Therefore, there is no reason to solely collecting the

flowers since a higher bioactive yield could be achieved by harvesting the upper

leaves with flower. The biomass yield and the yield of caffeic acid derivatives are

increasing with age of the plant until the third or fourth growth year. Harvest of

aerial parts yearly is recommended.

A high content of caffeic acid derivatives in aerial parts is achieved with a har-

vest in the flowers bud stage. However, a high content of alkamides and a high

yield of both alkamides and caffeic acid derivatives are achieved if the aerial

parts are harvested in the flowers wilting stage. We would recommend a harvest

of the aerial parts yearly in the flowers wilting stage, until the plant is three to

four years old.

Roots: The biomass and by that the content of caffeic acid derivatives are in-

creasing with age until the plant is three to four years old, which therefore is the

recommended plant age for harvesting roots. The most beneficial harvest time

in the season depends on the compounds of interest and the intention with the

field. Earlier it has been recommended to harvest roots in fall, but a harvest in

fall is not recommended for Danish grown E. purpurea, since this gives the low-

est content of all the bioactive compounds. High content of caffeic acid deriva-

tives and half of the alkamides are achieved with a harvest of roots in spring,

and a spring harvest give the farmer the opportunity to use the field for a new

crop the same year. If the aerial parts are to be harvested the same year, roots

should be harvested one week after harvest of aerial parts, since it will result in

the highest content of bioactive compounds, or alternatively the aerial parts and

the roots should be harvested simultaneous in the aerial parts wilting stage.

4.3 Nitrogen application

50


Nitrogen is important for plant growth, but the level of nitrogen does not only

determine the plant growth, it can also change the content of secondary metabo-

lites in plant tissues [Chishaki and Horiguchi 1997]. The caffeic acid derivatives

of interest in E. purpurea are all synthesized by the phenylpropanoid partway,

which is initiated by the enzyme phenylalanine ammonia lyase (PAL) [Winkel-

Shirly 2001]. Nitrogen deficiency have been shown to increase the activity of

(PAL) in multiple plants [Margna 1977; Kováčik and Bačkor 2007], since the

plant by activation of PAL can liberate ammonium from phenylalanine when it

is transformed to cinnamate (Figure 3.4) [Kováčik and Bačkor 2007]. As a con-

sequence of PAL activation, the content of phenolic acids increases when the

plants lack nitrogen [Margna 1977]. Alkamides, on the other hand, contains ni-

trogen in their chemical structure (Figure 3.1). Therefore, the plant cannot lib-

erate ammonium from the biosynthesis of alkamides and nitrogen deficiency

will therefore not increase the content of alkamides. In Echinacea species, a

higher application of nitrogen should therefore reduce the concentration of caf-

feic acid derivatives, while the concentration of alkamides should increase.

Several papers have investigated the influence of nitrogen application on

Echinacea plants [Berti et al. 2002; Dufault et al. 2003; El-gengaihi et al. 1998;

Montanari et al. 2008; Shalaby et al. 1997; Zheng et al. 2006a; Zheng et al.

2006b], and the investigations can be divided into two groups: one group inves-

tigated the best source for applied nitrogen, and another the most beneficial

amount. First, how is the source influencing the biomass yield?

The NO3-/NH4

+ ratio of the applied nitrogen has been investigated in relation to

biomass accumulation, and Montanari (2008) and Zheng (2006b) reports that

the NO3-/NH4

+ ratio generally does not affect the biomass accumulation in

roots, leaves or aerial parts of Echinacea species. In cases where NO3-/NH4

+

ratio had an effect, higher NO3-/NH4

+ ratio resulted in increased biomass yield

of roots and aerial parts. Both experiments were conducted in a greenhouse on

young plants and field experiment of that type on mature plants would be inter-

esting. Whether or not the root yield and aerial biomass are increasing with in-

creasing amount of applied nitrogen, it is important to point out, that higher

biomass production not necessarily result in a higher phytochemical production

[Zheng et al. 2006a], and that the effect on roots can differ from the effect on

aerial parts.


51

In the field experiment conducted in relation to this thesis (Paper III), nitrogen

was applied in the form of urea, organic bound nitrogen, which is metabolised

to NH4+ by mineralisation. The plants received either no nitrogen (0), 100 or

200 kg nitrogen/ha in late spring and the aerial parts were harvested in bloom.

The soil contained only low amounts (below 10 kg N/ha) of mineralised nitro-

gen at the start of the experiment.

