The extraction, stability, metabolism and bioactivity of ...

The extraction, stability, metabolism and bioactivity of the alkylamides in Echinacea spp.

Number of Volumes: 1

Submitted by Kevin Spelman, to the University of Exeter as a dissertation for

the degree of Doctor of Philosophy in Biology, October 2009.

This dissertation is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement.

I certify that all material in this dissertation which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other

University.

……………………………………………………………………………

Acknowledgements

The following people and institutions were crucially in the successful completion of this degree.

Joshua Beatty, University of North Carolina, Greensboro

Lena Bezman, Johns Hopkins University

Phillip Bowen, University of North Carolina, Greensboro

Nadja Cech , University of North Carolina, Greensboro

Jim and Peggy Duke, The Green Farmacy

Robin Dunn, Phoenix, AZ

Julia S. Hartenstein, National Institutes on Aging, National Institutes of Health

Billy Martin, Virginal Commonwealth University

Yash Patel, University of North Carolina, Greensboro

Shahnaz Qadri, University of North Carolina, Greensboro

Jason Redick, University of North Carolina, Greensboro

Cheryl Rockwell , University of Kansas Medical Center

Masa Sasagawa, Bastyr University

Nicholas Smirnoff, Exeter University

James Snow, Tai Sophia Institute

Tai Sophia Institute

The Tai Sophia Institute Library, especially Ginny Rodes, Stuart Rodes and Holli Richey

Ethan Will Taylor, University of North Carolina, Greensboro

Katrina Tutor, Bastyr University of Health Sciences

Chuck Van deZande, Greensboro, NC

Irving Wainer, National Institutes on Aging, National Institutes of Health

Cynthia Wenner, Bastyr University of Health Sciences

But especially Patrick Spelman, who gave me the courage to question dogma.

1

Abstract

The fatty acid amides, a structurally diverse endogenous congener of molecules active in

cell signaling, may prove to have diverse activity due to their interface with a number of

receptor systems, including, but not limited to cannabinoid receptor 2 (CB2) and PPARγ.

Select extracts of Echinacea spp. contain the fatty acid amides known as alkylamides.

These extracts were a previously popular remedy relied on by U.S. physicians, one of the

top sellers in the natural products industry and are currently a frequently physician

prescribed remedy in Germany. In the series of experiments contained within, Galenic

ethanolic extracts of Echinacea spp. root were used for the quantification, identification,

degradation and bioactivity studies. On extraction, depending on the ratio of plant to

solvent and fresh or dry, the data indicate that there is variability in the alkylamide classes

extracted. For example the acetylene alkylamides appear to extract under different

concentrations, as well as degrade faster than the olefinic alkylamides. In addition, the

alkylamides are found to degrade significantly in both cut/sift and powdered forms of

echinacea root. Human liver microsome oxidation of the major alkylamide dodeca-

2E,4E,8Z,10Z-tetraenoic acid isobutylamide generate hydroxylated, caboxylated and

epoxidized metabolites. The carboxylated metabolite has, thus far, shown different

immune activity than the native tetraene isobutylamide. Bioactivity studies, based on

cytokine modulation of the alkylamides have been assumed to be due to a classic CB2

response. However, the results of experiments contained herein suggest that IL-2 inhibition

by the alkylamide undeca-2E-ene-8,10-diynoic acid isobutylamide, which does not bind to

CB2, is due to PPARγ activation. These data, combined with data generated by other

groups, suggest that the alkylamides of Echinacea spp. are polyvalent in effect, in that they

modulate multiple biochemical pathways.

2

List of Contents

Chapter 1 Research on herbal medicine and review

of the alkylamides of Echinacea spp. ……………… ……………….

Introduction 7

An Example of a Medicinal Plant, Echinacea spp. 13

The phenylpropanoids 17

The alkylamides 21

Is PPARγ a target of alkylamides? 29

PPARγ is linked to immune function 31

PPARγ binds fatty acid derivatives 32

PPARγ is linked to IL-2 and other cytokines 32

Structural similarities between alkylamides and PPAR-

γ agonist 34

Thesis plan 35

Characterization, extraction and degradation of the

alkylamides in E. purpurea 35

Metabolism of the alkylamide isomers, dodeca-

2,4,8,10-tetraenoic acid isobutylamide 36

Bioactivity of the alkylamides 36

Hypothesis 36

Reference list 36

Chapter 2 Alkylamide characterization and yield in

fresh versus dry ethanolic extracts of E. purpurea ……………… ……………….

Introduction 50

Methods 56

Reagents 56

Plant Material 56

Plant Extractions 56

Sample Preparation 58

Determination of yield of dissolved solids 59

Preparation of alkylamide standard solutions 59

HPLC-ESI-MS 60

Statistical analysis 61

Results 61

Identification of alkylamides 62

Method validation 73 Comparison of alkylamide yield in various E. purpurea

extracts 75

Comparision of yield of dissolved solides in various E.

purpurea extracts 78

Extraction of the tetraene isomers as a function of

time 81

Discussion 84

Summary 86

Reference list 88

3

List of Contents, Continued

Chapter 3 An HPLC-ESI-MS evaluation of the

stability of alkylamides ……………… ……………….

Introduction 96

Methods 100

Preparation of Echinacea purpurea extracts 100

Analysis of Extracts 101


Results 103

Determination of alkylamide identity 103

Quantitative determination and comparison of

alkylamide content in aged ethanolic extracts 103

Comparision of alkylamide content in one year old

cut/sift, powder and ethanolic extracts 105

Discussion 105

Summary 112

Reference list 113

Chapter 4 Metabolism of the alkylamides in

Echinacea spp. by HLM & CYP450 ……………… ……………….

Introduction 117

CYP1A2 120

CYP2C9 121

CYP2C19 121

CYP2D6 121

CYP2E1 122

CYP3A4 122

Organic anion-transporting polypeptide 124

P-glycoprotein 124

Methods 125

Extract preparation 125

Liver microsome & CYP450 assays 125

HPLC–ESI-MS 126


Results 128

Metabolites of dodecatetraenoic acid isobutylamide 128

Metabolite formation as a function of time 131

CYP450 1A1 is responsible for the formation of

dodecatetraene-7-hydroxyl acid isobutylamide 134

Discussion 136

Summary 144

Reference list 144

4

List of Contents, Continued

Chapter 5 The activation of PPARγ by undeca-2E-

ene-8,10-diynoic acid isobutylamide ……………… ……………….

Introduction 152

Methods 156

Reagents 156

Immunodetection of PPARγ in Jurkat cells by Western

blotting 156

Mitogenic stimulation 157

Jurkat T cell culture and IL-2 ELISA 157

Cell survival by XTT assay 158

Reverse transcription PCR analysis 159

Fibroblast cell culture and differentiation 160


Results 161

PHA titration to determine submaximal IL-2

production 161

Determining optimal IL-2 production 163

Presence of PPARγ in Jurkat cells 164

A PPARγ selective antagonist attenuates the IL-2

inhibition of UDA 165

Cell survival of Jurkats after treatment does not

account for IL-2 modulation 167

Effect of PPARγ activation by UDA on transcriptional

activation of c-fos and c-jun 167

Treatment of 3T3-L1 preadipocytes with UDA as an

indicator of PPARγ involvement 170

Discussion 171

Summary 179

Reference list 180

Chapter 6 Discussion ……………… ……………….

Extraction 188

Metabolism 189

Degradation 190

Bioactivity: alkylamides as PPARγ activators 191

Are there previous investigations suggesting PPARγ

activity 193

Other possible targets of the alkylamides 197

The C/EBP protein 197

The RXR protein 198

The Vanilloid receptors 199

Synergic activity? 199

Future work 201

Reference list 203

5

List of Tables and Figures

Figure 1.1 Structure of common alkylamides in various Echinacea spp. 22 Table 2.1. Solvent and root ratios for the extracts 57 Table 2.2A. Alkylamides from E. purpurea 63 Table 2.2B. Alkylamides from E. purpurea 64 Figure 2.1. A & B Characteristic selected ion chromatograms obtained by

liquid chromatography-mass spectrometry analysis of an E. purpurea root

extract (1:5) 65

Table 2.3. MS/MS Analysis of Echinacea alkylamides 68 Figure 2.2. MS/MS Spectrum of Isomeric Alkylamides 49 Table 2.4. Calibration Parameters and Repeatability for Quantification of

Alkylamides 74

Table 2.5. Quantification of Alkylamides K, L & M; O; and J in Ethanolic

Extracts of E. purpurea root 76

Figure 2.3. A,B,C. Comparison of alkylamide concentrations in fresh 1:2, dry

1:11 and dry 1:5 E. purpurea root ethanolic extracts 79

Figure 2.4. Yield of dissolved solids in ethanolic extracts of Echinacea

purpurea roots 82

Figure 2.5. Relative concentration of dodecatetraenoic acid isobutylamides

(alkylamides K, L and M) in an E. purpurea root extraction over time 83

Figure 3.1. Quantified Alkylamides in E. purpurea preparations 102 Figure 3.2. Comparison of alkylamide stability in E. purpurea root ethanolic

extracts over one year’s time 104

Figure 3.3. Percentage of alkylamides remaining after one year’s storage in

ethanolic extract, cut/sift and powdered E. purpurea root. 106

Equation 4.1. The catalytic cycle of cytochrome P450 119 Figure 4.1 Selected ion chromatograms of the Z-tetraene and its metabolites 129 Table 4.1. Parent compound & metabolite structural assignment after

oxidation by human liver microsomes 130

Figure 4.2. Time dependent metabolism of the Z-tetraene. 132 Figure 4.3. Selected ion chromatograms of the Z-tetraene and metabolites as a

function of time 133

Figure 4.4 Metabolism of dodecatetraenoic acid isobutylamide by CYP450

1A1 134

Figure 4.4 Metabolism of dodecatetraenoic acid isobutylamide by CYP450

1A1 135

Figure 5.1. Undeca-2E-ene-8,10-diynoic acid isobutylamide (UDA) 160 Figure 5.2. PMA/PHA optimization for IL-2 response 161 Figure 5.3. IL-2 response between 3 to 18 hours 162 Figure 5.4. IL-2 response between 18 and 48 hours 163

Figure 5.5. Immunodetection of PPAR in Jurkat cells by Western blotting 164 Figure 5.6. PPAR-γ antagonist T0070907 attenuated the UDA induced

inhibition of IL-2 secretion by suboptimally stimulated Jurkat E6.1 cells. 165

Figure 5.7. Cell survival assay of Jurkat E6.1 cells treated with UDA and

T0070907 166

Figure 5.8. Relative quantitation in IL-2 protein and transcription factors c-fos

and c-jun in response to UDA 168

Figure 5.9. Dose-dependent response of 3T3-L1 cell differentiation by UDA 170 Table 6.1. Bioactive Fatty Acid Amides 191 Table 6.2. Ligands of CB2 and PPARγ 194

6

List of accompanying manuscripts

1. Cech NB, Tutor K, Doty BA, Spelman K, Sasagawa M, Raner GM, Wenner CA.

2006. Liver enzyme-mediated oxidation of Echinacea purpurea alkylamides:

Production of novel metabolites and changes in immunomodulatory activity.

Planta Med. 2006;72(14):1372-7.

2. Freeman C, Spelman K. 2008. A critical evaluation of drug interactions with

Echinacea spp. Mol Nutr Food Res 52:789-98.

3. Spelman K, Wetschler M. Cech NB. Comparison of alkylamide yield in ethanolic

extracts prepared from fresh versus dry Echinacea purpurea utilizing HPLC-ESI-

MS. J Pharm Biomed Anal 2009:49(5);1141-9.

4. Spelman K, Iiams-Hauser K, Cech NB, Taylor EW, Smirnoff N, Wenner CA. Role

for PPARγ in IL-2 inhibition in T cells by Echinacea derived undeca-2E-ene-8,10-

diynoic acid isobutylamide. Int Immunopharmacol 2009. Epub ahead of print.

Author's declaration: The contribution of the author to manuscript 1 is limited to the

discovery of the carboxylated metabolite which is illustrated in Figure 2 and utilized in

Figure 3.

Chapter 1 Introduction to the Literature

7

Chapter 1

Research on herbal medicine and review of the alkylamides of Echinacea spp.

Introduction

A History of Research on Medicinal Drugs

The hunt for active constituents in plants— the so called ―silver bullets‖ that

characterize modern –single chemical entity drugs—is just over two centuries old. With

multidrug resistance becoming a leading obstacle to curing infectious disease, resistance of

chemotherapy in oncology, and adverse events in medicine becoming a leading killer of

U.S. population, and the current research suggesting that multiple constituents may have

even greater efficacy than single isolates (Borisy et al., 2003), it is foundational for

medicinal plant researchers to understand single why isolated plant constituents were

chosen over complex mixtures of phytochemicals as the preferred therapeutic choice.

As chemistry was maturing in the 19th

century, the developing field of analytical

chemistry, with its ability to isolate and purify the active ingredients of plants, was

foundational in early drug research and thus, medicine (Drews, 2000). Alkaloids, as a

widely diverse group of constituents, were some of the first principals isolated from plants.

The historical record suggests Derosne was the first to extract plant alkali; he extracted a

mixture of two alkaloids from opium in 1803 (Bruneton, 1995). During the same time

period Sertürner was purifying constituents from opium and in 1817 succeeded in isolating

morphine. (Bruneton, 1995; Huxtable and Schwarz, 2001) In the next five years, Pelletier


8

and Caventou, two French pharmacist-chemists at the Ecole de Pharmacie of Paris,

isolated a number of noticeably active compounds including; caffeine, emetine from ipecac

(Cephaelis ipecacuanha, Rubiaceae) and strychnine from Strychnos nux-vomica

(Loganiaceae) (Bruneton, 1995). One of these alkaloids, the anti-malarial quinine, was to

be a drug that would change the political and economic landscape of Africa and other

tropical areas (Bruneton, 1995; Jarcho and Torti, 1993; Yarnell and Abascal, 2004).

In the same period, Francois Magendie, known as the father of experimental

pharmacology and the teacher of the renowned French physiologist Claude Bernard, began

experimenting with Javanese arrow-poisons and eventually found that the active

constituent was strychinine, elucidating much of its biochemical activity along the way.

Some years later, working on the mechanisms of vomiting and ipecac, Magendie and

Pelletier demonstrated that emetine was the primary active substance. Rather intriguingly,

they were unable to isolate a pure substance. It was later shown that that their emetine was

a mixture of at least three alkaloids (Singer and Underwood, 1962).

Nevertheless, perhaps due to Sertürner establishing the belief that isolation of a

particular constituent could capture the therapeutic activity of a medicinal plant (Huxtable

and Schwarz, 2001), Magendie took pharmacology further into a reductionist direction by

promoting the use of isolated principles from plants. In 1821 he published a pocket

formulary for practicing physicians entitled (translated from the French) ―Formulary for

the preparation and use of several new drugs, such as nux vomica, morphine, prussic acid,

strychinine, veratrine, the chinchona alkaloids, emetine, iodine‖(Singer and Underwood,

1962). This work was essentially a guide to using isolated alkaloids in clinical medicine.

The silver bullets of modern pharmacology had arrived.


9

Half a century later the physician Thomas MacLagan, successfully used salicylic

acid, a metabolite of salicin, in a clinical trial on patients with rheumatism (Sneader, 2000).

By the late 19th

century, clinical trials such as MacLagan‘s and the groundbreaking

experiments in physiology of Claude Bernard and François Magendie had fertilized the

medical sciences to the point that pharmacology, which had been seen as having limited

relevance to the medical sciences, was elevated to a respectable ranking among the medical

disciplines. During this period, Oswald Schmiedeberg and his students at the University of

Strasbourg laid many of the intellectual and experimental foundations of pharmacology

(Drews, 2000; Koch-Weser and Schechter, 1978). Friedrich Bayer (Sneader, 2000) and

Charles Frederic Gerhardt (Lafont, 1996) produced acetylsalicylic acid and laid the

foundation for the synthetic processing practices of what became the pharmaceutical

industry.

As a result of the combination of analytical chemistry‘s reductionistic influence,

Schmiedeberg‘s group, and Sertürner‘s and Magendie‘s work, one of the foundations of

pharmacology came to be the isolation and purification of constituents from plant

medicines that were already being used in various non-purified forms (Drews, 2000; Koch-

Weser and Schechter, 1978). About half of the United States Pharmacopoeia (USP) at the

beginning of the 20th

century were still ―impure‖ multi-constituent plant medicines

(Vickers, 2002). Many of the 19th

century and early 20th

century medical journals

documented case studies substantiating the effectiveness of plant medicines in their crude

form.

Early efforts in the development of pharmacological agents were based on the

observations of living systems. In other words the outcomes were observed and

conclusions were developed from observations. This strategy provided us with such drugs


10

as aspirin, as well as the Chinese and Ayurvedic pharmacopoeias, both of which are

currently being mined for therapeutics to life-threatening diseases. As our understanding of

pathogenesis advanced, research strategies moved to in vivo animal models, followed by in

vitro cellular models, producing such drugs as penicillin and cisplatin (Lansbury, 2004).

However, with increasing technology came sharper focus on single etiological agents;

currently most drug discovery is confined to single protein targets. Medicinal chemists

insist on single target-based screens because the alternative, studying multiple interactions,

was, until recently, not technologically possible and was considered too complex

(Nussbaum and Ellis, 2003).

Thus the medical sciences have drastically changed their focus over the last 200

years. Not only have we moved from complex molecular mixtures to single molecules, we

have also shifted our focus from disease models of decreasing complexity, from the living

to the inanimate (Lansbury, 2004). Lansbury (2004) suggest that despite the evidence that

the leap from cellular models to a single protein is an uncertain strategy, it is considered

intellectually superior. A similar attitude has been adopted for single molecules versus

multi-constituent extracts which are often called ―dirty‖ denoting a lack of selectivity.

Williamson points out that when complex extracts were simplified to one molecule

scientists did not realize until much later that the specific mode of activity and the adverse

side effects were altered, sometimes producing more serious adverse effects.

Critics of medicinal plants argue that the low concentration of any one

phytochemical in a plant creates a mixture of compounds too dilute to have an effect.

However, Rajapakse et al.(2002) have demonstrated that very low concentrations of any

one chemical will contribute to a chemical mixture‘s activity, even if that chemical does

not show activity when isolated. This particularly challenges the research on herbal


11

medicine that has suggested that some herbal medicines are void of activity because of

failure to find a single active constituent. This also challenges the research on medicinal

plants that equate activity of a plant with a single isolated chemical contained within a

plant or plant part.

The general conclusion drawn from a century of research on isolating actives from

medicinal plants, is that the occurrence of only one bioactive constituent is rare. Rather,

medicinal plants commonly contain numerous active constituents (Bruneton, 1995; Singer

and Underwood, 1962) and as a result may modulate numerous cellular pathways (Martin,

2006). Attempts as early as 1928 demonstrated that the pharmacological activity of

combinations of constituents often had different activity that could not be predicted by the

activity of the isolated constituents (Borisy et al., 2003). In other words, the efficacy of

medicinal plants often cannot be reduced to a single constituent. More recently, Borisy et

al. (2003) in screening combinations of active compounds found that unexpected activity

arose from chemical combinations that could not have been predicted by the constituent

properties. Thus much of the research on medicinal plants that seeks a pharmaceutical gem

from a jungle of phytochemistry is incomplete as it neglects the possibility of synergic,

additive or antagonist activity of multi-constituent remedies (Cech, 2003). Yet

pharmacological modeling has used isolation as a fundamental tenant of inducing

physiological change in humans.

The chemical complexity of matrices of constituents is not without disadvantages.

Medicinal plants/multi-component remedies represent a particular challenge to understand

in regard to molecular modes of activity. That this is a particular complex issue is

demonstrated by the attempts to use information theory to cope with the complexity of the

multi-component nature of herbal remedies (Gong et al., 2003; Liang et al., 2004). This


12

issue still remains to be solved and will continue to delay meaningful pharmacological

research on medicinal plants. Until modes of activity are clearly elucidated, however,

outcome studies can still provide meaningful data.

Unfortunately, modern pharmaceutics severed the connection between plants, foods

and medicines during their 20th

century journey in search for disease curing silver bullets.