Most investigations on the effect of the amount of nitrogen applied to Echinacea

species have been conducted with the purpose to investigate the effect on the

content of alkamides. The content of alkamides has been reported to increase in

roots of E. angustifolia and E. purpurea with increasing amount of nitrogen

applied [Berti et al. 2002; El-gengaihi et al. 1998]. Moreover, in our and Egyp-

tian grown E. purpurea the content of alkamides is clearly and significantly in-

creasing in the aerial parts with an increasing amount of applied nitrogen (Fig-

ure 4.12) [Paper III, El-Gengaihi et al. 1998]. Even at application of 200 kg

N/ha there was no sign of levelling off in the concentration of alkamides. There-

fore, both in the aerial parts and in roots the content of alkamides is increasing

with increasing amount of applied nitrogen and this makes sense, since alka-

mides contains nitrogen in their structure.

The effects on caffeic acid derivatives are not that well investigated. An investi-

gation on other plant species indicates that the content probably would decrease

with an increasing amount of applied nitrogen; however, this is not the case in

Echinacea [Gershenzon 1983; Margna 1976]. A North American investigation

with increasing amounts of applied nitrogen reports no significant effect on the

content of the dominating caffeic acid derivatives in E. pallida and E. purpurea

roots [Dufault et al. 2003]. Even so, they conclude that there is a tendency to a

decrease. In our investigation on aerial parts, there was neither a significant ef-

fect of amount of applied nitrogen on the content of cichoric acid – the major

caffeic acid derivative in E. purpurea [Paper III].


52

Figure 4.12. Content of alkamide 13, 14 in aerial parts is increasing with increas-

ing nitrogen application in both Danish and Egyptian grown E. purpurea. Error

bars are left out in order to make the pattern clear. For the Danish grown E. pur-

purea the error bars be seen in paper III [El-gengaihi et al. 1998, Paper III].

In summary, despite nitrogen deficiency should have a tendency to increase the

content of caffeic acid derivatives there are to our knowledge no investigations

where the increase has been significant in any Echinacea species, neither in

roots nor in aerial parts. The recommended amount of applied nitrogen is there-

fore solely based on the content of alkamides, and they are increasing with high-

er amounts of applied nitrogen. Therefore, it is recommended to growth

Echinacea species with a relatively high concentration of nitrogen in the soil (at

least 200 kg nitrogen/ha) to secure a high yield of alkamides. The cost-

effectiveness of this disposition is of cause dependent on the prizes of raw mate-

rial and if there is a bonus for high content of alkamides.

Applied nitrogen (Kg N/ha)

0 100 200Alk

am

ide 13

,14

co

nte

nt

(mg

/g D

W)

0.00

0.10

0.20

0.30

0.40

0.50

Danish grown - Pharmasaat

Danish grown - Rieger-Hoffmann

Egyptian grown

53

Chapter 5

Induced stress

The quality of plant material can be enhanced significantly by optimisation of

different cultivation techniques of the plant of interest, as has been shown earli-

er. However, can the accumulation of secondary metabolites be enhanced fur-

ther? When a plant is exposed to abiotic or biotic stress, the accumulation of

secondary metabolites is triggered and activated by different elicitors [Kessler

and Baldwin 2002; Zhao et al. 2005] and this knowledge might be used to en-

hance the quality of plant material even more. Either exogenous elicitors known

to trigger and activate the accumulation of secondary metabolites can be applied

to the plant or the plant can be exposed to external stress that indirectly causes

the plant to up-regulate production of secondary metabolites. Both methods are

examined in this section.

Alkamides influences morphological processes in Echinacea species, and they

are known to activates important stress related genes, which lead to an en-

hanced production of both H2O2 and jasmonic acid [Méndez-Bravo et al. 2011].

Moreover, it have been reported that exogenous application of methyl

jasmonate to the aerial parts, enhances the production of most alkamides in

roots of E. pallida, the aerial parts themselves were not investigated [Binns et

al. 2001].

The production of caffeic acid derivatives is initiated by the formation of cin-

namate from the amino acid, phenylalanine, and the formation is catalysed by

PAL (Figure 3.4) [Dixon and Pavia 1995; Winkel-Shirly 2001]. All elicitors acti-

vating this enzyme, therefore potentially activates the accumulation of caffeic

acid derivatives.

Four elicitors are investigated in this section: hydrogen peroxide (H2O2); methyl

jasmonate; salicylic acid and chitosan oligosaccharide. H2O2, methyl jasmonate

54

and salicylic acid are important elicitors used by plants to signal a stressed situ-

ation, an enhancement of either of them in the plant is a signal to defence [Zhao

et al. 2005], and all tree have been reported to activate PAL [De León et al.

2012; Kim et al. 2007; Kováčik et al. 2009; Oliver et al. 2009; Tierranegra-

Gracia et al. 2011; Zhao et al. 2005]. Chitosan oligosaccharide is used to activate

protection against plant-diseases [Yin et al. 2010] and it has been reported to

induce several plant defence responses [Kim et al. 2005]. As for the other three

elicitors, chitosan oligosaccharide has been reported to activate PAL [Ferri et al.

2009; Kim et al. 2005; Vander et al. 1998; Yafei et al. 2009; Yin et al.2012].