At the same time, the abandonment of searching natural products for drug leads is

accompanied by an inexorable rise in the cost of generating new drugs. Such methods as

high-throughput screening, have been reported to have not had a significant impact on the

derivation of new drugs (Proudfoot, 2002). Random search through combinatorial libraries,

according to one estimate, leads to hits at a rate of 1:10,000 (Jansen, 2003). Unfortunately,

combinatorial libraries are not generally based on biologically relevant properties but on

the basis of chemical accessibility and maximum achievable size (Martin and Critchlow,

1999). Conversely, combinatorial libraries based on natural products, many compounds

that, by default, have been selected for biological activity through the high-throughput

screening of the evolutionary process, increase the likelihood of finding active compounds

(Brohm et al., 2002). Similarly, ethnobotanical leads, which can be viewed as rudimentary

small-scale, but long-standing, clinical trials, have yielded positive activity in the order of

2 to 5 times higher than random screening (Lewis and Elvin-Lewis, 1994). Butler (2004)

rhetorically asks, is it from lack of natural products screening or from an increase in the

difficulty of drug discovery that is the key issue in the hunt for silver bullets? If the

problem lies in the rejection of natural products screening, a likely part of the issue, then an

obvious source of new drug discovery lies in natural products. It seems quite likely that the

increasing cost of generating new-to-nature molecules will generate a gap in medical care


13

that will reconnect plants and human health at a new level of technological sophistication

(Ray et al., 2006).

Comprehensive evaluations of medicinal plants are urgently needed before more

plant species are lost and knowledge of specific traditional medicines becomes

irretrievable. While the study of a medicinal plant and its many components -- some of

them unidentified or having unknown properties -- is theoretically, economically and

technically challenging, it should not be abandoned for the sake of investigative

expediency. Research into the multi-component nature of medicinal plant remedies offers a

segue way into more complex therapeutics. Thus, the issue of using herbal remedies to

alleviate human suffering is not one of merely assessing efficacy and safety (Vickers,

2002; Willcox et al., 2001), but a matter of the medical community‘s struggle to

understand a pharmacological paradigm that embraces the complexity of bio-molecular

networks interfacing with multi component remedies.

Jansen (2003) points out that pharmacological research based on multiple

perturbations of biological networks is likely to be more informative, and possibly

economical, than those in which biological systems are studied by varying one

environmental factor or gene at a time. Csermely et al. (2005) proposes that drug-design

directed at multiple targets will likely result in the development of more efficient

therapeutics than currently offered by single-target agents. These perspectives coupled

with the latest pharmacological models based on systems biology, build a paradigm in

which multicomponent remedies, such as medicinal plants, are recognized as sophisticated

pharmacological agents. Additionally these multi-component remedies may offer improved

efficacy and safety over isolated silver bullets (Ágoston et al., 2005; Wagner and Ulrich-


14

Merzenich, 2009). Yet current Food and Drug Administration regulations, limited by

economic politics, have generated obstacles to the development of multicomponent drugs.

An Example of a Medicinal Plant, Echinacea spp.

The current body of scientific literature on echinacea can be confusing due to the

multiple species in use – namely E. purpurea (L.) Moench, E. pallida (Nutt.) Nutt. and E.

angustifolia DC. (Asteraceae) - which have phytochemical similarities, but also have

notable differences, particularly around the identity and concentration of key constituents

(Bauer, 1996; Chen et al., 2005; Hudson et al., 2005). Even though 80% of the echinacea

products sold to consumers are made from E. purpurea (Li, 1998), all three 1Echinacea

species are often used interchangeably for the treatment of cold, flu, respiratory infection,

and inflammation (Bauer and Foster, 1991). Multiple species, plant parts, and preparations

are also used, each of which may have a different constituent profile (Williamson, 2006).

―Echinacea‖ may refer to the roots, aerial parts, whole plant or a combination of the above;

echinacea products can be made from fresh or dried plant parts, and may be prepared by

juicing, alcohol extraction, infusion, decoction, or consumed as tablets or capsules (Mills

and Bone, 2000). Most preparations are derived from the aerial parts of E. purpurea and

underground parts of E. purpurea, E. angustifolia, or E. pallida. Preparations from

Echinacea spp. plants have been used for more than a century by physicians for treatment

of a variety of infections (Couch and Giltner, 1920) and considerably longer if use by

indigenous people is considered (Moerman, 1998).

1 Echinacea is used as both a common name and a Genus. Therefore both non-italicized and italicized

nomenclature is used throughout this dissertation.


15

Echinacea is also one of the most frequently used medicinal plants in clinical

settings. For example, of the most prescribed drugs in Germany, echinacea preparations

have been in the top 200 for many years (Der Marderosian, 1991). There are more than 800

Echinacea products on the market (Sun et al., 2002). Despite the reluctance to use

echinacea products clinically in the U.S., German physicians write over 3 million

prescriptions annually for echinacea products for the treatment of upper respiratory tract

infections (Keller, 1991; Tyler, 1994). Research suggests that Echinacea species have

immunological effects, as well as anti-viral, antibacterial, antifungal, insecticidal and anti-

inflammatory properties (Kim et al., 2000).

Physiological effects of Echinacea include immunomodulatory and other activities,

such as stimulation of phagocytosis, induction of cytokines from macrophages and

antioxidant activity (Bauer, 1999a; Hashemi et al., 2008; Hudec et al., 2007). The

immunostimulating properties of extracts from Echinacea appear to be well established

(Burger et al., 1997; Maass et al., 2005; Melchart et al., 1995; Morazzoni et al., 2005; See

et al., 1997; Sun et al., 1999). Studies have reported that echinacea extracts have the ability

to activate human phagocytic function both in vitro and in vivo (Burger et al., 1997;

Melchart et al., 1995; Morazzoni et al., 2005; Percival, 2000; Roesler et al., 1991).

Although some studies have failed to find immunostimulatory activity in vivo (Schwarz et

al., 2002), other researchers have noted up-regulation of immune function in ex vivo

models in human immunodeficiency disorders (See et al., 1997).

A recent clinical trial by Turner et al. (2005) demonstrated no effect of echinacea

on the reduction of symptoms or duration of a cold. Conversely, recent Cochrane reviews

reach different conclusions; some echinacea preparations may be better than placebo

(Melchart et al., 2000), inconsistent evidence suggest that preparations based on the aerial


16

parts of E. purpurea may be effective for the early treatment of colds in adults (Linde et

al., 2006), in children E. purpurea was found to reduce the incidence of a second acute

respiratory tract infection (Del-Rio-Navarro et al., 2006).

A recent double-blind placebo-controlled trial failed to find immune stimulation

(Schwarz et al., 2002). However, Schoop et al. (2006) in a meta-analysis, reports that

standardized extracts of Echinacea were effective in the prevention of symptoms of the

common cold after clinical inoculation, as compared with placebo. Islam and Carter (2005)

after reviewing the evidence suggest that there is a beneficial effect, but like others suggest

that differences in products and doses make evaluation challenging (Melchart et al., 2000).

Finally, the most recent meta-analysis finds that the evidence supports Echinacea‘s benefit

in decreasing the incidence and duration of the common cold (Shah et al., 2007).

In spite of all of the aforementioned studies, there is still uncertainty as to which

constituents primarily contribute to the purported immunomodulatory action of Echinacea

species. The constituents of E. purpurea cover a wide range of polarity, from polar

polysaccharides and glycoproteins, to moderately polar caffeic acid derivatives, to the

rather lipophilic alkylamides (otherwise known as alkamides). Besides the alkylamides,

which are the focus of this dissertation, three other constituent groups may have

immunomodulatory activity; the phenylpropanoids (caftaric acid, caffeic acid, chlorogenic

acid, cichoric acid, cinnamic acid, cynarin, echinacoside etc.), polysaccharides and

glycoproteins (Bauer 1996; Bauer 1998). However, in extractions with ethanol

concentrations above 40%, only very low levels of polysaccharides are left in suspension,

and denaturing of proteins is expected (Brinker, 1999; Dalby-Brown et al., 2005). Thus,

the major constituents of ethanolic echinacea extracts are phenylpropanoids and

alkylamides. As a result of the ethanolic extracts that are used in the experiments herein the


17

following review of constituents of Echinacea spp. will be limited to the phenylpropanoids

and the alkylamides. The emphasis will be on the alkylamides as these are the subject of

the experiments contained herein.

Bauer and Wagner (1999b) have suggested that the lipophilic fraction, e.g.,

alkylamides, is mainly responsible for the immunological activity of alcoholic Echinacea

extracts. In addition there are claims of synergistic and polyvalent activity between

constituents in Echinacea and while intriguing, they are nevertheless largely

unsubstantiated (Wagner, 1999; Wills et al., 2000). They do, however, provide an

opportunity to explore the features of interaction between the diverse constituents of this

plant with various biochemical pathways and targets with reproducible and appropriate in

vitro models.

The phenylpropanoids

The phenylpropanoids, present in all Echinacea spp., are predominantly caffeic

acid derivatives (McCann et al., 2007). However the molecular species of these

compounds is dependent on which plant taxa are being analyzed; caftaric acid (2-O-

caffeoyltartaric acid; monocaffeoyltartaric acid), chlorogenic acid [3-O-(3,4-

Dihydroxycinnamoyl)-D-quinic Acid], cinnamic acid, cynarin (5-O-dicaffeoylquinic acid),

2-O-feruloyltartaric acid and 2-O-caffeoyl-3-O-coumaroyltartaric acid are found in all

Echinacea spp. (Bergeron et al., 2002). In E. purpurea, cichoric acid (2R,3R-O-

dicaffeoyltartaric acid) is found ca 1% of herb of E. purpurea and there are only traces in

angustifolia and pallida (Becker and Hsieh, 1985). Conversely, echinacoside and cynarin

are found in angustifolia and pallida only (Barnes et al., 2005).


18

Caffeic acid derivatives have been found to have antioxidant, antiviral,

phagocytosis stimulating and hylauronidase inhibitory activity. Specifically, cichoric acid

has demonstrated an inhibitory effect on hyaluronidase in the range of 50% inhibition

(IC50) at concentrations of 0.42 mM. In addition, the hyalurinadase IC50 of cynarine and

chlorogenic acid were found to be 1.85 and 2.25 mM (Facino et al., 1993).

The antimicrobial activity of the phenylpropanoids has been investigated due to the

antiviral activity of bee propolis and the subsequent finding that part of this activity was

due to caffeic acid derivatives (Hudson and Towers, 1999), as well as the finding that

caffeic acid is the main anti-HIV compound of Hyssop officinalis leaves (Kreis et al.,

1990). In addition, several esters of cinnamic acids, have been found to inhibit influenza

virus infection in chicken eggs (Serkedjieva et al., 1992). It should be noted that the

cichoric acid in Echinacea spp. is the levorotary isomer, while the dextrorotary isomer is

found in chicory (Cichorium intybus) and lettuce (Lactuca sativa). In the case of echinacea

antiviral activity has been observed on rhinovirus (RV) (Hudson et al., 2005), influenza

virus (FV) (Hudson et al., 2005), vesicular stomatitis virus (VSV) (Cheminat et al., 1988),

herpes simplex virus (HSV) (Binns et al., 2002; Hudson et al., 2005), human

immunodeficiency virus (HIV) (McDougall et al., 1998a; Neamati et al., 1997; Reinke et

al., 2004; Robinson, 1998; Robinson et al., 1996). L-cichoric acid has recently been found

to selectively inhibit HIV-1 integrase (McDougall et al., 1998a; Neamati et al., 1997;

Robinson, 1998; Robinson et al., 1996). In addition, the toxic concentration of cichoric

acid was found to be a 100 fold greater suggesting a reasonable therapeutic index (Reinke

et al., 2004). However, none of these models suggest notable antiviral activity except the

HIV syncitia cell-based assays which suggested that L-cichoric acid shows promise as an

anti-HIV lead with an EC50 (HIV-1 replication) of 2 μg/mL (McDougall et al., 1998b). An


19

E. purpurea HSV antiviral model, which combined UV-A light with echinacea extracts or

isolated constituents, found that both the lipophilic constituents (i.e. alkylamides,

ketoalken/ynes) as well as the more polar compounds (phenylpropanoids) were antiviral

suggesting multiple constituents may be responsible for echinacea‘s antiviral activity

(Binns et al., 2002).

Antibacterial activity has also been observed for echinacoside (Stall et al., 1950).

Bauer and Wagner (1991) found activity against Staphylococcus aureus, Corynebacterium

diphtheria and Proteus vulgaris. Of particular interest to the earlier argument of multiple

constituents offering promise as drug agents, Hudson and Towers (1999) suggest that

crude plant extracts are often more potent viral inhibitors than single pure compounds.

Although many have mistaken the use of echinacea extracts in colds and flus as an

interpretation that there must be antimicrobial activity, it should be kept in mind, however,

that thus far, the bulk of echinacea‘s activity against infections does not appear to be

related to its antimicrobial activity. Research suggests that immune modulation is the

primary activity responsible for the observed decrease in colds and flu symptoms

(Woelkart et al., 2008).

There has also been in vitro antioxidant/radical scavenging activity by the

phenylpropanoids demonstrated for E. purpurea and E. angustifolia extracts (Dalby-Brown

et al., 2005; Facino et al., 1995; Thygesen et al., 2007). Phenylpropanoids, especially

cichoric acid and caffeic acid, are some of the most efficient antioxidants from natural

sources (Hudec, Burdova, Kobida, Komora, Macho, Kogan, Turianica, Kochanova, Lozek,

Haban, and Chlebo 2007). Cichoric acid is also found in the aerial parts of E. pallida.

Other caffeic acid derivatives are found in the three main Echinacea species, especially in

the aerial parts (Bauer and Wagner, 1991).


20

While the activity of the phenylpropanoids is potentially interesting, it should not

be confused as being responsible for the main activity of echinacea extracts. Unfortunately,

in the haste to produce products appealing to the public and supported by scientific

evidence (although weak evidence), manufacturers have generated products standardized

to phenolic acids or echinacoside (in the case of E. angustifolia). These products have

been found to be inactive as immunostimulatory agents, although cichoric acid has been

found in vitro to stimulate phagocytosis (Bauer and Wagner, 1991) and modulate NF-κB,

TNF-α and NO levels (Aktan et al., 2003; Goel et al., 2002a). However, products

standardized to phenolic acids or echinacoside are able to maintain their anti-inflammatory

and antioxidant properties (Wilasrusmee et al. 2002).

Finally, it should be mentioned in regard to the phenylpropanoids that their stability

in extracts is an issue. Some, but not all, of the phenylpropanoids in Echinacea spp. are

heat sensitive. For instance, echinacoside concentrations in E. angustifolia are greatly

reduced with the addition of heat to the drying regime of the plant (Kabganian et al., 2002).

In addition, many of the caffeic acid derivatives are known to degrade rapidly in ethanolic

solution (Livesey et al., 1999). While in pressed juice products of E. purpurea, cichoric

acid is thought to be substantially reduced by enzymatic degradation via the polyphenol

oxidases (Nüsslein et al., 2000). However, 2-O-feruloyltartaric acid appears to be stable

(Bergeron et al., 2002). While the activities of the phenylpropanoids is interesting, they do

not appear to cross the gut membrane (Matthias et al., 2004).


21

The alkylamides

The widespread occurrence, metabolism and physiological significance in plants of

the fatty acid amides have only recently begun to be appreciated. The primary fatty acid

amides, the alkamides, even though restricted in distribution, are known to occur in 10

plant families, namely, Aristolochiaceae, Brassicaceae, Convolvulaceae, Euphorbiaceae,

Menispermaceae, Piperaceae, Poaceae, Rutaceae, and Solanaceae. In the above-listed

families, these metabolites may play a protective role (Molina-Torres et al., 2004). Plants

containing greater than > 0.5% of alkylamides include Echinacea spp. (Asteraceae),

Chrysanthemum spp. (Asteraceae), Anacyclus pyrethrum (L., Asteraceae), Spilanthes spp.

(Asteraceae), Piper spp. (Piperaceae), Zanthoxylum spp. (Rutaceae). In the Asteraceae

alkylamides are particularly prevalent, characteristically in the Heliantheae and the

Anthemideae tribes, in such plants as Achillea spp., Echinacea spp. and Spilanthes spp.

(Greger, 1984). Currently over 300 fatty acid amides have been characterized to date

(Gertsch, 2008).

The alkylamides are fatty acid derivatives whose general structure derives from the

condensation of an unsaturated fatty acid and an amine (Hofer et al., 1986). This class of

compounds is known to be bioactive in microbes, plants and animals. Farrell and Merkler

(2008) point out that an emerging theme arriving from research laboratories is that fatty

acid amides are common in numerous plant and animal species. Longer chain alkylamides,

specifically N-acylethanolamines, in low concentration are known to regulate animal

physiology, while low concentrations of medium chain alkylamides have been shown to

modulate plant physiology. In both plants and animals, the N-acylethanolamines (NAEs)

16:0, 18:1, and 18:2 acylethanolamides are common. However, plants appear to be


22

particularly responsive to low concentrations of medium chain NAEs, whereas, animal

physiology is largely regulated by low concentrations of long chain polyunsaturated NAEs

(e.g., NAE20:4), both of which constitute a relatively minor fraction of the NAE pool in

their respective systems. Because of their significant role in cell signaling, these molecules,

and their analogs, appear to have diverse activity due to their interface with a number of

receptor systems, including, but not limited to CB2 and PPARγ (Farrell and Merkler,

2008).

NH

O

NH

O

Figure 1.1. Structure of common alkylamides in various Echinacea spp.

The alkylamides are fatty acid derivatives formed through the condensation of a fatty acid with an amine.

A. An olefinic alkylamide, dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide.

B. An acetylenic alkylamide, undeca-2E-ene-8,10-diynoic acid isobutylamide.

Of relevance to this discussion, are the unsaturated alkylamides; acetylenic

alkylamides and the olefinc alkylamides (Figure 1.1). While both members of the

unsaturated class of alkylamides occur in the Asteraceae, the acetylenic alkylamides are

A

B


23

characterized as having an acetylenic tail (Figure 1.1B), with over 250 compounds

occurring in over 100 genera in the Heliantheae tribe alone (Christensen and Lam, 1991).

This is opposed to the purely olefinic alkylamides (contain double-bonded fatty tails)

identified in 6 genera in 5 subtribes: Ecliptinae (Wedelia): Galinsoginae (Acmella);

Helianthinae (Echinacea); Verbesininae (Salmea); Zinnininae (Heliopsis and Sanvitalia).

However, in the previous listed genera not all species contain alkylamides (Molinatorres et

al., 1996). Both class of alkylamides commonly occur with various degrees of

unsaturation. For example, Figure 1.1A illustrates one of the isomeric polyunsaturated

tetraenes, the most prominent alkylamides contained in E. angustifolia and E. purpurea

and Figure 1.1B shows the polyacetylene alkylamide undeca-2E-ene-8,10-diynoic acid.

Initial studies reported that the olefinic alkylamides, the class of alkylamides of

research interest in Echinacea spp., were found to be insecticidal (Crombie and Harper,

1949; Jacobson, 1948, 1954; Jacobson, 1967). This was later suggested to be due to effects

at the voltage-gated sodium channels (Ottea et al., 1989; Ottea et al., 1990). Like

veratridine, the N-alkylamides are reported to have greater activity with resistant strains of

insects than do the pyrethroids (Amanda et al., 1990). As would be expected for

allelochemicals, the plant signaling molecule methyl jasmonate has been found to

upregulate alkylamide production in young plants (Binns et al., 2001). Later work

demonstrated that the alkylamides promote growth and alter root development in

Arabidopsis seedlings (Ramirez-Chavez et al., 2004).

While the insecticidal properties of echinacea and other alkylamide containing

plants are indicative of biological activity and were likely exploited by native Americans,

the ethnobotanical documentation list uses such as anesthetic, antiseptic, alterative and

sialogogue for Echinacea spp. (Duke, 2009; Moerman, 1998). Other plants that were rich


24

in alkylamides were used similarly (Ekanem et al., 2007). For example, Spilanthes spp. are

also known as ‗toothache plant‘ due to traditional cultures exploitation of the anesthetic

properties of the alkylamides.