Moreover, it was investigated whether an applied stress situation could enhance

the production of secondary metabolites. E. purpurea has been reported to be a

salt sensitive plant [Niu and Rodriguez 2006] and exogenous application of salt

(NaCl) is therefore likely to stress the plant considerably.

In our investigation of effect of stress all four elicitors and the applied stress

(H2O2, methyl jasmonate, salicylic acid chitosan oligosaccharide and NaCl) were

exogenous applied in three different concentrations to six months old plants

grown in a greenhouse. The solutions were sprayed on the whole aboveground

part, there were eight replicates in each treatment and leaves and flowers were

harvested two days after treatment.

Chapter 5 - Induced stress

55

5.1 Hydrogen peroxide

Hydrogen peroxide (H2O2) is a vital signal compound in plants, which plays

many important roles in plant activities, including defence and protection

against abiotic and biotic stress [Garg and Manchanda 2009]. Hence, applied

stress has been reported to increase the content of H2O2 (rapeseed, tobacco, and

Greek oregano) [Li et al. 2009; Wang et al. 2008; Yin et al. 2012]. Moreover, it

has been reported that there is a close correlation between the content of sec-

ondary metabolites and accumulation of H2O2 [Wu et al. 2007]. However, be-

sides being a beneficial signal transduction molecule in plants, that easily can

diffuse into cells and activate important plant defences, H2O2 is a highly damag-

ing toxic molecule [Apel and Hirt 2004; Garg and Manchanda 2009]. Accumu-

lation of H2O2 in cells has been reported to increase damages to membrane li-

pids and epidermal cell nuclei and by that induce cell death [Vasil‟ev et al.

2009; Wang et al. 2008; Wu et al. 2007]. Therefore, by producing the right

amount of H2O2, the plants can defend itself by activating several defence

mechanisms including production of secondary metabolites. If the plant pro-

duces more H2O2 than the cell can use, it will result in accumulation of too high

concentrations of H2O2, which will result in a hypersensitive response and cell

death.

Exogenous application of H2O2 to the aerial parts of E. purpurea had no signifi-

cant effect on the content of caffeic acid derivatives in leaves measured after 2

days in our investigation [Paper IV]. Whereas, the content of caffeic acid deriva-

tives in flowers was increased with increased concentration of the applied H2O2

solution. The middle and high concentration resulted in a significantly higher

content of caffeic acid derivatives than the content in control plants (Figure 5.1).

With exogenous application of 100 ppm H2O2 solution, the concentration of caf-

taric acid, cichoric acid and total caffeic acid derivatives increased with 275, 100

and 150 %, respectively [Paper IV].

In flowers, exogenous application of H2O2 had no effect on the content of alka-

mides, whereas in leaves H2O2 treatment gave a significant decline in content of

alkamides (Figure 5.1) [Paper IV].

In summary, exogenous application of H2O2 resulted in a declining content of

alkamides in leaves and no effect on alkamides in flowers. Whereas, the same

concentrations of applied H2O2 solution resulted in no effect on the content of

caffeic acid in leaves, and an enhanced content of caffeic acid derivatives in

5.1 Hydrogen peroxide

56

flowers. This could indicate that the leaves are more sensitive to the exogenous

application of H2O2 than the flowers.

Figure 5.1 The effect of exogenous application of H2O2 on the content of alka-

mides in leaves and caffeic acid derivatives in flowers of E. purpurea. Bars (SE,

n=8) marked with * indicate that the content is significantly (p < 0.05) different

from control (0 ppm) [Paper IV].

Applied concentration of the eliticor H2O2 (ppm) to leaves

5C

on

trol

1 10

01

00

00

Alk

amid

e co

nte

nt

(mg/g

DW

)

0.0

0.5

1.0

1.5

2.0

6

Con

trol

1 10

01

00

00

13,14

Con

trol

1 10

01

00

00

Total alkamide

Con

trol

1 10

01

00

00

Applied concentration of the eliticor H2O2 (ppm) in flowers

Caftaric acid

0 1 10

01

00

00

Caf

feic

aci

d d

eriv

ativ

esco

nte

nt(

mg/g

DW

)

0

2

4

6

8

Cichoric acid

0 1 10

01

00

00

Total cafeic acid derivatives

0 1 10

01

00

00

* *

*

*

*

* *

**


57

5.2 Methyl jasmonate

Jasmonic acid and its volatile methylated form methyl jasmonate is known to be

a transducer of elicitor signals that stimulates the biosynthesis of secondary me-

tabolites in plants [Zhao et al. 2005]. Methyl jasmonate is a volatile, released

from stressed plants with the purpose of alerting healthy neighbour plants,

which thereafter may induce defence systems without even being exposed to

stress themselves [Mur et al. 1997]. Exogenous application of methyl jasmonate

might therefore be used to enhance the production of defence compounds such

as the bioactive compounds in Echinacea.

Jasmonic acid and methyl jasmonate have been reported to induce the activity

of PAL and to enhance the production of phenolic acids [De León et al. 2012;

Kim et al. 2007; Oliver et al. 2009; Tierranegra-Gracia et al. 2011]. Application

of jasmonic acid to irrigation water and spraying of methyl jasmonate directly

on the aerial parts have been reported to increase the content of alkamides in

roots of Echinacea species [Binns et al. 2001; Romero et al. 2009]. However,

nobody has investigated the effect on the aerial parts.

A weekly application of methyl jasmonate to lettuce sprayed directly on the

plant induced approximately 3 times higher content of phenolic acid than in

control plants [Tierranegra-Gracia et al. 2011], furthermore, spraying of methyl

jasmonate on lettuce once 2, 6 and 8 days before harvest likewise induced in-

creased content of phenolic acids [Kim et al. 2007]. We observed likewise a ten-

dency to an enhanced content of caffeic acid derivatives in leaves of E. purpurea

in our investigation. However, there was no significant effect on the content of

neither caffeic acid derivatives nor alkamides in neither leaves nor flowers by

spraying methyl jasmonate directly on the aerial parts of E. purpurea once 2

days before harvest [Paper IV]. The non-significant effect can have many expla-

nations. One explanation could of cause be, that aerial parts of E. purpurea are

not responding on application of methyl jasmonate. Another could be that E.

purpurea is responding, but the response as production of bioactive compounds

have either not taken place yet or is already over. An investigation in response

time from application to maximum response, would therefore be beneficial.

In summary, despite, methyl jasmonate has shown promising effects on the

content of alkamides and caffeic acid derivatives in roots of E. purpurea we did

5.2 Methyl jasmonate

58

not observe any significant effects in leaves or flowers with a single application

of methyl jasmonate two days before harvested.


59

5.3 Salicylic acid

Salicylic acid seems to have two conflicting functions regarding plant defence to

applied stress. Salicylic acid is known as an inducer of systemic acquired re-

sistance (SAR) in plant-pathogen interactions [Zhao et al. 2005], it has been

reported to induce PAL activity [De León et al. 2012; Kováčik et al. 2009], and

investigations have shown that continued application of salicylic acid to E. pur-

purea in the growth season for two years can enhance the content of cichoric

acid and caftaric acid in aerial parts [Kuzel et al. 2009]. However, other investi-

gations have revealed that salicylic acid can inhibit the jasmonic acid pathway

for secondary metabolite production [Zhao et al. 2005] and exogenous applica-

tion of salicylic acid can inhibit the accumulation of phenolic acids [Saltveit and

Choi 2007]. A single application of salicylic acid did not affect the concentration

of neither alkamides nor caffeic acid derivatives in flowers of six months old E.

purpurea in our investigation [Paper IV]. The same result was found in soy-

bean, where a single application of salicylic acid had no effect on the content of

most phenolic acids [Zhang et al. 2006]. However, all applied concentrations of

salicylic acid did significantly (p < 0.05) decrease the concentration of caffeic

acid derivatives and the decrease was approximately the same for all applied

concentrations in our investigation [Paper IV]. The plants treated with salicylic

acid contained approximately 40% less caffeic acid derivatives (caftaric acid,

cichoric acid and total caffeic acid derivatives) in leaves than control plant (Fig-

ure 5.2). This might imply that the applied concentrations of salicylic acid inhib-

ited the accumulation of caffeic acid derivatives in this plant part, that salicylic

acid inhibits accumulation of phenolic acids has also been reported for lettuce

[Saltveit and Choi 2007].

In summary, exogenous application of salicylic acid had no effect on the content

of alkamides in leaves and flowers of E. purpurea. The application did also not

effect the content of caffeic acid derivatives in flowers. However, in leaves, the

accumulation of caffeic acid derivatives was inhibited by salicylic acid and the

content was significant lower in the treated plants than in the control.

5.3 Salicylic acid

60

Figure 5.2 The effect of exogenous application of salicylic acid on the content of

caffeic acid derivatives in leaves of E. purpurea. Bars (SE, n=8) marked with * indi-

cate that the content is significantly (p < 0.05) different from control (0 ppm) [Pa-

per IV].

Caftaric acid

Cichoric acid

Total caffeicacid derivatives

Applied concentration of the salicylic acid (ppm) to leaves

Co

ntro

l0

.01

5 100

0

Co

ntro

l0

.01

5 100

0

Ca

ffei

c a

cid

der

iva

tiv

e co

nte

nt

(mg

/g D

W)

0

5

10

15

20

Co

ntro

l0

.01

5 100

0

* * *

* * * **

*


61

5.4 Chitosan oligosaccharides

Chitosan oligosaccharide is an elicitor used to induce protection against and

even treatment of plant-diseases [Yin et al. 2010]. Chitosan oligosaccharide is

produced by hydrolysis of chitin by degradation of the O-glycosidic linkage. Chi-

tosan is a polysaccharide found in cell walls of fungi and in the exoskeletons of

crustaceans, and it is currently obtained from the outer shell of crustaceans such

as crabs, krills and scrimps. Chitosan oligosaccharides is structurally composed

of several N-acetyl-D-glucosamine molecules linked with a β-1,4-glycosidic

bonds (Figure 5.3) [Bautista-Banos et al. 2006; Kim et al. 2005].