Continued research on alkylamides elucidated an oncolytic effect from the

alkylamides (Voaden and Jacobson, 1972). In vitro studies have shown various modes of

anticancer activity for complex extracts of echinacea (Mazzio and Soliman, 2008). Other in

vitro research has demonstrated inhibition of angiogenesis induced by lung and kidney

cancers by an alkylamide containing extract (Rogala et al., 2008). Past studies on structure

activity relationships of alkylamides have suggested that particular of the alkylamides

promote differentiation of leukemia to a benign state (Harpalani et al., 1993). In addition,

in a study of patients suffering from metastasizing advanced colorectal cancers, 7 of 15

patients showed no further progression of the cancer after using Madaus Echinacin (60

mg/m2 i.m. x 7 d). One patient had partial tumor regression (Lersch et al., 1992). In vitro

work using hexane extracts, which are rich in alkylamides due to the lipophilic nature of

these fatty acid derivatives, have demonstrated that a juice of E. angustifolia and E.

purpurea herba reduce the cell viability of a human colon cancer cell line (COLO320) and

a human pancreatic cancer cell line (MIA PaCa-2) (Chicca et al., 2007). Although the basis

of activity for the oncolytic activity has thus far not been clearly elucidated, stimulation of

natural killer (NK) cell numbers and activity appear to be a possible explanation. Mice

injected with leukemic cells followed by a daily dose of E. purpurea (Phyto Adrien

Gagnon, Santé Naturelle [A.G.] Ltée, La Prairie QC, Canada 0.45 mg/d) showed an

elevation of NK cells as compared to the control group. After a month of echinacea dosing

the untreated mice had died. By 3 months, 1/3 of the echinacea treated group were still

alive and went on to live full life spans with 2x the normal NK cell count as normal mice


25

(Currier and Miller, 2001). In a similar study, mice that were immunized against leukemia

5 weeks prior to injection with live leukemic cells, again had a survival rate of about 30%.

When echinacea was combined with the immunization, the survival rate went up to 60%

group (same dose and preparation). In this case the echinacea treated group had NK levels

3x the immunized mice. Other murine models have shown increases in NK cells (Sun et

al., 1999). In vivo research has shown in humans that the number of NK cells (CD16+ and

CD56+) increases after dosing with an echinacea product. These previously mentioned

studies utilizing various model systems is a step forward in validating the clinical

experience of the Ecletics who claimed that Echinacea angustifolia was useful in breast

and other cancers (Ellingwood and Lloyd, 1919). Unfortunately, the majority of these

studies do not provide clear evidence as to the specific activity of alkylamides due to the

use of multicomponent extracts.

However, specific immunological activity of the alkylamides has been researched

in vitro. Earlier studies by Bauer and Wagner (1985) showed that the chloroform fraction

of the root of E. purpurea, which concentrates the alkylamides, enhanced the phagocytic

activity of monocytes. In other studies murine macrophages, when exposed to select

alkylamides or a mixture of alkylamides exhibited a significant down-regulation of LPS-

mediated activation as demonstrated by a reduction in nitric oxide (NO) production at

ID50s of the majority of the alkylamides at 3-6 μg/mL (Chen et al., 2005). However,

Stevenson et al. (2005) demonstrated no effect on NO production by the acetylenic

isobutylamide undeca-2E-ene-8,10-diynoic acid (UDA, Figure 1.1B) and the olefinic

isobutylamide dodeca-2E,4E,8Z,10Z-tetraenoic acid (Z-tetraene, Figure 1.1A ≤ 2.0

μg/mL), but did show a significant reduction of NO by the mixture of alkylamides found in

E. purpurea and E. angustifolia root. Matthias et al. (2007) showed that a mixture of


26

alkylamides or UDA inhibited LPS induced production of NO, although only the mixture

was statistically significant. This group also showed that the Z-tetraene (2.0 ng/mL)

reduced levels of NFκB, while UDA had no effect (≤ 2.0 ng/mL). However, UDA (2.0

ng/mL) significantly increased TNF-α levels as compared to controls (Stevenson et al.,

2005).

Other in vitro studies have shown that in macrophages that are not primed for an

immune response only UDA has an effect. NFκB expression levels are reduced by UDA (2

ng/mL), although the Z-tetraene shows a trend towards reduction (Matthias et al., 2007). If

the macrophages are primed with LPS, NFκB expression is significantly reduced by Z-

tetraene or a mixture of alkylamides, while UDA shows a trend towards reduction. In this

same series of experiments, Matthias et al. (2007), showed that in macrophages that are

primed with phorbol myristate (PMA), TNF-α levels are reduced by cichoric acid (0.8

ng/mL) and Z-tetraene (2 ng/mL), but increased by a mixture of alkylamides. In

experiments with Jurkat cells (transformed T lymphocytes), when Jurkats are unstimulated

a mixture of alkylamides and UDA (both at 2.0 μg/mL) show a trend in the reduction of

NFκB expression, while the Z-tetraene showed a trend towards an increase in NFκB

expression levels. In LPS stimulated Jurkats the mixture of alkylamides and the Z-tetraene

(both at 2 μg/mL) showed a significant increase of NFκB. In PMA stimulated Jurkats there

were no effects by alkylamides except a strongly significant effect by UDA down-

regulating NFκB expression. The only other work to date that has utilized Jurkats with

isolated alkylamides is the study by Sasagawa et al (2006). They found that IL-2

production was inhibited by the isomeric mixture of the tetraenes (≥1.25 μg/mL), as well

as dodeca-2E,4E-dienoic acid isobutylamide (≥0.625 μg/mL).


27

Ex vivo studies show that the oral administration of the alkylamide fraction of E.

angustifolia and E. purpurea to mice improved phagocytic carbon clearance by factors of

1.5 and 1.7 respectively (Bauer et al., 1989; Bauer et al., 1988). Other studies show that the

alkylamides at a dose level of 12 mg/kg/day, had a significant influence on the phagocytic

activity of alveolar macrophages. In addition, the alkylamides caused a dose-dependent

increase in NO release from the alveolar macrophages on stimulation with LPS (Goel et

al., 2002b).

Most recently, a great deal of interest in the alkylamides has been generated on two

fronts: The recent reports that the alkylamides, unlike the phenylpropanoids, demonstrate

bioavailability in humans (Matthias et al., 2005) and the discovery that the alkylamides

function as cannabinoid ligands (Raduner et al., 2006; Woelkart et al., 2005). This is

significant because the endocannabinoid network, which consist of lipid messenger

molecules and their associated receptors, is now known to modulate such systems as the

nervous system, the endocrine network, the immune system, the gastrointestinal tract and

the reproductive system, as well as playing a key role in pathological conditions. As a

result, since the discovery of the cannabinoid receptor 1 (CB1) (Matsuda et al., 1990) and

cannabinoid receptor 2 (CB2) (Munro et al., 1993), G protein-coupled receptors found

predominantly in the CNS and the periphery, respectively, lipids such as (-)-Δ9

tetrahydrocannabinol and fatty acid derivatives have been scrutinized more intensely as

molecular messengers. Other CB ligands include the arachidonic derivatives such as

dihomo-γ-linolenylethanolamide, docosatetraenolylethanolamide, oleamide, and N-

oleolydopamine (Hanus, 2007; Kogan and Mechoulam, 2006; Pertwee, 2005). However,

the only endogenous ligands that thus far demonstrate cannabinoid binding at


28

physiological relevant concentrations are anandamide and 2-arachidonoyl glycerol

(Pertwee and Ross, 2002).

In reference to immunomodulation, CB2 is found in the spleen and on leukocytes

(monocytes/macrophages, dendritic cells and osteoclasts) and is well established to

modulate immune function (Berdyshev, 2000; Klein et al., 2000). Of particular relevance

to the immunological effects of Echinacea spp. is that specific alkylamides bind with

similar affinities to CB2 as the endocannabinoids (≈57 nM) (Matovic et al., 2007). Two

laboratories have confirmed binding to CB2 by select alkylamides (Raduner et al., 2006;

Woelkart et al., 2005). Using radioligand binding assays, Raduner et al. (2006)

demonstrated that the radioligand binding to CB2 of dodeca-2E,4E,8Z,10Z-tetraenoic acid

isobutylamide gave a 53% inhibition with a Ki of 57 nM. In the same series of experiments

dodeca-2E,4E-dienoic acid isobutylamide gave an inhibition of 52% to CB2 with a Ki of

60 nM. However, Woelkart and colleagues (2005) showed CB2 Ki‘s much higher. They

report Ki‘s for the isobutylamides dodeca-2E,4E-dienoic acid and dodeca-2E,4E,8Z,10E/Z-

tetraenoic acid of 5.5 and 9.7 μM respectively. Docking studies also indicate that the Z-

tetraene and dodeca-2E,4E-dienoic acid isobutylamide fit into the putative binding pocket

of CB2 (Raduner et al., 2006). Later work showed that of the three tetraene isobutylamide

isomers which make up the bulk of alkylamides in E. purpurea and E. angustifolia,

dodeca-2E,4E,8Z,10Z-tetraenoic acid (Z-tetraene) had the highest affinity (57 nM) as

opposed to dodeca-2E,4E,8Z,10E-tetraenoic acid (4500 nM) and dodeca-2E,4E,8E,10Z-

tetraenoic acid (9000 nM). The Z-tetraene makes up 32% of the tetraene isomers in E.

purpurea and 10% in E. angustifolia.

Besides binding assays, CB activity has been demonstrated in vitro. Gertsch et al.

(2004) showed that the isobutylamides dodeca-2E,4E-dienoic acid and dodeca-


29

2E,4E,8Z,10Z-tetraenoic acid (Z-tetraene) were able to significantly increase the induction

(5-12 fold) of tumor necrosis factor-α mRNA in macrophages. This effect was blocked by

the CB2 antagonist WR144528. Further studies demonstrated a significant up-regulation

(130-160%) of IL-6 in CB2 positive HL60 cells by the Z-tetraene and dodeca-2E,4E-

dienoic acid isobutylamide, but no effect by undeca-2E-ene-diynoic acid isobutylamide.

CB2 antagonist SR144528 (500 nM) was able to inhibit the up-regulation of IL-6. In the

same series of experiments an up-regulation of IL-8 expression by dodeca-2E,4E-dienoic

acid isobutylamide was shown, but no effect by the tetraenes or UDA isobutylamide on IL-

8 expression (Raduner et al., 2006).

While the CB2 activity of select alkylamides is a novel discovery, it has caused

some to suggest that the basis of activity for echinacea has finally been discovered

(Woelkart et al., 2005). Considering the previously discussed activity of the

polysaccharides and the phenylpropanoids, this is quite likely an overstatement. In

addition, it is important to recognize that the numerous effects of the endocannabinoids

may be mediated by other targets. For example, a CB3 receptor has been proposed (Ryberg

et al., 2007) and recent results also demonstrate that the endocannabinoids, anandamide

and 2-AG, bind to PPARγ (Rockwell and Kaminski, 2004; Rockwell et al., 2006; Sun and

Bennett, 2007).

Is PPARγ a target of alkylamides?

The peroxisome proliferator-activated receptors (PPAR) constitute a set of three

receptor sub-types which are members of the nuclear receptor superfamily of ligand-

activated transcription factors, which includes steroid hormone receptors. They are


30

encoded by distinct genes that function as lipid sensors that regulate gene expression in

many metabolically active tissues (Bishop-Bailey and Wray, 2003). The PPARs, so named

because the stimulation of PPARδ results in the increase of peroxisomes, have a significant

role in cellular energy balance, fuel utilization, the metabolism of fatty acids and other

lipids, the generation and remodeling of adipose tissue and fibrotic and hypertrophic

responses in the heart and vascular wall. Many of these actions are via interactions with

nuclear factors such as NF-κB and activator protein 1 (AP-1), thus modulating expression

of pro-inflammatory cytokines and adhesion molecules, and altering cell signaling

pathways (Touyz and Schifffrin, 2006). Thus, PPARs, in response to stressors, play a role

in the complex orchestration of adaptive cellular physiology working in a concerted mode

with the vitamin D receptor and the retinoic acid receptor (RXR). Thus, many

pharmaceutical companies are involved in PPAR research in areas such as diabetes,

obesity, cardiovascular disease and immunology with drug development in mind.

The endogenous ligands to the PPAR sites were originally unknown, earning the

PPARs the name ―orphan nuclear receptors‖. However, many ligands are now known:

PPARα is known to bind polyunsaturated fatty acids such as docosahexanoic acid (DHA)

and eicosapentaenoic acid (EPA), oxidized phospholipids, lipoprotein lipolytic products as

well as the lipid lowering fibrates (fenofibrate and gemfibrozil) (Desvergne and Wahli,

1999; Touyz and Schifffrin, 2006). PPARδ ligands include polyunsaturated fatty acids and

prostaglandins and its activity is postulated to be biochemically related to lipid levels,

wound healing and colon cancer, but the evidence for a connection to all of these pathways

is limited (Weindl et al., 2005). PPARγ binds fatty acid derivatives, such as

hydroxyoctadecadienoic acid (HODEs), prostaglandin derivatives, such as 15-deoxy-δ-

(12,14)-prostaglandin J2 (15d-PgJ2), and thiazolidinedione (TZDs) drugs, such as


31

pioglitazone and rosiglitazone (Touyz and Schifffrin, 2006). Nevertheless, the ―true‖

endogenous ligands are still debated due to these receptors demonstrating a significant

amount of promiscuity in ligand binding.

PPARγ is linked to immune function

A variety of types of immune cells contain the PPAR subtypes, α, δ, and γ. PPARs

are noted to generate transrepression via molecular association with, and the resulting

inhibition of, nuclear factor of the κ-enhancer in B cells (NFκB), nuclear factor of

activated T cells (NFAT), activator protein 1 (AP-1 – a heterogeneous dimer of c-fos and

c-jun) and other transcription factors (Blanquart et al., 2003; Chung et al., 2003; Yang et

al., 2000).

Specifically, PPARγ produces a variety of different immune effects, which

generally involves leukocyte down-regulation (Daynes and Jones, 2002). This results in the

inhibition of cytokine production, suppressed proliferation and circumstance dependent

modulation of apoptosis (Appel et al., 2005; Wang et al., 2002). PPARγ is known to affect

recruitment of monocytes in vascular endothelial cells and quench inflammation in a

number of disorders (Neve, 2000). Storer et al. (2005) observed inhibition of nitric oxide,

TNF, IL-1β and IL-6, as well as the chemokines MCP-1 from microglia and astrocytes by

the thiazolidinedione drugs, but noted that the endogenous ligand 15d-PgJ2 had stronger

anti-inflammatory activity than the TZDs. Other research demonstrates PPARγ mediated

protection from experimentally induced encephalitis (Iruretagoyena et al., 2006; Storer et

al., 2005) as well as significant anti-inflammatory effects on ex-vivo T cells from multiple

sclerosis patients (Schmidt et al., 2004). In addition, a 12 week human trial with Type 2


32

diabetic patients given troglitazone (a PPARγ agonist) lowered 2sCD40L resulting in a

novel finding of a mode of anti-inflammatory activity that may limit diabetes associated

arterial disease. Other groups have found similar activity in coronary artery disease (Marx

et al., 2003).

The above evidence suggests that PPARγ is involved in the signal transduction of

immune dynamics, and that activation of PPARγ is likely important in ameloriating

immune dysregulation. Thus agonists, inverse agonists and antagonist of PPARγ (and

possible PPARα & PPARδ ) likely offer insights into immune allostasis.

PPARγ binds fatty acid derivatives

An obstacle in molecular biology has been the determination of the endogenous

ligands of PPARs. It seems a number of naturally occurring compounds have been found

to activate PPARs. However, as of yet, none of the ligands of PPARs have been

completely accepted as endogenous ligands (Bishop-Bailey and Wray, 2003). This has

resulted in the speculation that PPARs may function as general lipid sensors. As previously

discussed polyunsaturated fatty acids and archidonate derivates – eicosanoids, have been

found to be endogenous ligands of PPARs (Burstein, 2005). PPARγ is currently known to

be tripped by prostaglandin derivatives such as 15-deoxy-Δ(12,14)-prostaglandin J2,

hydroxyoctadecadienoic acid and the exogenous pharmaceuticals known as the TZDs

(Touyz and Schifffrin, 2006).

2 Diabetic patients have elevated plasma levels of soluble CD40 ligand (sCD40L), which is proinflammatory.

This is independent of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein

cholesterol, triglycerides, blood pressure, body mass index, gender, C-reactive protein, and soluble ICAM-1.

Thus, CD40L is emerging to be a ―nontraditional‖ risk factor implicated in atherosclerosis.


33

PPARγ is linked to IL-2 and other cytokines

Although cytokine inhibition by leukocytes is speculated to be transduced by CB

receptors, non-cannabinoid modes of activity appear to also be involved (Klein, 2005;

Pertwee and Ross, 2002). IL-2 secretion is a characteristic response of T cell activation.

This cytokine is vital in adaptive immune response due to essential roles in T cell

proliferation, differentiation and cell survival. Rather conveniently, there is very little basal

expression of IL-2 but rapid secretion on T cell perturbation. This makes IL-2 a marker of

T cell activation. Central to the experiments contained in the PPARγ chapter, both

anandamide and 2-AG, in a number of models, demonstrate concentration dependent

inhibition of IL-2 (Rockwell et al., 2006). Moreover, anandamide induces a concentration-

dependent inhibition of interleukin-2 in primary splenocytes which is unaffected by

SR141716A and SR144528. In the same model arachidonic acid shows similar activity as

anandamide which is attenuated by pretreatment with nonspecific COX inhibitors as well

as COX-2 specific inhibitors. Pretreatment with the PPARγ antagonist T0070907 also

attenuated anandamide mediated suppression of IL-2 secretion (Rockwell and Kaminski,

2004).

Following the line of reasoning that IL-2 activity is at least partially mediated by

PPARγ, Yang et al. (2000) observed that PPARγ free Jurkat cells did not respond to

PPARγ ligands troglitazone and 15d-PgJ2, but PPARγ expressing Jurkats did exhibit IL-2

inhibition in response to PPARγ agonists. Of significance in regard to structural leads, the

PPARγ agonist Wy14643 did not show any IL-2 activity. Clark et al. (2000) and Rockwell

et al. (2006) also found PPARγ inhibitory activity on IL-2 secretion by the PPARγ agonists

ciglitazone and 15d-PgJ2. Rockwell et al. (2006) also ruled out CB1 and CB2 activity on


34

inhibition of IL-2 in their model of Jurkat cells and 2-AG ligand by demonstrating the

inhibition of transcriptional activity of the crucial IL-2 transcriptional factors, NFAT and

NFκB. Thus in the absence, but not the presence, of T0070907 IL-2 secretion was

inhibited. Finally, Rockwell et al. (2006) reported that 2-AG treatment doubled the

PPARγ/ PPAR response elements (PPRE) and suppressed mRNA levels for IFN-γ and IL-

4 in Jurkats.

Structural similarities between alkylamides and PPAR-γ agonist

As has been previously stated, PPARs are believed to be lipid sensors. Thus, select

fatty acids and many of their derivatives are recognized as PPAR ligands (Nakamura et al.,

2004; Rodriguez-Cruz et al., 2005). A structural lead to the PPARγ ligands comes from the

observation that saturated fatty acids do not appear to act as agonist for the PPARs

(Nakamura et al., 2004). As previously discussed, the alkylamides are fatty acid derivatives

formed by the condensation of a fatty acid with an amine (Hofer et al., 1986). In the case

of Echinacea spp. the alkylamides reported to be CB2 ligands are unsaturated: the Z-

tetraene as well as other isobutylamides such as dodeca-2E,4E-dienoic acid, dodeca-

2E,4E,8Z-trienoic acid are all reported to bind with similar Ki‘s making them viable

pharmacological ligands for CB2.

Of interest is that with few exceptions, the alkylamides that are reported to have

physiologically relevant Ki‘s for the CB2 are of the olefinic class of alkylamides. The

polyacetylenic alkylamides appear to be active in cell culture studies, but are reported to

have Ki‘s for CB2 that are too high for pharmacological activity in vivo. For example,

UDA contained in E. angustifolia (although mistakenly reported to occur in E. purpurea –


35

see Chapter 2), binds CB2 with negligible affinity (40-66 μM) (Raduner et al., 2006;

Woelkart et al., 2005). In addition, Chen et al. (2005) demonstrated that the

isobutylamides that have acytelenic tails reduced NO production and had lower ID50s than

the established CB2 ligands Z-tetraene and dodeca-2E,4E-dienoic acid (3 μg/mL vs. 6

μg/mL). PPARγ activation has shown to result in reduction in NO (Varga and Nagy,

2008).

Other discrepancies concerning CB2 being the sole target for alkylamides are

suggested by Raduner et al. (2006) who report that in CB2 negative HL-60 cells, IL-8

expression is inhibited by the isobutylamides Z-tetraene, dodeca-2E,4E-dienoic acid and

undeca-2E-ene-diynoic acid. Notably, PPARγ has been reported as a target for IL-8

inhibition (Chinetti et al., 2000; Tesse et al., 2008). Thus, the numerous effects reported for

the alkylamides raises the question, whether the plethora of effects merely reflect

alkylamide action on CB2 or whether there are unknown targets playing a role.

Research plan

Considering the proposed activity of alkylamides discussed above, the research

described in this thesis had the following aims:

1. Characterization, extraction and degradation of the alkylamides in E. purpurea.

The characterization and extraction of the alkylamides contained within E. purpurea will

provide details as to the availability of these compounds in ethanolic extracts. These

extracts, as well as alkylamide isolates, will then be utilized in subsequent metabolism and

bioactivity investigations. Degradation studies will also provide information on the


36

stability of the alkylamides. The quantification of various alkylamides occurring in the

extracts at different time points will also be useful data for the natural products industry.

2. Metabolism of the alkylamide isomers, dodeca-2,4,8,10-tetraenoic acid

isobutylamide.

The enzymatic transformation of the isomeric tetraenes will also provide meaningful data

on the bioavailability of the alkylamides during and after absorption.

3. Bioactivity of the alkylamides.

Specifically, the bioactivity of the isobutylamide undeca-2E-ene-8,10-diynoic acid will be

explored using an ELISA assay to measure IL-2 response in Jurkat cells. In addition, a

fibroblast adipocyte differentiation model utilizing 3T3-L1 cells will be explored.

Hypothesis

This thesis will investigate two hypotheses: First, that the alkylamides present in

Echinacea spp. are preserved during traditional preparative methods. Second, that specific

alkylamides from traditional preparations of Echinacea inhibit the release of IL-2 and

affect expression of key genes involved in the immune response, via the PPARy signalling

system.

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Chapter 2 Extraction of Alkylamides

50

Chapter 2

Alkylamide characterization and yield in fresh versus dry ethanolic extracts of Echinacea purpurea utilizing

HPLC-ESI-MS

Introduction

The analysis of medicinal plant extracts presents a number of analytical challenges.

Although many products have been shown to contain active constituents, they are

commonly obscured in biological matrices that generate confounding variables.

Unfortunately, such technical challenges may generate obstacles to chemical analysis

resulting in ambiguity of product content. Fortunately, hyphenated chromatographic

approaches such as gas chromatography mass spectrometry (GC-MS), liquid

chromatography mass spectrometry (LC-MS) and capillary electrophoresis mass

spectrometry (CE-MS) allow for analysis of challenging mixtures through enhanced

selectivity, chromatographic separation and measurement precision, as well as chemical

fingerprinting (Hansen, 2001; Li et al., 2007). In particular, electrospray ionization mass

spectrometry (ESI-MS), with its exceptional specificity, speed and sensitivity (Cech and

Enke, 2001; Xing et al., 2007) and its ability to ionize non-volatile molecules, is an ideal

first choice for characterization of chemically complex plant extracts.

The investigations described herein employ high performance liquid

chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS)

for the comprehensive characterization of several extracts from the medicinal plant

Echinacea purpurea (L.) Moench. Echinacea is widely used for the treatment of upper

respiratory infections, and is a global top seller. Three main species of echinacea are used


51

clinically and available to consumers, Echinacea pallida, E. purpurea and E. angustifolia.

Of these, E. purpurea represents 80% of commercial production (Li, 1998). E. purpurea

products range from the injectables prepared to rigorous European pharmaceutical

manufacturing standards, to the low tech ethanolic extractions or “tinctures” that follow

general manufacturing practices (GMPs) of the United States dietary supplements industry.

Although in Germany the aerial parts are preferred, ethanolic extracts of echinacea root

make up a large source of sales and clinical use in the United States. Manufacturing

practices generally dictate whether the starting plant material should be fresh or dry, but in

the case of echinacea species, both fresh and dry root extracts are commercially available.

To further complicate matters, these extracts are prepared with varying ratios of

plant:solvent depending on the manufacturer. Currently, there are few investigations

comparing the efficiency of extracting active constituents under these various extraction

conditions. There is currently a lack of information regarding differences in chemical

composition among extracts prepared using fresh versus dried Echinacea. One of the

goals of the studies conducted herein was to provide such information.

Of the four constituent groups currently believed to be the source of activity in the

echinacea genus; alkylamides (alkamides), phenylpropanoids (caffeic acid derivatives),

polysaccharides, and glycoproteins (Bauer, 1996, 1998), to date, human pharmacokinetic

studies of Echinacea spp. suggest that the alkylamides are the major constituent group

circulated in plasma (Matthias et al., 2005a; Woelkart et al., 2006).

Alkylamides, found in other medicinal plants besides Echinacea spp., have been of

pharmacological interest since the tingling and numbing effect from chewing plants rich in

these compounds were noted (Greger, 1984). This anesthetic property was utilized by

Native Americans (Moerman, 1998) and eventually by physicians in the early 20th

century


52

for a variety of purposes including as a sialogogue, antitussive and for toothache.

Alkylamides were later recognized as insecticidal (Greger, 1984; Jacobson, 1948;

Jacobson, 1967; Jondiko, 1986; Meisters and Wailes, 1966; Towers and Champagne,

1988) and oncolytic (Voaden and Jacobson, 1972). Recent investigations have

demonstrated immunomodulatory activity of alkylamides in vitro (Dalby-Brown et al.,

2005; Matthias et al., 2007; Merali et al., 2003; Muller-Jakic et al., 1994; Raduner et al.,

2006; Sasagawa et al., 2006; Sharma et al., 2006) and in vivo (Goel et al., 2002), as well as

direct antiviral activity (Hudson et al., 2005; Vimalanathan et al., 2005). Of late, these fatty

acid derivatives have become a subject of renewed interest due to their recent identification

as cannabinoid receptor 2 (CB2) ligands (Gertsch et al., 2006; Gertsch et al., 2004;

Raduner et al., 2006; Woelkart et al., 2005b) and most recently, PPARγ activity has been

demonstrated (Christensen et al., 2009; Spelman et al., 2009).

Even though there is a renewal of pharmacological investigations in the

alkylamides, there is a paucity of research on the extraction of alkylamides (Gafner and

Bergeron, 2005; Stuart and Wills, 2000; Stuart et al., 2004), especially of the abundant

ethanolic extractions that make up a significant share of the echinacea products purchased

by consumers. These galenicals are prepared by maceration or percolation of the starting

plant material in a variable ratio of ethanol and water dependent on plant species, plant part

and manufacturing customs. The few studies investigating the extraction of alkylamides

have generally utilized dried root, while alkylamide extraction of fresh roots has scarcely

been studied (Sun et al., 2002). Notably, the Germans prefer E. purpurea aerial parts,

where most phytotherapists in the United States prefer preparations from the root.

Regardless, traditional preparation of echinacea tincture is often from fresh plant material

(Moore, 2003).


53

Given that the alkylamides appear to be one of the key constituent classes

responsible for pharmacological activity of E. purpurea, the studies described herein

focused on this class of constituents. Due to the convenient chromophoric nature of the

secondary amide groups and olefinic double bounds of the alkylamides, much of the past

characterization of alkylamides has been generated with LC-UV (Bauer et al., 1988a;

Bauer and Remiger, 1989; Bauer et al., 1988b; Bauer et al., 1988c, 1989; Bauer et al.,

1988d). LC with gradient elution provides reproducibility, good linear range and, key to

the complex environments presented by phytochemical matrices, allows for analyzes of

multiple constituents from plant extractions (Ong, 2004). While initial reports had used

longer analysis times (Bauer and Remiger, 1989), Bergner demonstrated that the use of a

short C18 reverse phase column with small particle size (75 x 4.5 mm i.d.; 3μm) allows for

improvement of the separation and shortens the time of analysis of alkylamides (Bergeron

et al., 2000).

However, comprehensive profiling of echinacea alkylamide content using LC-UV

alone has been challenging. These compounds are present at widely different

concentrations, and many of them are isomeric. Consequently, co-elution of structurally

similar alkylamides is common, and UV detectors may not detect minor alkylamide

constituents because of low concentrations and/or co-elution with other compounds. Mass

spectrometry (MS) provides a distinct advantage over UV detectors due to its sensitivity

and the ability to select by mass the ions corresponding to the compounds of interest (He et

al., 1998; Hudaib et al., 2002; Matovic et al., 2007). As the data presented here will

demonstrate, this advantage makes HPLC-ESI-MS an ideal technique for the

comprehensive analysis of the isomeric alkylamide content in Echinacea purpurea.


54

LC-ESI-MS provides further advantages such as greater sensitivity and resolution

and as a result less need for separation of chromophores. Additionally, LC-MS/MS

fragmentation has also been used to identify the characteristic fragmentation pattern of

alkylamides (Bauer et al., 1988b; Cech et al., 2006a; Cech et al., 2006b). Although several

investigators have previously employed HPLC-ESI-MS to the analysis of Echinacea (Cech

et al., 2006a; He et al., 1998; Luo et al., 2003; Sloley et al., 2001; Woelkart et al., 2005a;

Woelkart et al., 2006) many of these previous investigations have not been quantitative

(He et al., 1998; Luo et al., 2003; Sloley et al., 2001). Furthermore, because of the

abundance of isomeric alkylamides, even with the use of MS detectors, mis-identification

or incomplete identification of alkylamides has been common (Bauer et al., 1987; He et al.,

1998; Jacobson, 1967; Wills and Stuart, 1999).

The investigations described here employ HPLC-ESI-MS with an ion trap mass

spectrometer for the quantitative and qualitative analysis of alkylamide content in various

Echinacea purpurea extracts, filling a gap in the current literature. The objective of these

studies is to employ comprehensive analysis with HPLC-ESI-MS to compare the

alkylamide content of various fresh and dry Echinacea purpurea extracts. In contrast to

previous reports (Bauer and Wagner, 1991; Hudaib et al., 2002; Luo et al., 2003; Perry et

al., 1997; Wills and Stuart, 1999), we report that the species Echinacea purpurea contains

at least 15 different alkylamides that are detectable in ethanolic extractions of the root.

Utilizing the capability of the ion trap mass analyzer to generate MS-MS fragments, we are

able to report what is, to date, the most comprehensive list of alkylamides present in

traditionally prepared ethanolic extractions of E. purpurea root. In addition, our results

demonstrate the optimal length of maceration time for extracting alkylamides in ethanol,

and demonstrate differences in alkylamide yield in extracts prepared using various


55

plant:solvent ratios, and with fresh and dry plant roots. The results presented here are

relevant to researchers engaged in the chemical characterization of plant extracts in that

they demonstrate the power of MS-MS characterization for distinguishing structurally

similar constituents. More broadly, these results are relevant to the dietary supplements

industry because they demonstrate methods that can be employed to yield extracts with

significant content of active constituents, in this case alkylamides. Using novel

pharmaceutical and biomedical analysis we were able to identify new alkylamides and a

correction of a previously published error. Thus, using LC-ESI-MS we have generated the

most comprehensive list of the alkylamides to date, many of which are proving to be

actives of Echinacea purpurea root extracts.

Methods

Reagents

The following chemicals and reagents were used: Acetonitrile (high-performance

liquid chromatography (HPLC) grade (Honeywell Burdick and Jackson, Muskegon, MI),

acetic acid (Fisher Chemical, Fairlawn, NJ), alkylamide standards (Chromadex Inc., Santa

Anna, CA) ethanol (AAPER, Shelbyville, KY), nanopure water (Nanopure Diamond

D11931, Barnstead International, Thermolyne, Dubuque, IA).


56

Plant Material

Cultivation of E. purpurea took place in Grants Pass, OR at Pacific Botanicals.

Fresh, dormant roots of E. purpurea were harvested in March 2007. Species was verified

by Richard Cech (Horizon Herbs, Williams, OR) and voucher specimens were submitted

to the University of North Carolina Herbarium in Chapel Hill, NC (accession numbers

583416 and 583417). The roots were two-years-old at time of harvest.

Plant Extractions

A typical protocol (Adams and Tan, 1999) for the manufacture of ethanolic extracts

was followed in all extractions, except that post washing, the roots were briefly soaked (5

min) in 70% ethanol as a disinfectant, and blown partially dry with compressed air. A loss

of the isomeric dodeca-2,4,8,10-tetraenoic acid isobutylamides (tetraenes) of 1.4% (against

final fresh root concentrations) was calculated from the initial rinse. The roots were then

cut into small pieces (≤ 1 cm wide) and treated using three different extraction techniques,

fresh root extraction (1:2 w:v) and dry root extraction at two different root to solvent

ratios, 1:11 and 1:5 (w:v) as shown in Table 2.1. All ratios are expressed as mass raw plant

material (E. purpurea roots) in weight (g) per volume (mL) of extraction solvent.

To prepare fresh root extracts, samples of the cut roots (65 g) were blended using a

Waring Blender (Tarrington, CT) in a solvent of 95% ethanol (AAPER, Shelbyville, KY)

at a ratio of 1 g roots:2 mL solvent. Samples of root from the same batch were dried in an

oven at 50 °C and water content was determined to be 74.5% (Table 2.1). Dry root

extractions were carried out with the same method as the fresh except that the solvent


57

consisted of 74.5% ethanol and 25.5% water (to account for the plant water removed upon

drying). To make these dry root extracts, 16.6 g of dried root was added to 179 mL of

solvent (74.5% ethanol) for a ratio of 1:11 and 16.6 g of dried root was added to 83 mL of

solvent (74.5% ethanol) for a ratio of 1:5 (Table 2.1). Four replicate extracts were prepared

at each extraction ratio (fresh 1:2, dry 1:11 and dry 1:5).

Table 2.1. Solvent and root ratios for the extracts

Designation E. purpurea root (g)

Ethanol (mL) a

Water (mL)b

Final ratio dry plant material:solvent

(mg:mL)c

% alcohold

Fresh 1:2 65.0 130 48 17:178 or 1:11 69

Dry 1:11 16.6 130 49 17:179 or 1:11 69

Dry 1:5 16.6 60 23 17:83 or 1:5 69 a. This value represents the quantity of ethanol (95%) added to the plant material in the extraction

process.

b. This value represents the quantity of water added to the plant material in the extraction process. For

the fresh extract, additional water is contributed by the plant roots.

c. This ratio, the ratio of plant material to solvent in the final extract, is calculated by dividing the dry

weight of the plant material by the total volume of solvent (including water contributed by the fresh

plant material, which contains 74.5% water).

d. This percentage represents the amount of alcohol in the final extract. It includes both the quantity of

water used to prepare the extract and the quantity of water contributed by the fresh plant material.

To prepare fresh root extracts, samples of the cut roots (65 g) were blended using a

Waring Blender (Tarrington, CT) in a solvent of 95% ethanol (AAPER, Shelbyville, KY)

at a ratio of 1 g roots:2 mL solvent. Samples of root from the same batch were dried in an

oven at 50 °C and water content was determined to be 74.5% (Table 2.1). Dry root

extractions were carried out with the same method as the fresh except that the solvent

consisted of 74.5% ethanol and 25.5% water (to account for the plant water removed upon

drying). To make these dry root extracts, 16.6 g of dried root was added to 179 mL of

solvent (74.5% ethanol) for a ratio of 1:11 and 16.6 g of dried root was added to 83 mL of

solvent (74.5% ethanol) for a ratio of 1:5 (Table 2.1). Four replicate extracts were prepared

at each extraction ratio (fresh 1:2, dry 1:11 and dry 1:5).


58

Aliquots (dry root 1:11, 200 µL) for the extraction as a function of time study were

taken on a daily basis (days 2-33) during the process of maceration and stored at -70° C

until the time of analysis for alkylamide content. After maceration for one month, the

solvent was removed from all of the extracts using a hydraulic press. The extracts were

then aliquoted into 1 mL portions in polypropylene microcentrifuge tubes and kept in the

dark at room temperature until needed for analysis. Previous investigations have

established stability of alkylamides under these conditions (Livesey et al., 1999). All

extractions were macerated at 24 °C.

Sample preparation

Prior to analysis, samples were removed from storage and allowed to reach room

temperature. Aliquots (500 µL) from all extractions were centrifuged at 14,000 rpm

(Savant Speedvac Sc110, Farmingdale, NY) for 5 minutes. Supernatant was then diluted in

the same solvent used for extraction (70% ethanol), and pipetted (300 µL) into autosampler

vials (Agilent Technologies, Santa Clara, CA) for LC-MS analysis. Several dilutions were

prepared from each extract to provide samples for which the alkylamides of interest were

present at concentrations within the linear dynamic range of the method. Neat samples

were used for determination of dodeca-2E-ene-8,10-diynoic acid, 100-fold dilutions were

used for analysis of dodeca-2E,4E-dienoic acid , and1000-fold dilutions were used for

analysis of the isomers of dodeda-2,4,8,10-tetraenoic acid isobutylamide.


59

Determination of yield of dissolved solids

Polypropylene microcentrifuge tubes (1.5 mL) were weighed before addition of

500 µL aliquots of centrifuged extracts. After dehydration in the speedvac for 39 hours at

24 °C, the mass of dissolved solids for each sample was determined. The ratio of the mass

of dissolved solids to the amount of dry root used in the equivalent volume of extract was

then calculated, providing a measure of the quantity of dissolved solids extracted per mass

of Echinacea root (extract yield). For the fresh extract, the dry weight of the root used for

the extract was calculated by subtracting the mass of the water contained in the roots from

the total mass of the fresh roots.

Preparation of Alkylamide Standard Solutions

The alkylamide standards were purchased from Chromadex (Santa Anna, CA) with

certificates of analysis verifying identity by NMR and HPLC. Primary stock solutions of

undeca-2E-ene-8,10-diynoic acid isobutylamide (molecular weight 231.34, lot #21235-

501), dodeca-2E-ene-8,10-diynoic acid isobutylamide (molecular weight 245.37, lot #

04950-601), dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (molecular weight

247.38, lot # 04953-102) and dodeca-2,4-dienoic acid isobutylamide (molecular weight

251.41, lot # 04951-101) were prepared in ethanol and stored at 4 ○C. The stock solution

was then diluted in ethanol to produce final concentrations of 0.1, 10, 50, 100, 500 µM,

depending on the instrument sensitivity to the particular compound.


60

HPLC-ESI-MS Analysis

An ion trap mass spectrometer with electrospray ionization source (LCQ

Advantage, ThermoFisher, San Jose, CA) was employed. The solvent gradient, which was

a minor variation on that previously published (Cech et al., 2006a), was as follows, where

solvent A is aqueous acetic acid (17mM, original pH 2.74) and solvent B is neat HPLC

grade acetonitrile. For t = 0 to 4 min, a constant composition of A-B (90:10 v/v); for t = 4

to 15 min, a linear gradient from A-B (90:10, v/v) to A-B (60:40, v/v); for t = 15 to 30 min,

a linear gradient from A-B (60:40,v/v) to A-B (40:60, v/v); for t = 30.1 to 35 min, a

constant composition of A-B (0:100,v/v); for t = 35.1 to 43 min, a constant composition of

A-B (90:10, v/v). The mass spectrometer was operated in the positive ion mode with a scan

range of 50.00-2000.00. Spray, capillary, and tube lens offset voltages were 4.5 kV, 3 V

and -60V, respectively. MS/MS studies were performed by programming the MS to the ion

masses shown in Tables 2.2A and B.

Constituents in the extracts were identified according to their molecular weights,

HPLC retention times, and previously established MS–MS fragmentation patterns (Cech et

al., 2006a; Hiserodt et al., 2004). Quantitation was accomplished using the previously

published methods (Cech et al., 2006a; Sasagawa et al., 2006) based on the available

alkylamide standards. Briefly, calibration curves were plotted for each alkylamide as the

peak area of the selected ion chromatogram for the protonated molecular ion of the

alkylamide of interest versus concentration. A concentration range of 0.1 to 100 µM was

used for all alkylamides except dodeca-2,4-dienoic acid isobutylamide, which was plotted

over a range of 10-500 µM due to poor instrument sensitivity to this molecular species.

The structure of the alkylamides that functioned as standards are noted with an asterisk in


61

Tables 2.2A and 2.2B. For the extraction as a function of time study, samples from daily

collections were analyzed in a single run within 10 weeks of the initial sample collection.

Statistical analysis

The means standard error of the mean (SEM) were determined for each set of

concentrations or peak areas. Data is expressed as the mean ± SEM and means were

compared using a two-tailed t-test for paired data when differences were observed. The

mean values were considered significantly different if p < 0.05. Where appropriate,

outlying data points were rejected on the basis of the Q-test. Statistical analyses were

performed with Microsoft Excel (2003).

Results

In this section, several types of results are presented. First, a comprehensive profile

of alkylamide constituents in E. purpurea is listed, with a description of how these

compounds can be identified using HPLC-ESI-MS. Second, quantities of dissolved solids

and specific alkylamides present in various E. purpurea extracts are compared. The

extracts analyzed here were prepared using several different procedures commonly

employed in the dietary supplement industry. Analysis of these extracts provides insight

into the similarities and differences in extract composition that result from these variations

in extraction technique.