O

O

NH2OH

CH2OH

OOH

O

NH2OH

CH2OH

O

NH2OH

CH2OH

OH

n

Figure 5.3 The chemical structure of chitosan oligosaccharides.

Contrary to chitin and chitosan, chitosan oligosaccharide is water soluble due to

the shorter chain length and free amino groups, which makes it easier to use in a

wide range of industries, such as cosmetology, food, biotechnology, pharmacol-

ogy, medicine, and agriculture [Bautista-Banos et al. 2006]. Chitosan oligosac-

charides are known as useful elicitors to protect and treat plants from various

diseases [Yin et al. 2010] and it has been reported to induce several plant de-

fence responses [Kim et al. 2005] including enhancement of PAL [Kim et al.

2005; Ferri et al. 2009; Vander et al. 1998; Yafei et al. 2009; Yin et al. 2012]

and induce production of hydrogen peroxide [Li et al. 2009; Wang et al. 2008;

Yin et al. 2012]. Moreover, it has been reported that application of chitosan oli-

gosaccharides can enhance the content of phenolic acids (sweet basil and Greek

oregano) [Yin et al. 2012; Kim et al. 2005].

Exogenous application of chitosan oligosaccharide in our experiments had no

significant effect on the concentration of alkamides in leaves and flowers of E.

purpurea or on the concentration of caffeic acid derivatives in flowers. Whereas,

it had a significant (p < 0.05) declining effect on cichoric acid and total caffeic

acid derivatives in leaves of E. purpurea (Figure 5.4) [Paper IV]. The plants

5.4 Chitosan oligosaccharides

62

treated with a middle (100 ppm) concentration of chitosan oligosaccharides

contained approximately 25 % less cichoric acid and total caffeic acid deriva-

tives, and the plants treated with a low (1 ppm) concentration of chitosan oligo-

saccharide contained approximately 34 % less cichoric acid and total caffeic acid

derivatives than control plant.

A non-significant tendency to decrease could also be observed in the content of

alkamides in the leaves (Figure 5.4). This could indicate that the decreased con-

tent was not a consequence of an inhibited biosynthetic pathway. Investigations

reports that chitosan/oligochitosan can induce programmed cell death (pea

leaves and tobacco cells) [Vasil‟ev et al. 2009, Wang et al. 2008], and that might

explain the declining content of both alkamides and caffeic acid derivatives in

leaves of E. purpurea. As in the investigations with H2O2 and salicylic acid, the

leaves seem to be more sensitive than the flowers.

In summary, application of chitosan oligosaccharides has no significant effect

on the content of alkamides in leaves and flower, neither on the content of caf-

feic acid derivatives in flowers. There was however, a significant decrease in the

content of caffeic acid derivatives in leaves, and since the alkamides also showed

a non-significant decrease, it is believed that the applied concentration of chi-

tosan oligosaccharides might have caused cell dead, which also has been ob-

served in other plant species treated with chitosan oligosaccharides.


63

Figure 5.4 The effect of exogenous application of chitosan oligosaccharide on the

content of caffeic acid derivatives in leaves of E. purpurea. Bars (SE, n=8) marked

with * indicate that the content is significantly (p < 0.05) different from control (0

ppm) [Paper IV].

Applied concentration of the chitosan oligosaccharide (ppm) to leaves

5

Co

ntro

l1 10

010

00

0

alk

am

ide

con

ten

t (m

g/g

DW

)

0.0

0.5

1.0

1.5

2.0

6

Co

ntro

l1 10

010

00

013,14

Co

ntro

l1 10

010

00

0

Total alkamide

Co

ntro

l1 10

010

00

0

Caftaric acid

Co

ntro

l1 10

010

00

00

Ca

ffei

c a

cid

der

iva

tiv

e co

nte

nt

(mg

/g D

W)

0

4

8

12

16

20

Cichoric acid

Co

ntro

l1 10

010

00

00


Co

ntro

l1 10

010

00

00

Applied concentration of the chitosan oligosaccharide (ppm) to leaves

** *

5.5 Sodium chloride

64

5.5 Sodium chloride

Sodium (Na) is a very common metal and it is the sixth most abundant element

on earth. Together with chloride (Cl), it forms sodium chloride (NaCl) table salt.

Sodium is often not essential in plant except for C4 plants and studies have

shown that high salinity often is harmful to plants, and reduces growth and

yield [Shillo et al. 2002].