62

Identification of Alkylamides

Tables 2.2A and 2.2B show the structures and relevant data for alkylamides that

have previously been identified from Echinacea purpurea. The references in the farthest

right column refer to the publications in which these identifications were made. This is the

most comprehensive listing of alkylamides of E. purpurea root to date. Most reports and

reviews list some, but not all, of the alkylamides present in this species (Bauer et al.,

1988b; Gotti et al., 2002; Gray et al., 2003; Wills and Stuart, 1999). Past estimates suggest

the presence of eleven alkylamides in the roots of E. purpurea (Bauer, 1998; Pietta et al.,

2004). Tables 2.2A and 2.2B are more comprehensive, listing a total of 17 compounds,

although, as described later on, some of these identifications are only tentative and one

compound (C) was not detected in our samples of E. purpurea.

Figure 2.1 illustrates two compilations of selected ion chromatograms obtained

from analysis of the 1:5 dry root E. purpurea extract. The ions plotted correspond to the

m/z of the protonated (MH+) forms of various alkylamides in E. purpurea, with the letters

above the peaks corresponding to the designations of structure in Tables 2.2A and 2.2B.

The higher intensity ions, m/z 230, 250, and 248 (Figure 2.1A), were plotted

separately from the lower intensity ions 244, 252, 258, 262 (Figure 2.1B) for ease of

visualization.

A number of the alkylamides in Tables 2.2A and 2.2B are isomers, and their MH+

ions, therefore, are detected at the same m/z values. Such isomeric alkylamides can be

distinguished on the basis of retention time (Binns et al., 2001; Cech et al., 2006a) and/or

MS-MS fragmentation (Cech et al., 2006a; Sloley et al., 2001). In Figure 2.1, undeca-

2E,4Z-diene-8,10-diynoic acid isobutylamide (A) has a distinct peak, as does its


63

Table 2.2A. Alkylamides from E. purpurea.

Desig- nation

Alkylamide nomenclature

Structure Amide Moiety

Total MW

Ref

A

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

isobutylamide

O

N

Isobutyl 229.32 (Bauer et al.,

1988b)

B

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

isobutylamide

O

N

Isobutyl 229.32

(Bohlmann and

Grenz, 1966)

C*

*Undeca-2E-ene-8,10-diynoic

acid isobutylamide

O

N

Isobutyl 231.34 (Binns et al.,

2002b)

D

Undeca-2E,4Z-diene-8,10-

diynoic acid 2-methylbutyl

amide

N

O

2-methyl butyl

243.35 (Bauer et al.,

1988b)

E

Undeca-2Z,4E-diene-8,10-


amide

N

O

2-methyl butyl

243.35

F

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

isobutylamide

N

O

Isobutyl 243.35

(Bohlmann and

Grenz, 1966)

G

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

isobutylamide

N

O


1988b)

H

Dodeca-2E,4E,10E-

triene-8-ynoic acid

isobutylamide

N

O

Isobutyl

245.37

(Bauer et al.,

1988b)


64

Table 2.2B. Alkylamides from E. purpurea continued.

a. Bohlman & Grenz, 1966 identified the mixture of the tetraenes K, L & M (in root)

b. Bauer et al, 1988 determined the stereochemistry of K & L;

c. Matovic et al, 2007 identified the third tetraene isomer M.

d. Compounds C, J, L and O, marked with an asterisk (*) were utilized as standards.

Desig-nation

Alkylamide nomenclature

Structure Amide Moiety

Total MW

Ref

J*

*Dodeca-2E-ene-8,10-

diynoic acid isobutylamide

N

O

Isobutyl 245.37 (Binns et al.,

2002b)

Kab

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

N

O


1988b)

Lab*

*Dodeca-2E,4E, 8Z,10Z-

tetraenoic acid isobutylamide

O

N

O

N


1988b)

Mac

Dodeca-2E,4E, 8E,10Z-

tetraenoic acid isobutylamide

N

O

Isobutyl 247.38 (Matovic et al., 2007)

N

Dodeca-2E,4E,8Z-

trienoic acid isobutylamide

N

O


1988b)

O* *Dodeca-2E,4E-

dienoic acid isobutylamide

O

N

Isobutyl 251 .41

(Bauer and

Remiger, 1989)

P

Trideca-2E,7Z-diene-8,10-diynoic acid

isobutylamide

N

O

Isobutyl 257.38

(Bauer and

Remiger, 1989)

Q

Dodeca-2E,4Z-diene-8,10-


amide

N

O

2-methyl butyl

257.38 (Cech et al.,

2006a)

R

Dodeca-2,4,8,10-

tetraenoic acid 2-methylbutyl

amide

N

O

2-methyl butyl

261.41 (He et

al., 1998)


65

Figure 2.1. A & B Characteristic selected ion chromatograms obtained by liquid chromatography-mass

spectrometry analysis of an E. purpurea root extract (1:5).

The designating letters correspond to the alkylamides in Tables 2.2 A and B. The chromatogram in (A) was

derived by plotting a combination of the selected ion chromatograms for ions with m/z 230 (alkylamides A

and B) m/z 248 (alkylamides K, L & M), and m/z 250 (alkylamide N). Chromatogram B was obtained by

plotting selected ion chromatograms for some of the minor alkylamide ions, m/z 244 (alkylamides D, E, F &

G), m/z 252 (alkylamide O), m/z 258 (alkylamides P and Q), and m/z 262 (alkylamide R) From these

chromatograms, it is apparent that the 8,10 alkyne alkylamides A/B undeca-2E/Z,4E/Z-diene-8,10-diynoic

acid isobutylamide; D/E undeca-2E/Z,4E/Z-diene-8,10-diynoic acid 2-methylbutylamide ; F/G dodeca-

2E/Z,4E/Z-diene-8,10-diynoic acid isobutylamide; P/Q dodeca-2Z,4E-diene-8,10-diynoic acid 2-

methylbutylamide elute before the olefinic alkylamides that function as CB2 ligands – dodeca -

2E,4E,8Z,10Z-tetraenoic acid isobutylamide (L); dodeca-2E,4E-dienoic acid isobutylamide (O);

dodeca-2E,4E,8Z-trienoic acid isobutylamide (N).

isomer, undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide (B), consistent with reports

by Cech et al. (Cech et al., 2006a). The isomeric alkylamides D/E (both with MH+ values

of 244) elute as a single peak, as do the isomeric pair F/G (also with MH+ 244). By relying

on MS-MS fragmentation spectra (Figure 2.2), it is possible to determine that each peak

corresponds to a pair of isomers. Additionally, as shown in Figure 2.2, MS-MS spectra

can also be helpful for further structural assignment of isomeric compounds that are


66

resolved chromatographically, such as the isomers P and Q (both with MH+ ions at m/z

258).

The isomeric compounds D, E, F and G all demonstrate MH+ ions at m/z 244 and

compounds P and Q both have MH+ ions with m/z 258. Nonetheless, each alkylamide is

uniquely associated with its retention time and mass spectrum, and is readily

distinguishable. Table 2.3 illustrates the primary fragments that result from collisionally

induced dissociation of several isomeric alkylamides. Structurally similar fragments have

been grouped with designations of i, ii, iii, iv and v for ease of reference.

One of the major groups of fragments formed by collisionally induced dissociation

is the acyllium ion (fragment group i in Table 2.3). The formation of acyllium fragments

from alkylamides was recently reported by Hiserodt et al. (Hiserodt et al., 2004). These

ions form due to a charge-remote hemolytic cleavage that yields a resonant distonic radical

cation, which subsequently undergoes hydrogen rearrangement. Alkylamides F and G

(Figure 2.2 D and E) show an acyllium ion at m/z 171, while alkylamides D and E (Figure

2.2 A and B) result in the acyllium ion at m/z 157. The acyllium ion and the first

generation fragment resulting from the subsequent loss of CO, indirectly leads to the

elucidation of the alkyl moiety length. Utilizing MW of these fragments, it is implicit that

an alkyl moiety with 12 carbons for F, G & Q is present as compared to 11 carbons (D, E)

or 13 carbons (P) is present Table 2.3.

Two additional fragments useful for elucidation of alkylamide structure are the

group ii and group iii fragments (Table 2.3). The group ii fragments result from the loss of

the amide portion alkylamide, and correspond to the remaining alkyl chain. The group iii

fragments are observed at an m/z value 2 amu above the group ii fragments. Past research

has tended to ignore these ions, even though they are of high intensity, perhaps due to


67

uncertainty as to how they are formed. Recent work utilizing deuterated alkylamides

suggests that in the diene alkylamides, these fragments are formed when an unsaturated

bond is lost and the remaining double bond shifts to the 3 position (in 2,4-dienes), with a

subsequent gain of 2 hydrogens (Hiserodt et al., 2004).

In combination, the group ii and iii ions can be used to determine 1) whether the

alkylamide is a diene; 2) how many carbons are present in the akyl chain; and 3) the

identity of the amide moiety (isobutyl versus 2-methylbutyl). Isobutylamides will have

two fragments corresponding to a loss of 101 (group ii) and 99 (group iii) from the MH+

precursor ion (Figure 2.2 D, E, and F). For 2-methylbutylamides, the fragments will

reflect the additional carbon in the amide moiety, and fragment ions corresponding to a

loss of 115 (group ii) and 113 (group iii) from the MH+ precursor ion will be observed

(Figure 2.2 A, B and C).

The group iv fragments (Table 2.3) correspond to the MH+ ion of the protonated

alkylamide that remains after loss of the N-alkyl group. Abebe et al (1997) suggest that

ionized amides lose their N-alkyl substituent group with moderate facility. Loss of the N-

isobutyl group results in a significant fragment (relative intensity 26-60%) at m/z 188 for

compounds F and G and 185 for compound Q, while loss of the N-(2-methylbutyl) group

results in a fragment with m/z 174 for compounds D and E (relative intensity 56-100%)

and a fragment with m/z 202 for compound P. This confirms the presence of an isobutyl

group (MW 56) in the structure of compounds F & G [(M+H) – (M – N-alkyl); 244 – 188

= 56] as does P [258 – 202 = 56]. This is compared to D & E [244 – 174 = 70] which

contain a N-2-methylbuyl amide moiety (70). This also shows that alkylamide Q contains a

2-methylbutyl amide [258 – 188 = 70]. The mass that is lost to form the group iv fragment

serves as additional confirmation to distinguish isobutylamides from 2-methylbutylamides.


68

The final fragments that result from collisionally induced dissociation of

alkylamides are group v in Table 2.3. They are formed by cleavage of various C-C bonds

on the N-alkyl substituent (Figure 2.2). The group v fragments are useful for verifying

whether the N-alkyl substituent is a 2-methylbutylamide or isobutylamide.

Table 2.3. MS/MS Analysis of Echinacea alkylamides. Fragments produced by collision-induced

dissociation of selected parent ions and their suggested structures are shown

Designation, name, m/z

group i

4

RI

3 30-

90%

group ii

5

RI 30-90%

group iii

6

RI 60-100%

group iv

7

RI 26-100%

group v 8 RI < 3-20%

D 244

1

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

metylbutyl amide 157 129 131 174

188,202,

216

E 244

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

2

157 129 131 174 188,202

, 216

F 244

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

isobutylamide 171 143 145 188

202, 216

G 244

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

isobutylamide 171 143 145 188

202, 216

P 258

Trideca-2Z,7E-diene-8,10-diynoic acid

isobutylamide 185 157 159 202 216,230

Q 258

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

171 143 145 188 202, 216

1. The number beneath the letter designation indicates the m/z value for the MH+ ion.

2. Proposed structure for compound E based on retention time and MS/MS fragmentation.

3. RI corresponds to relative Intensity.

4. The group i fragments correspond to acyllium ions as shown in Figure 2.2.

5. The group ii fragments are carbocations that correspond to the alkyl chain of the alkylamide and are

formed by loss of the amide portion (isobutylamide or 2-methylbutylamide).

6. The group iii fragments correspond to the alkyl chain of the alkylamide and are formed by the loss of the

amide portion of the molecule and saturation of one of the double bonds on the akyl chain.

7. The group iv fragments correspond to the protonated alkylamide minus the N-alky group

8. The group v fragments correspond to the protonated alkylamide minus various portions of the N-alkyl

group (see Figure 2.2)


69

2-methylbutyl amides Isobutyl amides

Figure 2.2. D, E, F, G, P Q. MS/MS Spectrum of Isomeric Alkylamides. The above Diagrams illustrate the utility of MS/MS

under low-energy collisional induced dissociation. This generates a characteristic pattern of relatively few primary fragments

that are 1) structurally diagnostic; 2) easily interpreted due to uncomplicated spectra; and 3) have enhanced relative intensity.

Thus the isomeric alkylamides of MW 244 and 258 (MH+) can be distinguished with ease between isobutyl amides and 2-

methylbutyl amides. In addition the number of carbon atoms in the alkyl moiety is elucidated by the peaks for the acyllium ion

and the loss of the carbonyl and amide groups. Note that the proposed structure for alkylamide E follows the fragmentation

pattern of alkylamide D and the elution order of the alkylamides F and G.


70

An example of the utility of MS-MS for structural elucidation of alkylamides can

be demonstrated for the specific case of alkylamide P, trideca-2E,7Z-diene-8,10-diynoic

acid isobutylamide. The MS-MS spectrum of the precursor ion at m/z 258 for this

compound is shown in Figure 2.2 F. In Figure 2.2 F, the acyllium ion (group i fragment) is

present at an m/z value of 185 m/z. This suggests a thirteen carbon alkyl chain. (An alkyl

chain with eleven carbons would produce an acyllium ion with m/z 157 and one with 12

carbons would yield an acyllium ion at m/z 171.) The group ii fragment at m/z 157

provides further confirmation of the 13-carbon alkyl chain length. The presence of the

group iii fragment (m/z 159) indicates that the compound is indeed a diene alkylamide,

and, further, the characteristic loss of 99 to form this fragment (258-159 = 99) suggests that

the compound is an isobutylamide rather than a 2-methylbutylamide. The high intensity

group iv fragment at m/z 202 further confirms that the N-alkyl group is an isobutylamide.

The low intensity group v fragments also confirm an N-isobutyl group rather than an N-2-

methylbutyl group; additional group v fragments would be observed for a 2-

methylbutylamide. Thus, based on the MS-MS spectrum (Figure 2.2F) the identity of the

compound is proposed to be trideca-2E,7Z-diene-8,10-diynoic acid isobutylamide, which

has previously been shown to occur in E. pallida, but rarely is reported as occurring in E.

purpurea (Bauer and Remiger, 1989). It should be noted that the MS-MS spectra do not

provide confirmation of E/Z stereochemistry. The E/Z assignments have been made based

on comparison of the relative retention times observed in this study with those reported

previously (Bauer and Remiger, 1989), and are only tentative without NMR confirmation.

With HPLC-ESI-MS and MS-MS data such as those shown in Figure 2.1 and

Figure 2.2, all of the previously identified alkylamides from E. purpurea in Tables 2.2 A

and B except undeca-2E-ene-8,10-diynoic acid isobutylamide (compound C) were


71

identified in the extracts prepared in this study. Although several papers cite the presence

of undeca-2E-ene-8,10-diynoic acid isobutylamide in E. purpurea root (Binns et al.,

2002b; Matthias et al., 2005b), this compound was not detected in the echinacea extracts

used for these studies. A commercially available standard of this compound was readily

detectable with limit of detection of 0.15 μM, therefore, it can be concluded that undeca-

2E-ene-8,10-diynoic acid isobutylamide was not present in the extracts at concentrations

above 0.15 μM. When selected ion monitoring is used with LC-MS, a peak with m/z 232

(the MH+ ion of 231) is apparent, but it corresponds to the MH

+ (+ 1) isotope of undeca-

2E,4Z-diene-8,10-diynoic acid isobutylamide (A) and undeca-2Z,4E-diene-8,10-diynoic

acid isobutylamide (B) (Figure 2.1A). The presence of this isotope ion may have mislead

some investigators analyzing echinacea with LC-MS and could be responsible for some

literature reports of the presence of this compound in E. purpurea (Binns et al., 2002b;

Hudaib et al., 2002). Another possible reason for conflicting results concerning the

presence of undeca-2E-ene-8,10-diynoic acid isobutylamide may be differences in the

genetics of plant material, or the environmental conditions under which it was grown.

Binns et al. (Binns et al., 2002a), using solely UV spectra and retention time, identified

this compound in E. purpurea roots of wild plants but not cultivated germlings. Hence, it is

possible that some genetic strains of E. purpurea contain undeca-2E-ene-8,10-diynoic acid

isobutylamide while others do not. It is also possible that cultivation conditions may result

in expression patterns different than that for plants surviving in a less controlled

environment. However, our laboratory has investigated over 20 different US sources of E.

purpurea and thus far not detected this compound (data not included). Another possibility

is that previous reports of the presence of undeca-2E-ene-8,10-diynoic acid isobutylamide

in E. purpurea were due to improperly identified plant material. Another Echinacea


72

species, E. angustifolia, does produce significant levels of undeca-2E-ene-8,10-diynoic

acid isobutylamide, and the misidentification of echinacea species has often been

documented (Bauer et al., 1988a; Foster, 1985; Rogers et al., 1998).

In addition to aiding in structural elucidation of known alkylamides, with HPLC-

ESI-MS it was possible to tentatively identify a new alkylamide, the structure of which has

not been previously published. This compound is undeca-2Z,4E-diene-8,10-diynoic acid

2-methylbutylamide (compound E in Table 2.2A). Identification of this compound was

based on rentention time and the correlation between MS-MS fragmentation pattern and

alkylamide structure. The mass and fragmentation pattern for compound E (Figure 2.2)

confirms that it is a 2-methylbutylamide, and indicates the level of saturation and length of

the carbon chain. The mass spectral data do not indicate stereochemistry or bond position;

however, relative retention time does suggest that this compound is the 2Z/4E isomer of

compound D. For the previously identified alkylamide isomers that vary by the 2E/4Z and

2Z/4E stereochemistry, such as compounds A/B and F/G, we have demonstrated (Figure

2.1) that the 2E/4Z isomer elutes before the 2Z/4E isomer. Thus, it is logical to assume a

similar relationship in stereochemistry between compounds D and E. However, as noted

earlier, without NMR confirmation, the reported stereochemistry of this new alkylamide is

only tentative.

Bauer et al. previously demonstrated that with reversed phase HPLC, alkylamides

with terminal alkynes elute early in the separation followed by tetraene alkylamides (Bauer

and Remiger, 1989). For the purposes of this discussion, compounds A-G, J, P and Q are

designated as polyacetylene amides (A-E and P are also known as terminal alkynes), while

the term “tetraenes” refers to the isobutyl amide isomeric compounds K (dodeca-

2E,4E,8Z,10E-tetraenoic acid), L (dodeca-2E,4E,8Z,10Z-tetraenoic acid) and M (dodeca-


73

2E,4E,8E,10Z-tetraenoic acid ) which contain for alkene groups in their fatty acid moiety.

As shown in Figure 2.1, the observed elution orders for alkylamides in our study are

consistent with those published by Bauer. The polyacetylene amides as a group (A, B, C,

D/E, F/G, H, J, P, Q) elute early in the separation, between 25 and 31 minutes, followed by

the tetraenes (K, L and M, N, O). These findings are significant in that alkynes,

specifically 8,10 terminal alkynes, have been shown to modulate CYP 450 function

(Matthias et al., 2005b), while tetraene isomer L and alkylamides N and O are ligands of

the CB2 receptor (Raduner et al., 2006). The results in Figure 2.1 suggest preparatory

scale HPLC could be used to separate groups of alkylamides with differing physiological

and pharmacological activity.

Method Validation

Table 2.4 illustrates the linear regression equations and statistical data for the

alkylamide standards. Calibration curves were plotted as area of the selected ion

chromatogram for the protonated alkylamide versus concentration. The linear range of the

calibration curves was from 3 to 100 µM, except for dodeca-2,4-dienoic acid

isobutylamide (compound O) which demonstrates lower sensitivity with this analytical

method than do the other alkylamides. The linear range for this compound was 5 -500 µM.