E. purpurea has been reported to be a salt sensitive plant, with a low visual

score and a not acceptable appearance even with a low irrigation of saline water

(2 ds/m ≈ 1200 ppm) [Niu and Rodriguez 2006]. Hence, in commercial medici-

nal plant production visual appearance is not the main purpose. The aim is to

produce a high concentration of the bioactive compounds. The caffeic acid de-

rivatives of interest in E. purpurea are all synthesized by the phenylpropanoid

partway, which is initiated by PAL activity [Winkel-Shirly 2001], and it has been

shown that the PAL activity is positively affected by salinity stress in E. angusti-

folia [Montanari et al. 2008]. A step from 290 ppm (5 mol/m3) to 2900 ppm

(50 mol/m3) increases the PAL activity significantly, hence the concentration of

the caffeic acid derivatives also increased. When E. angustifolia was grown as a

soilless culture and NaCl (50 mol/m3) was applied directly to the growth media,

the concentration of chlorogenic acid, cynarin and cichoric acid were signifi-

cantly enhanced in the roots, and the total dry weight was significantly lower

[Montanari et al. 2008]. However, the concentration of caffeic acid derivatives

detected in leaves (the only aboveground part measured) was not affected sig-

nificant. To our knowledge, it has not been investigated whether or not direct

application of a saline solution can affect the concentration of the bioactive

compounds in leaves and flowers of E. purpurea.

Applied saline stress had no effect on the content of caffeic acid derivatives or

the alkamides in leaves of E. purpurea in our investigation [Paper IV]. The con-

centration of caffeic acid derivatives in flowers increased relatively to the control

(0 ppm NaCl) when the NaCl solution of 1 and 100 ppm was applied. The con-

centration of caftaric acid, cichoric acid and the total caffeic acid derivatives was

significantly (p < 0.05) enhanced from 0.18 ± 0.03 to 0.62 ± 0.06 mg/g, from

2.26 ± 0.36 to 4.73 ± 0.28 mg/g, and from 2.21 ± 0.54 to 6.54 ± 0.61 mg/g, re-

spectively, by application of 100 ppm NaCl solution (Figure 5.5) [Paper IV]. Ap-

plication of the very high NaCl solution (10000 ppm NaCl) did not increase the

concentration and the level was comparable with the control.


65

Figure 5.5 The effect of application of NaCl on the content of bioactive com-

pounds in flowers of E. purpurea. Bars (SE, n=8) marked with * indicate that the

content is significantly (p < 0.05) different from control (0 ppm) [Paper IV].

5

Co

ntro

l1 10

010

00

0

Alk

am

ides

co

nte

nt

(mg

/g D

W)

0

1

2

3

4

5

6C

on

trol

1 100

100

00

13,14

Co

ntro

l1 10

010

00

0

Total alkamide

Co

ntro

l1 10

010

00

0

Caftaric acid

Co

ntro

l1 10

010

00

0

Ca

ffei

c a

cid

der

iva

tiv

e co

nte

nt

(mg

/g D

W)

0

1

2

3

4

5

6

7

Echinacoside

Co

ntro

l1 10

010

00

0

Cichoric acid

Co

ntro

l1 10

010

00

0


Co

ntro

l1 10

010

00

0

Applied concentration of the NaCl (ppm) to flowers

Applied concentration of the NaCl (ppm) to flowers

*

*

*

*

*

*

5.5 Sodium chloride

66

In flowers a very low application of salinity stress (1 ppm NaCl) resulted in a

significant increase in the concentration of alkamide 5, alkamide 13, 14 and to-

tal alkamides. The concentration of alkamide 5, alkamide 13, 14 and total alka-

mides was significantly (p < 0.05) enhanced from 0.07 ± 0.01 to 0.22 ± 0.06

mg/g, from 0.2 ± 0.05 to 0.71 ± 0.05 mg/g and from 1.28 ± 0.29 to 3.61 ± 0.79

mg/g by application of a low (1 ppm) NaCl solution. Application of higher con-

centrations of NaCl solutions had no significant effect on the concentrations of

alkamides in E. purpurea [Paper IV].

In summary, although application of NaCl had no effect on the content of bioac-

tive compounds in leaves of E. purpurea, a low concentration of NaCl showed a

significant effect on the content of both alkamides and caffeic acid derivatives in

flowers.


67

5.6 Discussion of induced stress

Alkamides are known to be affected be abiotic and biotic stress and to activate

important stress related genes, such as genes encoding for biosynthetic enzymes

for jasmonic acid production [Méndez-Bravo et al. 2011, Binns et al. 2001, Sa-

bra et al. 2012]. However, few applied stress investigations have been conducted

with the purpose of enhancing the content of bioactive compounds. It has been

reported that exogenous application of methyl jasmonate can enhance the al-

kamide content in roots of E. purpurea [Binns et al. 2001; Romero et al. 2009],

however, the aerial parts were not investigated. The experiments conducted

here showed, that exogenous application of elicitors had nearly no effect on the

biosynthesis of alkamides when the aerial parts were harvested two days after

treatment. Salicylic acid and methyl jasmonate had no effect, application of chi-

tosan oligosaccharides had a non-significant declining effect, which probably

was caused by cell death and not by interfering with the biosynthesis of alka-

mides. Only application of H2O2 had a significant and decreasing effect on the

content of alkamides in leaves. The overall result was that application of elici-

tors two days before harvest had no beneficial effect on the content of alka-

mides.

Caffeic acid derivatives are represented in many more plant species than alka-

mides. The biosynthesis of caffeic acid derivatives and the potential enhance-

ment of the content are therefore more investigated. All the elicitors examined

in this investigation are known to enhance the activity of PAL, the first enzyme

in the biosynthesis of caffeic acid derivatives, they do therefore all have the po-

tential to enhance the content of these compounds [De León et al. 2012; Ferri et

al. 2009; Kim et al. 2005; Kim et al. 2007; Kováčik et al. 2009; Oliver et al.

2009; Tierranegra-Gracia et al. 2011; Vander et al. 1998; Yafei et al. 2009; Yin

et al. 2012; Zhao et al. 2005]. Even though exogenous application of methyl

jasmonate has been reported to enhance the content of phenolic acids in lettuce

[Kim et al. 2007; Tierranegra-Gracia et al. 2011], neither application of methyl

jasmonate, salicylic acid or chitosan oligosaccharides had an effect on the con-

tent of caffeic acid derivatives in the flowers of E. purpurea. However, the exog-

enous application of H2O2 had a significant (p < 0.05) increasing effect on the

content of caffeic acid derivatives in flowers with increasing concentration of the

applied H2O2. In leaves on the other side, application of methyl jasmonate and

H2O2 had no significant effect. Whereas, application of both salicylic acid and

chitosan oligosaccharides had a declining effect on the content of caffeic acid

5.6 Discussion of induced stress

68

derivatives in leaves. The negative effect of application of salicylic acid has been

observed before in lettuce [Saltveit and Choi 2007] and is probably caused by

inhibition of the jasmonic acid partway for secondary metabolite production

[Zhao et al. 2005]. Whereas, the negative effect of applied chitosan oligosaccha-

rides might be the result of cell death more than inhibition of the caffeic acid

derivatives accumulation [Vasil‟ev et al. 2009; Wang et al. 2008].

Application of a direct stress situation in form of a saline shock had contrary to

application of elicitors a clear effect. The content of both alkamides and caffeic

acid derivatives was enhanced significantly (p < 0.05) in flowers of E. purpurea

and the low applied concentrations had the highest effect. The enhancement in

content of alkamides by application of NaCl solutions confirms that alkamides

probably are involved in the biochemical defence of the plant in response to abi-

otic stress, even though exogenous application of elicitors had no effect.

Exogenous applications of elicitors on aboveground parts of E. purpurea, two

days before harvest had only minor effect on the content of bioactive com-

pounds in our investigation [Paper IV]. The missing effect can have several ex-

planations, one explanation could of cause be that aerial parts of E. purpurea

not is affected by exogenous application of elicitors. Another could be that E.

purpurea is responding, but the response as production of bioactive compounds

have either not taken place yet or is already over. An investigation in response

from application to maximum response would therefore be beneficial.

The leaf seems to be more sensitive than the flowers, and all significant ob-

served effects on leaves were a declining content of the bioactive compounds.

Only application of H2O2 showed a positive effect and only on the content of

caffeic acid derivatives in flowers.

Application of direct stress as a NaCl solution had on the other hand a positive

effect on the content of bioactive compounds, both alkamides and caffeic acid

derivatives, in the flowers of E. purpurea. The enhancement of alkamides and

caffeic acid derivatives confirms that they are involved in the biochemical de-

fense or environment adaption of the plants as a response to abiotic stress, even

though exogenous application of elicitors had minor effects in our investigation.

69

Chapter 6

Conclusion

“The hypothesis was that cultivation techniques and induced

stress during cultivation could lead to a higher content and/or change the

profile of bioactive compounds in Echinacea plants and hence improve

the quality of raw plant material for medicinal preparations”