Correlation coefficients (R2) of the alkylamide standards ranged from 0.996-1.000. The

limit of detection (concentration required to give a signal to noise ratio, S:N, of 3:1) for

isobutylamides dodeca-2E-ene-8,10-diynoic acid, dodeca-2,4,8,10-tetraenoic acid and

dodeca-2E,4E-dienoic acid were 0.051, 0.99 and 1.2 μM respectively. Limits of

quantitation (based on S:N of 10:1) were 1.7, 3.3 and 4.1 μM, respectively. Repeatability,


74

which was assessed by calculating the standard deviation for triplicate injections of the

same extract, ranged from 0.2 to 0.5 for all compounds quantified. It should be noted that

for these studies, all samples that were quantitatively compared were analyzed on the same

day by the same operator in a single run. Therefore, the repeatability of the method is a

true measure of the limitations of the method for quantitative comparison, and there can be

no concern that differences in instrumental response from run to run (poor reproducibility)

were responsible for any of the differences in alkylamide quantity described in the

following sections.

In order to keep the analysis of the alkylamides in linear range of detection, 10, 100

and 1000 fold dilutions were necessary, depending on the alkylamide of interest.

Quantitative analysis was possible only for the alkylamides that were available as

standards, which at the time of the analysis included compounds C, J, the tetraenes (K,L

and M) and compound O. Alkylamide C is not listed in Table 2.4 because it was not used

for quantitation, as this particular alkylamide could not be found in the extracts

investigated.

Table 2.4. Calibration Parameters and Repeatability for Quantification of Alkylamides

Isobutylamide Slope

(±SEa)

Intercept

(±SEa)

R2 Linearity (in µM)

Repeatability Limit of

Detection (in μM)

Limit of Quantitation

(in μM)

Dodeca-2E-ene-8.10-diynoic acid

2.93 x107

(±1.3 x106)

2.0 x 108

(±1.3 x 108) 0.996

0.100 – 100

0.5

0.051 1.7

Dodecatetraenoic acid

4.447 x106

(±2.8 x104)

3.3 x 106

(±1.4x 106) 1.000

1.0 – 100

0.2

0.99 3.3

Dodeca-2E,4E-dienoic acid

4.386 x104

(±2.2 x102)

1.95 x 105

(±6.4 x 104) 1.000

10.0 – 500

0.2

1.23 4.1

a. SE – Standard Error

b. Repeatability is calculated as the standard deviation of the calculated concentration for triplicate injections

of the same sample within a single analysis.


75

Comparison of alkylamide yield in various E. purpurea extracts

Three extracts were chosen for comparison of alkylamide content. One of these

was prepared from fresh E. purpurea roots using 1 g roots for every 2 mL of solvent. This

extract is designated “fresh 1:2” in Table 2.5, consistent with the convention for expressing

plant:solvent ratios in the dietary supplements industry. The other two extracts were

prepared from dry E. purpurea roots with different ratios, one using 1 g dried roots per 11

mL solvent (1:11) and the other1g dried roots per 5 mL solvent (1:5). As demonstrated in

Table 2.1, all of three extracts contain the same percentage of ethanol (69%). The extracts

differ only in the nature of starting material (fresh or dry root) and the ratio of root:solvent.

Once the mass of the fresh roots is adjusted to account for water content, the fresh 1:2 and

dry 1:11 extract have equivalent ratios of dry weight plant material:mL solvent (Table 2.1).

Therefore, by comparing the composition of the fresh 1:2 and dry 1:11 extracts, it should

be possible to determine how extract composition differs depending on whether fresh or

dried roots are used for extraction. The two extracts prepared from dried E. purpurea

differ only in the ratio of g root:mL solvent (1:11 versus 1:5), therefore, by comparing the

composition of the dry 1:11 and dry 1:5 extracts, it should be possible to determine

whether changing the root:solvent ratio has an effect on extract composition.

Utilizing available alkylamide standards, the quantities of particular alkylamides in

the fresh and dry E. purpurea root extracts were determined. Table 2.5 displays these

results in terms of concentrations of the isomeric tetraenes (compounds K, L and M),

dodeca-2E,4E-dienoic acid isobutylamide (compound O) and dodeca-2E-ene-8,10-diynoic

acid isobutylamide (compound J) per mL of solvent. The three different Echinacea

extracts, fresh 1:2, dry 1:11 and dry 1:5, all contained detectable amounts of these


76

alkylamides. However, as shown in Table 2.5, the dry 1:5 extract contained the greatest

amount of these compounds. This is to be expected given the lower ratio of g roots:mL

solvent used in the preparation of the 1:5 extract.

One notable result from the quantitative analysis of akylamide content shown in

Table 2.5 is that the concentrations of compound O (dodeca-2E,4E-dienoic acid

isobutylamide) are similar to the concentrations of tetraenes. Previous investigations have

not reported dodeca-2E,4E-dienoic acid isobutylamide as a major constituent of E.

purpurea root (Binns et al., 2002b; Qu et al., 2005; Wills and Stuart, 1999). The

importance of this compound may have been overlooked due to poor sensitivity of various

Table 2.5. Quantification of Alkylamides K, L & M; O; and J in Ethanolic Extracts of E. purpurea

root.

Concentrations are recorded in mM and mg/mL except in the case of compound J, for which concentrations

are expressed in μM and µg/mL.

Extract Concentration of

Type Alkamides K, L & M

mg/mL mMa SD

b

Fresh 1:2 1.9 e7.6 1.0

Dry 1:11 2.1 e8.3 0.5

Dry 1:5 6.9 28.0 5.0

ConcentratIon of

Alkamide O

mg/mL mMa SD

b

Fresh 1:2 1.8 c7.1 3.7

Dry 1:11 2.1 d8.4 2.0

Dry 1:5 6.2 25.0 5.7

Concentration of

Alkamide J

μg/mL μMa SD

b

Fresh 1:2 6.17 c25.2 2.5

Dry 1:11 9.2 f37.4 1.0

Dry 1:5 15.6 63.4 2.8

a. The mean concentration was calculated for four replicate extractions

b. SD represents standard deviation of the concentrations in mM (μM for J).

c. Represents statistically significant difference as compared to the 1:5 root extraction, p <0.05

d. Represents statistically significant difference as compared to the 1:5 root extraction, p <0.01

e. Represents statistically significant difference as compared to the 1:5 root extraction, p <0.005

f. Represents statistically significant difference as compared to the 1:2 root extraction, p <0.001


77

analytical methods to this particular compound (Woelkart et al., 2005a). The finding that

dodeca-2E,4E-dienoic acid isobutylamide is a major constituent of E. purpurea may be of

future significance given that this compound, like the tetraene isomers, is known to be a

CB2 ligand (Gertsch et al., 2004; Raduner et al., 2006; Woelkart et al., 2005b).

In order to easily compare how efficiently alkylamides were extracted in the three

different E. purpurea extracts, the quantity of each alkylamide (mg) was divided by the dry

weight of E. purpurea root (g) used to prepare an equivalent volume of extract. The

resulting value is referred to as “alkylamide yield” (Figure 2.3).

As discussed previously, the only difference in the fresh 1:2 versus the dry 1:11

extracts is whether fresh or dry root was used in their preparation. Therefore, assuming no

loss of alkylamide during the drying process, alkylamide yield would be expected to be

very similar for these two extracts. Indeed, the alkylamide yield for the fresh 1:2 versus

the 1:11 were comparable for the tetraenes (K, L and M-Figure 2.3A) (20.4 +2.8 vs. 22.2

+1.5 mg/g, respectively) and compound O (Figure 2.3B- 19.4 +1.7 vs. 22.6 +1.1 mg/g,

respectively). For alkylamide J, the yield was somewhat lower in the 1:2 extract as

compared to the 1:11 extract (0.0667 + 00.0067 vs. 0.0989 + 0.0027 mg/g, respectively).

This difference could be due to differences in particle size between the extracts, which may

have given rise to slightly more efficient extraction in the dry as compared to the fresh

extract. Importantly, comparison of alkylamide yield for the 1:2 and 1:11 extracts does not

indicate any significant degradation of alkylamides due to drying. The extracts in this

study were prepared from roots immediately after completion of oven drying. Kabganian

et al. (2002) came to the same conclusion finding no degradation of alkylamides in roots

that were oven dried. Whether or not there is a loss in alkylamide content in E. purpurea

roots stored for long periods of time would be a worthy subject of a future investigation.


78

In comparing the extraction yield of the alkylamides between the 1:11 versus 1:5

dry root extracts, a logical prediction, provided a 1:5 extract is not saturated, would be that

the alkylamide yield would be the same in the two extracts. As can be seen in Figure 2.3,

however, this is not the case. While alkylamide yield is similar in the two extracts, there

are statistically significant differences, and the nature of these differences varies depending

on the alkylamide examined. For alkylamides K, L, M and O (Figure 2.3 A and B),

extraction is more efficient in the 1:5 as compared to the 1:11 extract. This is the opposite

of what would be expected if the solution were saturated in the case of the 1:5 extract;

therefore, saturation is not the cause of the differences. Conversely, for alkylamide J,

extraction is more efficient in the 1:11 extract than the 1:5 extract (Figure 2.3C). Previous

studies have established that solvent interactions of N-alkylamides differ depending on

molecular structure (Liu and Murphy, 2007; Martińez et al., 2002) and the surrounding

phytochemical matrix (Liu and Murphy, 2007). Therefore, it is plausible that extraction of

certain alkylamides is favored in more dilute extracts, while concentrated extracts favor the

extraction of structurally different species.

Comparison of yield of dissolved solids in various E. purpurea extracts

Although the analysis of alkylamides is particularly relevant due to their

pharmacological importance, it was also of interest to provide a broader perspective on

how composition of E. purpurea root extracts differs depending on extraction conditions.

This was accomplished by comparing the amount of total dissolved solids in the three

extracts described in Section 3.3. The quantity of total dissolved solids is determined by

removing the solvent from the extract and weighing the residue. The resulting mass is a


79

measure of how much material overall (alkylamides as well as other compounds) was

dissolved in the original extract. When the mass of dissolved solids is divided by the dry

weight of the plant material used to produce an equivalent volume of extract, the resulting

value is referred to as “extract yield.” The extract yield is a measure of how much of the

initial starting material was converted into extract.

Figure 2.3 displays the extract yield (mg dissolved solids/g dry root) for the three

E. purpurea extracts under investigation. Overall, the extract yield was similar for all three

extracts. However, the yield for the fresh root extract (196.6 ± 2.7 mg/g.) was slightly

lower than the two dry root extracts (269 ± 12 and 290 ± 24 mg/g for the 1:11 and 1:5

extracts, respectively, p < 0.001). As mentioned previously, a similar effect was observed

for alkylamide yield. Again, this difference could possibly be attributed to differences in

particle size in fresh versus dry extractions. Between the two dried extracts, there was no

statistically significant difference in extract yield. This similarity between 1:11 and 1:5

extracts indicates that it is possible, by doubling the quantity of root used for the

extraction, to double the amount of material dissolved in the solvent, at least up to a ratio

of 1:5. It should be pointed out that because of the greater amount of root used to prepare

the 1:5 extract, this extract does, overall, contain a greater concentration of dissolved solids

then the 1:11 extract. However, there is no significant difference between the two extracts

when the amount of dissolved solids is expressed relative to the mass of root used in the

extract.


80

Figure 2.3. A,B,C. Comparison of alkylamide concentrations in fresh 1:2, dry 1:11 and dry 1:5 E. purpurea

root ethanolic extracts. Each panel represents a different set of alkylamides, with the tetrane isomers

dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide and dodeca-2E,4E,8E,10Z -tetraenoic acid


81

isobutylamide (alkylamides K, L & M) displayed in Figure 2.3A, dodeca-2E,4E-dienoic acid isobutylamide

(alkylamide O) in Figure 2.3B, and dodeca-2E-ene-8,10-diynoic acid isobutylamide (alkylamide J) in Figure

2.3C. As illustrated a higher concentration of tetraenes and dodeca-2,4-dienoic acid (alkylamide O) results

from the 1:5 vs. the 1:11 root extracts, but this is limited to ≈36% and 28% increase respectively, despite

extracting twice the plant material. However, for dodeca-2E-ene-8,10-diynoic acid isobutylamide

(alkylamide J), the 1:11 extraction appears to be more efficient than the 1:5 with a 20% increase in extraction

efficiency. The fresh root extractions (1:2) appear to be roughly equivalent to the dry 1:11 extractions, except

for alkylamide J. Note that the tetranenes and alkylamide O occur in similar concentrations in these extracts.

All concentrations represent the mean from four replicate extractions at room temperature. Error bars denote

SEM. Comparisons were made between Fresh 1:2, Dry 1:11 and 1:5 extracts, with * indicating p<0.05; **

indicating p<0.005; and *** indicating p<0.001

Extraction of the tetraene isomers as a function of maceration time

Lastly, the quantity of the isomeric dodeca-2,4,8,10-tetraenoic acid isobutylamides

present in a macerating E. purpurea extract against time was measured (Figure 2.5). The

results are all displayed relative to that achieved on the first day that concentration was

measured (day 2). These tetraene isobutylamides are significant because they typically

compose from 30 – 70% of the total alkylamides in echinacea products (Modarai et al.,

2007). The results in Figure 2.5 demonstrate that the extraction of the dodeca-2,4,8,10-

tetraenoic acid isobutylamides is complete by day 2. Thus, in terms of the extraction of

specifically these compounds, maceration beyond day 2 should not be necessary. While

commonly used long maceration times seem to have little effect on alkylamide content,

long maceration times could actually be detrimental if some compounds degrade during

maceration. Future investigations of the optimal maceration time for producing an extract

with maximal concentrations of all desirable constituents are warranted.

Thus far, we are unaware of any other reports comparing extraction efficiency of

fresh and dry root ethanolic extractions of E. purpurea. Previous work by Gafner et al.

(2005) has found that a heat treatment in the initial stage of extraction (70 ○C for 30 min)

of fresh E. purpurea aerial parts decreases the enzymatic degradation of the tetraenes as


82

well as the caffeic acid derivatives. As a result, this was found to improve extraction

efficiency.

Figure 2.4. Yield of dissolved solids in ethanolic extracts of Echinacea purpurea roots.

Mass of dissolved solids was determined by evaporation of the ethanol/water solvent from aliquots of

extracts, and this value was ratioed to the quantity of root (dry weight) used to prepare an equivalent volume

of extract to calculate dissolved solids yield. Yield of dissolved solids in the 1:11 extraction does not

statistically differ from 1:5 extraction. The fresh root extraction (1:2) differs from the 1:11 and the 1:5

extraction by 26.8% and 32.4% respectively. *indicates p<0.005 ** indicates p<0.001.

Ultimately, extraction efficiency depends on extraction conditions, the constituent or group

of constituents in question, the plant part and whether the starting plant material is fresh or

dry.

Discussion

The previous data reports the alkylamide content of ethanolic extractions from

fresh versus dry root and dry 1:11 versus dry 1:5 (root:menstruum) E. purpurea. We report

relatively no differences in the extraction of alkylamides in fresh versus dry 1:11 E.

purpurea root ethanolic extraction and variable gains in concentrations of alkylamides for

only the tetraene isomers J, K & L (dodeca-2E,4E,8Z,10E/Z-tetraenoic acid & the newly


83

identified dodeca-2E,4E,8E,10E-tetraenoic acid isobutylamide) in dry 1:5 versus dry 1:11

ethanolic extraction. Thus far, we are unaware of any other reports comparing different

strengths of dry root ethanolic extractions. Previously, differences have been found in the

extraction of fresh versus dry plant echinacea. Sun et al. (2002) utilizing a supercritical

extraction technique found about 3.5 fold increases in alkylamide extractions from dry

versus fresh E. angustifolia. It should be noted though, that the use of carbon dioxide with

significant pressure (>34 MPa) affords significantly different extraction conditions. This

technique is not practiced by the majority of the manufactures in the natural products

industry. A past analysis of fresh versus dry echinacea extractions by Stuart et al. (2004)

found a significant difference in extraction of polysaccharides in fresh versus dry E.

purpurea, perhaps partly because of degradation of the polysaccharides in dry plant

material but, alkylamide content was not assessed.

Figure 2.5. Relative concentration of dodecatetraenoic acid isobutylamides (alkylamides K, L and M) in an

E. purpurea root extraction over time. Relative concentrations were calculated by dividing the concentration

of each sample by the concentration at day 2 and converting to percent. Samples were taken daily over 28

days from dry root (1:11) ethanolic maceration of E. purpurea. Results show that maximal extraction of

dodecatetraenoic acid isobutylamide is achieved by day 2.


84

In addition, these data show for the first time that alkylamide extraction from E.

purpurea root is not improved by longer steeping times in ethanolic macerations. The

tetraene extraction from E. purpurea radix is complete by day 2 (Figure 2.5). Curiously, it

appears that this conclusion had already been realized by previous generations: The

nineteenth edition of the United States Dispensatory (Wood et al., 1907), the 1942

National Formulary (Committee On The National Formulary VII, 1942) and the 1961

Remington’s Practice of Pharmacy (Remington, 1961) state that the appropriate time for a

macerating tincture of medicinal plant to steep was 3 days or until the soluble matter is

dissolved. Many manufacturers of medicinal plant products, as well as phytotherapists,

currently suggest that the appropriate time for a maceration to steep is 2 – 4 weeks,

depending on the part of the plant. According to the above results, this could be excessive.

Due to the poor stability of the Echinacea spp. phenylpropanoids (caftaric acid, cichoric

acid, cinnamic acid, echinacoside etc.) in alcohol (Gafner and Bergeron, 2005; Livesey et

al., 1999), longer steeping times may produce inferior products due to the oxidation of

these constituents (Livesey et al., 1999). Considering that the lipophilic constituents are

generally the least extractable by ethanol and that the tetraenes are lipophilic and appear to

be fully extracted in 2 days, the suggestion by manufacturers that the strength of an

echinacea maceration improves due to longer steeping times needs to be reconsidered.

Furthermore, if upcoming research elucidates in vivo phenylpropanoid mediated immune

or antioxidant activity, reconsideration of the shelf life of echinacea products may prove

advantageous to optimizing the pharmacological activity of these remedies. More research

is needed on the optimization of echinacea and other medicinal plants extractions.


85

These results demonstrate that extraction of particular alkylamides is more efficient

when the ratio of solvent to plant material is smaller, as in the case of the fresh root 1:2 or

dry root 1:11 extractions as compared to the dry root 1:5 extraction. This suggests that

increasing the ratio of plant to solvent does not necessarily result in an extract with all

alkylamides represented. Accordingly, we show that increasing the ratio of root to solvent

changes the qualitative profile (and expectedly, the quantitative profile) of alkylamides

present in an ethanolic extraction. This may be significant because differing alkylamides

have demonstrated different activities; cannabinoid activity (Gertsch et al., 2006; Gertsch

et al., 2004; Matovic et al., 2007; Muller-Jakic et al., 1994; Raduner et al., 2006; Woelkart

et al., 2005b), lipoxygenase activity (Merali et al., 2003; Muller-Jakic et al., 1994),

cyclooxygenase activity (Muller-Jakic et al., 1994), fungicidal activity (Merali et al., 2003)

and antiviral activity (Hudson et al., 2005; Vimalanathan et al., 2005).

Consequently, the qualitative chemical character of ethanolic extracts needs to be

considered when investigating biological activity of complex chemical extracts such as

echinacea remedies. The generally agreed upon observation that several constituent groups,

besides the alkylamides, are responsible for the pharmacological activity of echinacea

(Bauer, 1998; Islam and Carter, 2005; Vimalanathan et al., 2005) and the lack of clinical

studies undertaken with pure compounds (until recently), beyond a preliminary

investigation with polysaccharides (Melchart et al., 1993), create a situation where the

native extracts of echinacea must still be regarded as the “active principles” (Bauer et al.,

2005). Thus, biological activity of dilute versus concentrated tinctures may vary beyond

just what might be expected for a more concentrated product.

The above issues exponentially add to the challenge at hand in research

laboratories, clinical settings and commerce in regard to extractions of medicinal plants:


86

The extraction of bioactive phytochemicals in a complex phytochemical matrix is

inherently difficult to investigate in terms of extraction, stability, degradation, and

ADMET (absorption, distribution, metabolism, excretion and toxicity) properties.

Summary

With these investigations, it has been demonstrated that HPLC-ESI-MS is an

excellent technique for comprehensive analysis of alkylamides from Echinacea purpurea.

Using HPLC-ESI-MS, a more comprehensive alkylamide profile was obtained than is

typically possible with other analytical approaches. By relying on collisionally induced

dissociation, it was possible to distinguish between isomeric alkylamides, and to

tentatively identify a new E. purpurea alkylamide, undeca-2Z,4E-diene-8,10-diynoic acid

2-methylbutylamide.