This thesis and the papers included make it clear that cultivation techniques can

have a major impact on the bioactive compounds in Echinacea. The content of

caffeic acid derivatives and alkamides varies between the three Echinacea spe-

cies of medicinal interest, between plant parts (roots vs. aerial parts), and even

from one seed population to another grown under the same conditions. The

choice of Echinacea species, plant part used and seed population has therefore a

major influence on the quality of raw Echinacea material for the medicinal plant

preparations of Echinacea. To obtain a high yield of bioactive compounds, aerial

parts of Danish grown E. purpurea should be harvested in the late flowering

stage the first three to four years of cultivation. The highest yield of bioactive

compounds in roots was found in the third to fourth growth year. The most ben-

eficial harvest time during a season varied with the compound of interest, how-

ever, the highest yield of caffeic acid derivatives and half of the alkamides was

achieved when roots were harvested in spring. The content of alkamides and

caffeic acid derivatives in roots was negatively influenced by prior harvest of

aerial parts in the same season. If aerial parts must be harvested the same year

as the roots, the roots should be harvested one week after harvest of aerial parts

or alternatively, aerial parts and roots should be harvested simultaneous. It is

not recommended to harvest Danish grown E. purpurea in fall as generally rec-

ommended [Li 1998; Marquard and Kroth 2001]. High application of nitrogen

fertilization resulted in a higher biomass yield and a significantly higher concen-

70

tration of alkamides in aerial parts, whereas, the concentration of caffeic acid

derivatives was unaffected.

Induced stress as direct applied stress in the form of a saline shock two days

before harvest did significantly enhance the content of secondary metabolites in

flowers on E. purpurea. The content of alkamides increased significantly (p <

0.05) compared to the control by application of 1 ppm NaCl solution and the

content of caffeic acid derivatives increased significantly (p < 0.05) by applica-

tion of a 100 ppm NaCl solution. Exogenous application of different elicitors to

E. purpurea with the purpose of mimicking a stress situation in the plant did

generally not have a positive effect on the content of alkamides and caffeic acid

derivatives. The exception from this pattern was H2O2, which at 100 ppm and

10000 ppm exogenous applications enhanced the content of caffeic acid deriva-

tives significantly (p < 0.05) in flowers of E. purpurea. Induced stress as a way

to enhance the quality of Echinacea plant material was possible, however only

following an applied stress with NaCl or with exogenous application of H2O2.

71

Chapter 7

Perspective

The review presented in this thesis as well as paper I - III indicates that there is

a large potential for improvement of the quality of raw material of Echinacea by

optimising cultivation techniques. We showed that plant parts; plant age; har-

vest strategies and nitrogen fertilisation, all could be optimised and they result

in higher quality plant material. The guidelines will be of benefits for farmers

and producers of medicinal preparations. There are, however, still many other

aspects in cultivation of Echinacea that could be optimised. Other plant nutri-

ents, such as phosphor, potassium and sulphur, would be worth investigating.

The influence of different soil types, the pH of soil and of course, climate should

be investigated, especially since the quality regarding caffeic acid derivatives

differs so greatly in results of different investigations around the world.

The induced applied stress on E. purpurea evaluated in this study, indicated

that many exogenous applied elicitors neither affect the content of alkamides

nor caffeic acid derivatives in aerial parts. However, prior investigations have

reported that elicitors such as salicylic acid and methyl jasmonate could affect

the content of both alkamides and caffeic acid derivatives in roots [Binns et al.

2001, Kuzel et al. 2009] and a more comprehensive investigation in this area

could be of great interest. Application of a stress situation in the form of a saline

shock had a significant effect on the content of both groups of bioactive com-

pounds. This indicates that both caffeic acid derivatives and alkamides are in-

deed part of the plants defence mechanism, and this makes applied stress worth

investigating further.

Many parameters in the effects of applied stress on the content of bioactive

compounds in E. purpurea are still unknown. It is known that the activities of

enzymes are highest only a few hours after application of elicitors [Oliver 2009],

however, biosynthesis and production of bioactive compounds takes time. The

72

question is therefore when is it most beneficial to harvest the treated plants, the

same day, the day after or a week after? In addition, should external stresses be

applied in short intervals to maintain a continued up-regulation of synthesis of

these compounds? As a result of a preliminary investigation, we harvested the

treated plants two days after elicitor treatment; however, a more comprehensive

investigation of harvest timing for the different compounds should be undertak-

en. Several of the applied elicitors investigated in this study have a damaging

effect on the plant when applied in high concentrations, like salicylic acid and

H2O2. A potential stress inducer must therefore be investigated with many con-

centrations in order to make a dose/response curve and by that identify the

most beneficial concentration for treatment.

Investigations in optimisation of the quality of Echinacea species would be

more accessible, if we knew more about the functions of the bioactive compound

in the plants. Knowledge such as where and how the bioactive compounds pro-

duced in the plants, are the bioactive compounds transported in the plant or

produced locally, and why do the plants produce these compounds. These re-

search areas still lack a lot of knowledge, and in-depth investigations in future

research would be helpful.

The review presented in this thesis as well as the four papers reveals that the

quality of Echinacea plant material could be improved with respect to content of

bioactive compounds. The subject is however not at all fully explored, and sev-

eral other cultivation techniques could be studied. The very great difference in

content of caffeic acid derivatives from on investigation to another is an ambi-

guity worth investigating. Application of induced stress could have a great po-

tential, however, this area needs more attention before it is fully understood.

73

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