All three extraction techniques investigated here (fresh 1:2, dry 1:5 and dry 1:11)

resulted in very similar alkylamide profile, and gave similar yields of alkylamides and of

total dissolved solids. The similarity in alkylamide content in fresh 1:2 and dry 1:11

extracts indicates that drying of root material at 50 °C does not result in a loss of

alkylamides. It appears that either fresh or dried roots can be used to prepare extracts with

high alkylamide content, although the overall yield was slightly lower for fresh extracts.

Lastly, the analysis of a 1:2 fresh root ethanolic extract suggest that the maximum

concentration of the tetraenes (dodecatetraenoic acid isobutylamides) is achieved by day 2

in an ethanolic extraction.

Although alkylamide yields were, overall, similar with the three extraction

techniques, there were some statistically significant differences in quantities of


87

alkylamides extracted. Notably, several alkylamides quantified (the isomeric tetraenes and

dodeca-2,4-dienoic acid isobutylamide) were extracted more efficiently with a root to

solvent ratio of 1:5 (w:v) as compared to a ratio of 1:11, while another alkylamide (dodeca-

2E-ene-8,10-diynoic acid isobutylamide) was extracted more efficiently with a ratio of

1:11. Given that the biological activity of alkylamides differs depending on structure, these

suggest that pharmacological activity of Echinacea purpurea extracts could differ

depending on the ratio of root:solvent used in extraction. Ultimately, in vitro and in vivo

studies are needed to elucidate the differences in pharmacokinetic and pharmacodynamic

activity of various Echinacea extracts. The results presented in this paper do, however,

suggest that it would be erroneous to assume that all ethanolic extracts of Echinacea

purpurea result in equivalent phytochemical profiles.

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Chapter 3 Degradation of Alkylamides

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Chapter 3

An HPLC-ESI-MS evaluation of the stability of alkylamides in ethanolic extract, cut/sift and powdered E.

purpurea root

Introduction

Recent sales of medicinal plant products in the U.S. reached 4.8 billion dollars

(Cavaliere et al., 2009). The National Health Interview Survey estimates that 10% of the

US population, about 29 million Americans, uses herbal products (Pleis and Lethbridge-

Cejku, 2006). A significant portion of these products come in either powdered form (in

bulk or in capsules), cut and sift (cut/sift-small cuttings of plant material sold in bulk) or

ethanolic extracts. Regardless of the form of plant material purchased ultimately, much of

this material becomes galenic ethanolic extracts, prepared by maceration or percolation of

the starting plant material in a variable ratio of ethanol and water depending on plant

species, plant part and manufacturing practices. While there is little research that elucidates

the stability of herbal products, some have suggested that the shelf life of these products is

unlimited (Low Dog, 1991). Of the investigations that have been done, in some cases

shelf-lives of less than 6 months have been reported for popular herbs in ethanolic extract

form (Bilia et al., 2002a). Unfortunately, storage conditions are a commonly unassessed

and unreported factor in research assessing medicinal plant efficacy, leaving a gap in the

assessment of the research on medicinal plants and a possible explanation for

inconsistencies in outcomes in such popular herbal remedies such as Echinacea spp.

Echinacea products reach sales well over U.S. 15 million in 2008 (Cavaliere et al.,

2009) and 182.9 tons of echinacea were wild harvested in 2004 (American Herbal Products

Association, 2007). Echinacea spp., are now grown in Austria, Germany, Russia, New


97

Zealand, Ukraine, Yugoslavia, and the Republic of South Africa (Letchamo et al., 2002),

as well as in Australia, where the annual production is more than 40 tons (Stuart et al.,

2004). Additionally, Asia, Latin America and the Middle East are involved in cultivation,

processing and marketing of Echinacea (Letchamo et al., 2002). E. purpurea is the

dominant species used in global production representing 80% of commercial production

(Li, 1998).

Currently it is known that of the constituents considered active in E. purpurea

products, alkylamides, phenylpropanoids, polysaccharides, and glycoproteins (Bauer,

1996, 1998), that not all of these constituents are stable in ethanolic extracts. As

mentioned in Chapter 2, in ethanolic extractions above 40%, there are very few

polysaccharides left in suspension and denaturing of proteins is expected (Dalby-Brown et

al., 2005), however these compounds extract well in glycerin (Bergeron and Gafner, 2007)

but are unstable in fresh plant material (Stuart et al., 2004). The phenylpropanoids (caffeic

acid derivatives such as cichoric acid (2R,3R-dicaffeoyl tartaric acid) (Bauer et al., 2001;

Hudec et al., 2007) are antioxidants known to be highly efficient (Hudec et al., 2007) and

active in immunological assays (Bauer, 1999; King et al., 1999; McDougall et al., 1998;

Nusslein et al., 2000). These, constituents are known to degrade rapidly in both cut/sift and

ethanolic extracts depending on the form of the preparation (Gafner and Bergeron, 2005;

Stuart and Wills, 2000). Accordingly of the four constituents considered active in E.

purpurea the only remaining constituent after a few weeks on the shelf in ethanolic

extracts are the alkylamides.

Thus far, of the constituents considered active in Echinacea spp. pharmacokinetic

studies suggest that alkamides are the predominant constituent circulated in human plasma

(Matthias et al., 2005; Woelkart et al., 2006) although in vitro investigations of


98

immunological activity have suggested multiple active constituents of Echinacea spp.

(Bauer, 1998). A recent small trial (n = 8) of the pharmacokinetics of E. purpurea (95%

herb, 5% root) demonstrated that the alkylamides from the tincture (65% ethanol) were

shown to be more bioavailable than from the capsule. However, equivalent immune

activity was shown for both preparations (Woelkart et al., 2006).

The stability of the highly unsaturated alkylamides in Echinacea spp. are an issue

that has yet to be adequately addressed, especially considering that they are likely to

oxidize (Bauer et al., 1988; Livesey et al., 1999). Specific to the alkylamides in E.

purpurea root, Stuart and Wills (2000) suggest stability of various alkylamides in dry root

and aerial material at 20 °C for 4 months, while Livesey et al.(1999) suggest stability up to

7 months regardless of temperature (-20, 25 or 40 °C) of the isomeric dodeca-2,4,8,10-

tetraenoic acid isobutylamides from roots. Conversely, Rogers et al. (1998) found levels of

alkylamides fell by 16% over 8 weeks. Perry et al. (2000) found a decline in alkylamide

over 16 months storage regardless of temperature (-18 & 24 °C) with the degree of

degradation depending on the molecular species of alkylamide. Finally, McCann et al.

(2007) demonstrated an increase of various alkylamides in E. purpurea at -20 over two

year’s time. Considering the previous data there appears to be a wide variance in results for

the stability of the alkylamides.

As for the stability of the alkylamides in medicinal plants whether in ethanolic

extracts, powder or cut/sift, the few investigations that have examined the stability of these

fatty acid derivatives offer conflicting information on their stability. Although Bohlmann

(1973) suggested over 3 decades ago that alkylamides were unstable in medicinal plants,

Stuart and Wills (2000) suggest that the alkylamides are stable in ethanolic extracts at 20

°C for 4 months in E. purpurea radix. Livesey et al. (1999) suggests stability up to 7


99

months at -20 °C, 25 or 40 °C in the E. purpurea root of the lipophilic constituents with an

80% decrease under the same conditions of the cichoric acid. Kabganian et al. (2002)

found that the isobutylamides undeca-2E-ene-8,10-diynoic acid and the isomers of dodeca-

2,4,8,10-tetraenoic acid (the tetraenes) in the roots of E. angustifolia, dried from 23 to 60

°C, were stable. while McCann et al. (2007) found an increase in alkylamide

concentrations in E. purpurea, but no change in E. angustifolia and E. pallida.

Conversely, in powdered root of E. purpurea root the tetraenes degraded by 80%

over 7 months at 24 °C and a 95% reduction was observed at 40 °C (while the cichoric

acid did not) compared to samples at -18 °C (Livesey et al., 1999). Perry et al. (2000)

found that in cut/sift form the dodeca-2,4-ene-8,10-diynoic acid isobutylamide isomers

were significantly decreased in 48 hours as compared to unchopped roots, but no other

alkylamides were changed. Rogers et al. (1998) found that E. angustifolia root powder loss

of 13% in 8 weeks (total alkylamides). Perry et al. (2000) found that concentrations of

dodeca-2,4-diene-8,10-diynoic acid isomers decreased in cut/sift (48 hr drying time) and

they reported declines of 80% or more, depending on the alkylamide, in cut/sift root stored

at 24 °C and 60% declines in alkylamide levels over 16 months at -18 °C. Yang (2008)

found degradation of the hydroxylated alkylamide, hydroxyl-sanshool, in 80% ethanol

with a 70% decrease in 24 hours and complete degradation in 4 hours due to UV light

exposure. Interestingly, these authors also suggested that the taste of huajiao (fruit of

Zanthoxylum bungeanum or Z. schinifolium), in this case relating to the characteristic

alkylamide induced tingling sensation, was not significantly changed after one year of

storage at room temperature (Yang, 2008). In the case of extracts from Echinacea spp.

traditional healers have suggested that the quality of the extract is proportional to the

amount of tingling produced by a few drops on the tongue.


100

Of particular concern is that the in vitro immunological activity of E. purpurea

extracts have been found to change after storage (McCann et al., 2007; Senchina et al.,

2005). This is unfortunate as E. purpurea and E. angustifolia are reported to be one of the

most frequently used medicinal plants in clinical settings. Of the most commonly

prescribed drugs in Germany, echinacea preparations have been in the top 200 for a

number of years (Der Marderosian, 1991). Considering the documented changes in

immunological effects as well as the global use of echinacea products by laboratories,

clinicians and consumers and the differing results in the stability studies, there is a need for

further evaluation of the degradation of the alkylamides of the echinacea species. The

following data demonstrates the degree of degradation of alkylamides in E. purpurea root

in various preparations (ethanolic extractions, cut/sift, powder) over one year’s time.

Methods

Preparation of Echinacea purpurea Extracts

E. purpurea roots were cultivated in Grants Pass, OR at Pacific Botanicals and

harvested in a fresh, dormant state at two years of age. Species verification was performed

by Richard Cech (Horizon Herbs, Williams, OR). Voucher specimens are available at the

University of North Carolina Herbarium in Chapel Hill, NC (accession numbers 583416

and 583417). Water content was determined to be 74.5% after samples were dried at 50

°C. To prepare the extracts, roots were washed thoroughly, briefly soaked (5 min) in 70%

ethanol as a disinfectant, and blown partially dry with compressed air. Approximately 16.6

g of dried root was added to 83 mL of solvent (74.5% ethanol + 25.5% distilled water) for


101

the 1:5 extractions within 6 hours of drying and within 72 hours of harvest. This

constitutes a ratio of 1g plant material: 5 mL solvent, hitherto referred to as 1:5. The same

root material was then stored in powdered or in cut/sift form. One year later the stored

material was then extracted at 1:5 in the same manner as previously mentioned. All

extracts were macerated for one month at which time the solvent was removed using a

hydraulic press. Four replicate extracts were prepared for each extraction (original extract,

one year aged cut/sift, one year aged powder).

Analysis of extracts

An aliquot of each sample (500 µL) was centrifuged to remove particulate matter

and the supernatant was run undiluted and diluted (1000-fold) in 75% ethanol and analyzed

using a validated liquid chromatography-mass spectrometry (LC-MS) method (Spelman et

al., 2009b). An ion trap mass spectrometer with electrospray ionization source (LCQ

Advantage, Thermo Finnigan, San Jose, CA) was employed. The solvent gradient and

instrument conditions have been previously published (Spelman et al., 2009b). All

analyses for the comparison of cut/sift and powder storage forms against the one year old

ethanolic extract of E. purpurea root were performed in a single run to minimize

variability due to fluctuation in instrument response over time. Quantification was

accomplished using a validated method based on alkylamide standards.(Spelman et al.,

2009b) Calibration curves were generated for available alkylamides (Chromadex, Santa

Anna, CA) dodeca-2E-ene-8,10-diynoic acid isobutylamide (MW 245.37) and dodeca-

2E,4E,8Z,10Z-tetraenoic acid isobutylamide (MW 247.38) as previously described

(Spelman et al., 2009b). Calibration curves consisted of concentration ranges of 1.0 to 100


102

µM for the dodeca-2,4,8,10-tetraenoic acid isobutylamides (R2 > 0.992) and 1.0 to 500 μM

for dodeca-2E-ene-8,10-diynoic acid isobutylamide (R2 > 0.998). Plant extracts were

analyzed within 24 hours of pressing (separation of plant material from solvent).

Statistics analysis

The standard error of the mean (SEM) was determined for each set of

concentrations. Data is expressed as the mean ± SEM and comparison of means was

conducted using a two tailed t-test for paired data when differences were observed. The

mean values were considered significantly different if p < 0.05. Statistical analysis was

performed with Microsoft Excel (2007).

NH

OA

NH

O

B

Figure 3.1. Quantified Alkylamides in E. purpurea preparations.

A. Dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide (one of 3 isomeric tetraenes)

B. Dodeca-2E-ene-8,10-diynoic acid isobutylamide (a polyacetylinic alkylamide)


103

Results

Determination of alkylamide identity

Figure 3.1 illustrates the structures of the alkylamides quantified in this study. The

identity of the isobutylamides dodeca-2,4,8,10-tetraenoic acid (tetraenes, an isomeric

mixture MW 247.3) and dodeca-2E-ene-8,10-diynoic acid (MW 245.3) were confirmed by

retention times and MW weights of the standards. A number of other alkylamides for

which standards were not commercially available were also identified in the E. purpurea

root extracts. The alkylamide identities in E. purpurea root have been extensively reported

elsewhere (see Spelman et al., 2009b and Chapter 2).

Quantitative determination and comparison of alkylamide content in one year old

aged ethanolic extract vs. freshly made ethanolic extract

Figure 3.2 exhibits the data comparing the concentrations of the isomeric tetraenes

and dodeca-2E-ene-8,10-diynoic acid isobutylamide in the newly made (within 72 hours of

harvest) vs. the same extract one year later of E. purpurea root extracts. Figure 3.2A shows

the tetraene concentration at 6.27 mg/mL (± 0.73 mg/mL) in the newly made extract. The

tetraene concentration, in the aged extract, shows no statistically significant difference.

Conversely, Figure 3.2B illustrates a substantial loss of the diactylene isobutylamide

dodeca-2E-ene-8,10-diynoic acid. The newly made extract contained 27.1 μg/mL (± 4.0

μg/mL) and one year later the concentration of this alkylamide had declined to 13.3 μg/mL

(± 0.41 μg/mL).


104

Figure 3.2. Comparison of alkylamide stability in E. purpurea root ethanolic extracts over one year’s time.

A. Dodeca-2,4,8,10-tetraenoic acid isobutylamides remain at the original concentration after one year’s storage.

B. Greater loss of alkylamide dodeca-2E-ene-8,10-diynoic acid isobutylamide is observed in the same extract with

a loss from 27.1 (± 4.0) μg/mL to 13.3 (± 0.41) μg/mL over one year’s storage. All concentrations represent the

mean from four replicate extractions (dry 1:5) at room temperature displayed in mg/mL (tetraenes) and μg/mL

(dodeca-2E-ene-8,10-diynoic acid isobutylamide). Error bars denote standard error of the mean (SEM).

Comparisons are made between newly made extract and one year old extracts with * indicating p<0.05.


105

Comparison of alkylamide content in one year old E. purpurea root in cut/sift,

powder and ethanolic extracts

Figure 3.3 exhibits the percentage remaining of the two quantified alkylamides in

root samples that were stored at room temperature in the dark. These samples, all

originating from the same batch of E. purpurea root, were stored as powder, cut/sift and in

the original extraction. Thus, a comparison is possible for extracts that have aged one full

year. A substantial loss of tetraenes is seen for both the cut/sift and the powdered root in

Figure 3.3A. The cut/sift shows a tetraene loss of 42.2% (57.8% ± 3.1% remaining) while

the powder form has lost 89.2% (10.8% ± 1.3% remaining) of its tetraenes as compared to

the original ethanolic extraction. Figure 3.3B illustrates a 50.5% (49.5% ± 1.5%

remaining) loss in the ethanolic extract and 75.7% (24.3% ± 0.23% remaining) and 82.9%

loss (17.1% ± 0.36% remaining) of dodeca-2E-ene-8,10-diynoic acid in the cut/sift and

powder, respectively.

Discussion

Bohlmann et al. (1973) initially reported alkylamides to be unstable and this is

supported by work with the isolates, in which oxidation, photodegradation, isomerization

and hydrolysis are known to occur under normal storage conditions (Livesey et al., 1999;

Yang, 2008). However, research has resulted in conflicting data on the stability of

alkylamides in ethanolic extracts, we show a reduction of alkylamides in all storage forms

of E. purpurea root investigated; ethanolic extract, cut/sift and powder, with the exception

of the tetraenes in a dry 1:5 extract. While the presented results for the dry 1:5 ethanolic


106

Figure 3.3. Percentage of alkylamides remaining after one year’s storage in ethanolic extract, cut/sift and

powdered E. purpurea root.

A. Dodeca-2,4,8,10-tetraenoic acid isobutylamides remaining in cut/sift vs. powdered root is 57.8 (± 3.1)% and

10.9 (± 1.3) % after one year’s storage, respectively. B.Dodeca-2E-ene-8,10-diynoic acid isobutylamide remaining

after one year’s storage of extract, cut/sift and powdered root is 49.1 (± 1.5) %, 24.3 (± 0.22) % and 17.1 (± 0.36)

% respectively. All concentrations represent the mean from four replicate extractions at room temperature, stored

at room temperature in the dark. Error bars denote standard error of the mean (SEM). Comparisons were made

between newly made dry 1:5 extract, one year old dry 1:5 extract and one year old cut/sift and powdered root.

Comparisons are made between each newly made extract (original) and each bar (one year old extracts for cut/sift

and powder) with * indicating p<0.05; ** indicating p<0.005.


107

extracts concur with previously published investigations (Perry et al., 2000; Rogers et al.,

1998), they stand in stark contrast to others (Livesey et al., 1999; Stuart and Wills, 2000;

Wills and Stuart, 2000). The presented results for dry forms of echinacea are comparable to

other reports on the stability of alkylamides (Livesey et al., 1999; Perry et al., 2000; Wills

and Stuart, 2000). Though, following the degradation of alkylamides in E. purpurea root in

various forms over one year, are novel.

The alkylamides in E. purpurea are mainly derived from undeca and dodecanoic

amides, with varying degrees of unsaturation and different double bond configurations in

the alkyl moieties (E/Z). The alkylamides quantified in these investigations, the tetraenes

and dodeca-2E-ene-8,10-diynoic acid isobutylamide (Figure 3.1), comprise 60% of the

total alkylamides in roots of E. purpurea. While in the aerial parts, the tetraenes alone

constitute about 76% of total alkylamides in E. purpurea, the vast majority from the flower

(Wills and Stuart, 1999). Other investigations have evaluated the less prominent

alkylamides (Liu and Murphy, 2007; Perry et al., 2000). Liu and Murphy (2007) showed

that the undeca-2,4-dienes with a diacetylinic tail degraded faster than the dodeca-2,4-

dienes with acetylenic tails, the triene compound, dodeca-2E,4E,10E-trien-8-ynoic acid, or

the tetraenes in both dry films and DMSO. Both Perry (2000) and Liu and Murphy (2007)

reported that the 2-methylbutyl amide, dodeca-2E,4E-diene-8,10-diynoic acid, had the

slowest degradation kinetics. Of interest, the terminus of the alkyl group, either H or CH3,

affects the degradation rate. Alkylamides with a polyacetylene alkyl moiety that ends in an

H degrades faster than the polyacetylene moiety terminating in a CH3. The degradation rate

of all alkylamides followed apparent first-order reaction kinetics for both Liu and Murphy

(2007) and Perry et al. (2000).


108

The presented data demonstrates that the olefinic tetraene isobutylamides degraded

at a lesser rate than the acetylenic isobutylamide dodeca-2E-ene-8,10-diynoic acid in

cut/sift and ethanolic extract. In the cut/sift the polyacetylene alkylamide measured appears

to degrade faster than the tetraenes at 42.2 (± 3.1) and 75.7 (± 0.23%) % loss, respectively

(Figure 3.3). The measured alkylamides in the powdered echinacea root appear to degrade

at a similar rate. Perry (2000) reports that dodeca-2,4-diene-8,10-diynoic acid

isobutylamide, a similar structural theme as the measured dodeca-2E-ene-8,10-diynoic acid

isobutylamide, degraded in 48 hours of drying time (32 °C) as compared to the tetraenes.

Kabganian et al. (2002) found that the isobutylamides undeca-2E-ene-8,10-diynoic acid

and the isomers of dodeca-2,4,8,10-tetraenoic acid (the tetraenes) in whole roots of E.

angustifolia were stable regardless of drying temperatures from 23 to 60 °C over 10 days.

Although ethanol extracts represent the common solvent used for preservation in

the natural products industry, differing solvents appear to influence stability for the

alkylamides (Liu and Murphy, 2007; Martińez et al., 2002). The presented data show a

50.5% loss of the polyacetylene alkylamide dodeca-2E-ene-8,10-diynoic acid

isobutylamide, as compared to no loss of olefinic tetraenes in ethanolic extracts (Figure

3.2). Alklylamides in aqueous or acidic extractions have previously shown to breakdown

rapidly. Isolated hydroxylated alkylamides in an 80% ethanolic solution are reported to

degrade by 70% in 24h (Yang, 2008). Conversely, others report that alkylamides were

stable for 7 months in ethanol (Livesey et al., 1999). McCann et al. (2007) found no

change in E. angustifolia radix extracts, but an increase in alkylamide concentrations in E.

purpurea radix. The increase was speculated to be due to solvent evaporation over two

years. Alkylamides from lyophilized ethanolic extracts of E. purpurea root reconstituted in

DMSO degraded faster than the dry film of E. purpurea root extracts (Liu and Murphy,


109

2007), while alkylamides in 70% methanol are reported to be stable at least 5 months

(Gafner and Bergeron, 2005). Perry et al. (2000) suggest that alkylamides are stable in

acetonitrile for at least 2 years. In regard to laboratory research, considering the solubility

issues reported for the alkylamides (Cech et al., 2006), this issue should be carefully

considered in regard to experimental protocols. The commonly used DMSO is not a

favorable solvent for alkylamide solubility/dissolution perhaps due to its polar nature.

Besides solvents, various constituents in E. purpurea extracts also appear to

influence the degradation rate of alkylamides. Previous results have suggested that the

phenylpropanoids retard degradation of the alkylamides. Cichoric acid, the major

phenylpropanoid in E. purpurea, and echinacoside, from E. angustifolia have been

reported to be the most potent antioxidants of the phenylpropanoids (Dalby-Brown et al.,

2005; Pellati et al., 2005). The phenylpropanoids (cichoric acid, caftaric acid, caffeic acid,

chlorogenic acid) in dry E. purpurea extracts have been reported to retard the degradation

of the alkylamides as compared to phenylpropanoid free extracts (Liu and Murphy, 2007).

However, this does not hold true for all solvents. In DMSO extracts, phenylpropanoid rich

extracts degraded faster than phenylpropanoid-poor extracts (Liu and Murphy, 2007).

Adding to the complexity, the phenylpropanoid content, common to many medicinal plant

species, have been reported to vary remarkably in echinacea products, ranging from 0.0 to

8.3 mg/g (Wills and Stuart, 1999). This is likely due to wide variations of quality in the

starting plant material. To complicate matters further, the phenylpropanoids are suggested

to breakdown quickly during the extraction process (Gafner and Bergeron, 2005; Nüsslein

et al., 2000), which may facilitate the degradation of alkylamides in ethanolic extracts (Liu

and Murphy, 2007). These issues should be carefully considered before making blanket

statements concerning the stability of echinacea products.


110

As previously mentioned, oxidation and hydrolysis of alkylamides are known to

occur under normal storage conditions (Livesey et al., 1999; Yang, 2008). Hence, an

interesting direction to explore would be the addition of antioxidants to alkylamides in

solution. This strategy has been shown to be effective for the stabilization of

phenylpropanoids in Echinacea spp. but has not been explored for the alkylamides. For

example, the addition of citric acid, malic acid or hibiscus extract were effective at

retarding the degradation rates of phenylpropanoids (Bergeron et al., 2002). Ascorbate has

also been shown to preserve the phenylpropanoids (Nusslein et al., 2000). Retarding the

degradation of the phenylpropanoids, would logically lead to the stabilization of the

alkylamides.

Considering that recent work has demonstrated activity in both the polyacetylene

alkylamides and the olefinic alkylamides (Raduner et al., 2006; Spelman et al., 2009a),

these results are of significance to laboratory outcomes and the efficacy of echinacea

products in the natural products industry. Importantly, the presented data, as well as the

reviewed data, suggest that prudence must be observed when echinacea extracts are

evaluated for biological activity due to rapid degradation of particular alkylamides.

The degradation of key constituents in medicinal plant preparations are not unique

to echinacea. Other species of medicinal plant extractions have also shown significant loss

of key constituents. Previous investigations have demonstrated that ethanolic extracts of

calendula flower (Calendula officinalis), milk-thistle (Silybum marianum), passionflower

(Passiflora incarnata), hawthorn (Cratagaegus oxycantha), hawkweed (Hieracium

pilosella) St. John’s Wort (Hypericum perforatum) and artichoke (Cynara scolymus) have

short shelf-lives (t90; i.e., 10% potency loss from the initial value of a compound) due to

the degradation of active constituents or marker compounds (Bilia et al., 2002a; Bilia et al.,


111

2002b; Bilia et al., 2007). For example, the napthodianthrone compounds hypericin, of

Hypericum perforatum, have a shelf-life of only 2 months. The flavolignans of silymarin in

ethanolic extracts; silybin, silychristin and silydianin (Bilia et al., 2002a), and the luteolin

flavonoids of Cynara scolymus (Bilia et al., 2002b) have shown shelf-lives (25 ○C) as short

as three months depending on water content of the extract and/or concentration of the

extract. Reports for Calendula officinalis and Passiflora incarnata suggest shelf-lives of 4

and 6 months respectively, as assessed by the flavonoid content (Bilia et al., 2002a).

Hawthorn is reported to have a shelf-life of only 7 months as measured by

proanthocyanidin concentrations (Bilia et al., 2007). Besides normal aging processes at

room temperature, extracts of medicinal plants have been shown to be highly sensitive to

changes in storage conditions such as humidity, temperature, and light (Kopleman et al.,

2001; Yang, 2008).

While assuring the quality of medicinal plant preparations is important, attempting

to quantify the quality of medicinal plant products with 1 or 2 marker compounds does not

ensure chemical equivalence nor necessarily equivalent pharmacological activity of

complex extracts (Kopleman et al., 2001). Gafner and Bergeron (2005) point out that the

future of understanding these compounds lies in embracing these extracts as patterns of

chemical information in contrast to more stringent legal requirements based on a single

marker compound. Indeed there have been recent attempts at using information theory to

aid in the understanding of complex extracts (Gong et al., 2003; Liang et al., 2004). And

since many of the secondary metabolites, considered superfluous to activity, have been

shown to induce a physiological response, pharmacological understanding of these

compounds must come from a global view as offered by systems biology or network

pharmacology.


112

Summary

Medicinal plant extractions are carried out with various methods in the research

laboratories and the natural products industry and these methods may affect quality.

Besides handling and extraction methods, solvents used and storage conditions such as

temperature, exposure to light, co-occurring constituents and the addition of antioxidants

influence the stability of these preparations. Specifically, in the case of Echinacea

purpurea the presented data demonstrate that there is a remarkable loss of alkylamides in

cut/sift and powder forms of root. In addition, there is a 50.5% loss of dodeca-2E-ene-

diynoic acid isobutylamide over 12 months in the ethanolic extracts but the tetraenes are

stable. These investigations suggest that the best preservation of the alkylamides is in

ethanolic extracts.

These data also show that for the ethanolic extracts of echinacea the alkylamides

are relatively stable as compared to the cut/sift or powdered form of E. purpurea roots. It

should be noted that a significant portion of herbal products that are not extracted with

ethanol are stored and sold as powder and cut/sift. The presented results demonstrate that

storage in powder form and cut/sift results in significant degradation of alkylamides. This

may result in suboptimal concentrations of alkylamides available for research purposes and

producing efficacious products. It is reasonable to extrapolate these data to storage of other

medicinal plant species containing alkylamides with a similar structural theme.

Stabilization of medicinal plant preparations is key to providing high quality material to

research laboratories, clinicians and consumers. The presented data suggest that mass

marketed herbal products should contain a harvest date and a processing date, as well as a


113

shelf-life expiration date. These results emphasize the problems for quality control of

medicinal plant preparations, in which the biologically active components may be unstable.

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Wills, R. B. H., and Stuart, D. L. (2000). Effect of handling and storage on alkylamides

and cichoric acid in Echinacea purpurea. J Sci Food Agric 80, 1402-1406.

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and Bauer, R. (2006). Bioavailability and pharmacokinetics of Echinacea purpurea

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96.

Chapter 4 Metabolism of the Alkylamides

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Chapter 4

Metabolism of the alkylamides in Echinacea spp. by Human Liver Microsomes and CYP450 isoforms

Introduction

A large variety of phytochemicals are found in foods, beverages, spices and

medicinal plants commonly consumed in the human diet. Many of these phytochemicals

are seen as having a health promoting or disease preventing activity (McCarty, 2004). In

addition, many of the same phytochemicals are known to change their activity after

metabolism as well as influence the metabolism of other exogenous agents such as

pharmaceutical drugs (Ioannides, 1999). Interestingly, the health effects of phytochemicals

are still considered debatable despite substantial in vitro, in vivo and epidemiology data for

many phytochemical groups. Few health claims on such phytochemicals have so far been

approved by regulatory

authorities. Conversely, the scant data on metabolism of

phytochemicals has spawned a sizable body of literature known as herb-drug interaction,

and has generated a staunch dogma on the dangers of phytochemical mixtures. This is in

spite of a long evolutionary precedent of human food sources including substantial

quantities of medicinal and food plants and their inherent phytochemical mixtures.

An example of medicinal plants with a long history of human use is the taxa

Echinacea, which Native Americans introduced to European settlers. Shortly thereafter, it

became one of the most popular remedies used by the Eclectic physicians for a variety of

conditions, including septicemia, typhoid fever and catarrhal conditions of the respiratory

and reproductive tracts (Felter, 1922; King et al., 1898). Additionally, physicians


118

practicing from the 19th

century through the mid 20th

century used echinacea to treat other

infections and counteract venomous snakebites (Barrett, 2003b). Echinacea was included

in the National Formulary from 1906 until 1950, eventually falling out of favor as

pharmaceuticals came to dominate clinical therapeutics. Currently, the aerial portion of E.

purpurea and the roots of E. pallida are approved for colds and infection of the respiratory

and urinary tracts by the German Commission E (Blumenthal et al., 1998).

Some of the Echinacea spp. phytochemicals, such as polyphenols, and alkylamides,

have raised considerable interest for their favorable effects on health and the prevention of

various diseases. Early research suggests that polyphenols, including the phenylpropanoids

found in echinacea, have antioxidant activity and may prevent cardiovascular diseases,

cancers, diabetes, osteoporosis,

and neurodegenerative diseases (Hudec et al., 2007;

Scalbert et al., 2005).

Alkylamides, although not as widely known, also have considerably broad effects,

including anesthetic, (Greger, 1984), insecticidal (Greger, 1984; Jacobson, 1948; Jacobson,

1967; Jondiko, 1986; Meisters and Wailes, 1966; Towers and Champagne, 1988), anti-

parasitic (Douvres et al., 1980; Foster et al., 1996; Lozano et al., 1984) and oncolytic

(Voaden and Jacobson, 1972), and most recently anti-plasmodial activity (Spelman et al.,

2009a). The alkylamides are also known to have particularly intriguing immunological

activity (Gertsch et al., 2006; Goel et al., 2002; Maass et al., 2005; Spelman et al., 2009b).

Consequently, Echinacea spp. preparations are one of the best sellers in the dietary

supplements industry and regularly prescribed in Germany to boost immune function in

cases of upper respiratory tract and urinary tract infections (Blumenthal et al., 1998).

Despite a long history of use in medicine, there is relatively little information on

the metabolism in mammalian cells of the constituents of Echinacea spp., especially the


119

alkylamides. At the same time, there appears to be a plethora of literature that is suggestive

of the effects of echinacea on the metabolism of exogenous agents, primarily concerning

the cytochrome P450 system.

The bulk of the available data on possible drug-echinacea interactions concerns the

cytochrome P450s (CYP450), an ancient superfamily of primarily microsomal and

mitochondrial heme-thiolate proteins. It has been estimated that 400 million years ago a

dramatic expansion of this enzyme family driven by several factors including the

introduction of plant allelochemicals into the diet (when aquatic organisms moved to land)

(Danielson, 2002). CYP 450 genes code for enzymes that play a role in the metabolism of

drugs and foreign chemicals as well as arachidonic acid metabolism and formation of

eicosanoids, cholesterol metabolism, bile acid synthesis, steroid metabolism, vitamin D

and vitamin A metabolism (Nebert and Russell, 2002). CYP450s are crucial in

understanding the clinical pharmacology of drug interactions in addition to inter-individual

variability in drug metabolism.

Though detoxification is a limited aspect of the CYP450 overall function, particular

families of the CYP450 system (CYP2, CYP3 , CYP4 and CYP6) do play a crucial role in

Phase I detoxification (Danielson, 2002). Although Phase I detoxification is generally

associated with hepatic tissue, these enzymes are found in many other tissue types (Nebert

and Russell, 2002). Reactions such as epoxidation, hydroxylation, heteroatom oxidation,

and N- or O- dealkylation, are common strategies for the oxidation of generally unreactive

hydrocarbons (Equation 4.1).

P450[Fe+3

]-RH + O2 + NADPH + H+ → P450[Fe+3

] + ROH + H2O + NADP+

Equation 4.1. The catalytic cycle of cytochrome P450


120

The above redox reaction takes place by initial activation by the heme group of

cytochrome P450 of the oxygen molecule. The first step in the process involves two

electrons from NADPH being passed to the heme by way of a reductase. These electrons,

via oxygen, form a molecule of water. The highly charged remaining oxygen is then

primed to oxidize the substrate.

Even though, there appears to be abundant literature on the Echinacea spp. effects

on metabolism through the CYP450 system, in reality there are only 10 papers in the

primary literature that provide original data. The metabolic influence of Echinacea spp.

and the alkylamides mainly focuses on the predominant alkylamides known to be

responsible for drug interactions with the CYP1, CYP2 and CYP3 families.

CYP1A2

In vitro investigations have found no overall effect of the tetraenes (isomers of

dodeca-2,4,8,10-tetraenoic acid isobutylamide) and dienoic (dodeca-2E,4E-dienoic acid

isobutylamide) isobutylamides on CYP1A2 even at high extract concentrations (up to 200

mM), but found that an extract of E. purpurea herb and root did have an inhibitory effect

(IC50 = 30.21 μg/mL) (Modarai et al., 2007). Both Gurley et al. (2004) and Gorski et al.

(2004) report human pharmacokinetic results involving 1A2. In a study of 12 volunteers

who took 1600 mg/d Echinacea purpurea root for 8 days, Gorski et al. (2004) found

significant reduction in oral caffeine clearance by 27%. The authors postulated that

inhibition at this level could affect 1A2 substrates with narrow therapeutic index such as

theophylline, and clozapine. Gurley et al. (2004) found no significant inhibition of 1A2 in


121

healthy volunteers after 28 days of 1600 mg/d of whole-plant extract of Echinacea

purpurea, although a 13% decrease in 6 hr paraxanthine/caffeine values suggested a

possible inhibitory effect of E. purpurea whole plant extract on 1A2. This effect, however,

was not statistically significant and was not considered clinically significant by the authors.

CYP2C9

Yale and Glurich (2005) using an unrevealed concentration of a remarkably

concentrated 50:1 E. purpurea aerial extract, reported mild in vitro inhibition. Gorski et al.

(2004) reported an 11% reduction in clearance of tolbutamide, a substrate of 2C9, after 8

days of E. purpurea root supplementation. Gorski and colleagues (2004) suggest this

interaction is not clinically significant.

CYP2C19

Modari et al. (2007) found an IC50 on the CYP2C19 isoform at 60.87 μg/mL for

an extract of E. purpurea herb and root and 18.9 μg/mL for the tetraenes.

CYP2D6

CYP2D6 is one of the most important mixed-function oxidases involved in the

metabolism of a wide range of xenobiotics. Interestingly, CYP2D6 also shows the largest

phenotypical variability, due to genetic polymorphisms of all of the CYPs. Evidence from

the Gurley et al. (2004) and Gorski et al. (2004) studies presented above, as well as in


122

vitro evidence from Yale and Gurlich (2005) and have shown no interaction between

Echinacea purpurea (aerial parts or root) and CYP2D6. However, Modari et al. (2007)

showed activity of an E. purpurea extract of herb and root had an inhibitory concentration

at 50% IC50 of 69.40 μg/mL and specific alkylamides (tetraenes) had an IC50 as low as

6.8 μg/mL. Both human studies concluded that no interactions were expected between

Echinacea purpurea and substrates of CYP2D6.

CYP2E1

Gurley et al. (2004) found no significant effects on CYP2E1 after 28 days of 1600

mg/d E. purpurea whole plant extract. In vitro data from Raner et al. (2007), suggest

inhibition of CYP2E1 by an ethanolic extract of E. purpurea root, reporting a

concentration of 2 µL/mL of a 95% EtOH extract producing a 27 – 29 % inhibition. Of

particular interest was the finding that the same concentration of a 33% EtOH extract of E.

purpurea root did not demonstrate 2E1 inhibition.

CYP3A4

Conflicting data were reported for 3A4; in vitro research, some at concentrations

not obtainable with oral dosing, suggested mild to strong inhibition on 3A4 while human

trials show no overall 3A4 activity. Budzinski et al., using benzyloxyresorufin as a

substrate and extract of 1) the E. purpurea aerial portion or 2) a mixture of E. purpurea

aerial portions and E. angustifolia root (1:1), observed moderate inhibition of 3A4

(unspecified extract ratio, IC50 >5% and <10%). Using solely the roots of both


123

angustifolia and purpurea resulted in higher inhibition of 3A4 (unspecified extract ratio,

IC50 >1% and <5%) (2000). The concentrations of extracts used in these assays, if typical

of commercial extracts, are unobtainable by oral dosing.

Further in vitro research by Budzinski et al. (2000) reported a significant difference

in the effects of E. purpurea versus E. angustifolia roots on 3A4. While E. angustifolia

demonstrated a strong inhibitory effect on 3A4 activity, E. purpurea was found to be a

mild inhibitor of CYP3A4 for 7-benzyloxy-4-trifluoromethylcoumarin metabolism and had

no activity on resorufin benzyl ether metabolism, except at non-relevant physiological

concentrations (resulting in mild inhibition) (Gurley et al., 2004). Another in vitro

investigation demonstrated hydroethanolic extracts of E. purpurea aerial portion and a

combination of E. purpurea and E. angustifolia (plant parts unspecified) moderately

inhibited CYP3A4-mediated 7-benzyloxyresorufin metabolism while root extracts of E.

purpurea and E. angustifolia demonstrated strong inhibition (Budzinski et al., 2000).

Unfortunately, not enough data was provided to calculate the concentration of plant

material that generated these results. Finally, more recent work showed that all echinacea

extracts studied (E. angustifolia, E. pallida, E. purpurea) had the potential to inhibit

CYP3A4. However, the investigators commented that the inhibitory potency was overall

weak and varied considerably between extracts. The effects of isolated alkylamides, the

tetraenes and diene isobutylamides were reported to be 1.9 and 5.2 μg/mL, respectively.

Human investigations by Gorski et al. (2004), found no changes in the metabolism

of midazolam, a 3A4 substrate, after participants ingested 1600 mg of E. purpurea root

daily for 8 days. While these investigators observed an 85% increase in intestinal

availability of midazolam, a 15% reduction of hepatic availability (p < 0.003) was also

noted. The authors postulated that the induction of hepatic 3A4 counteracted inhibition of


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intestinal 3A4, leading to little to no effect in midazolam metabolism overall (Gorski et al.,

2004). Using E. purpurea whole plant extract (aerial and root combined) in a human trial

Gurley et al. (2004) found no statistically significant differences in 3A4 phenotypic ratios.

CYP 450 phenotypic ratios have been shown to provide a practical method for predicting

CYP mediated drug interactions (Gurley et al., 2004).

Organic anion-transporting polypeptide (OATP-B)

Fuchikami et al. (2006) found inhibition of OATP-B by the aerial parts of E.

purpurea in vitro. The clinical significance of this finding is unclear, as few drugs are

metabolized via this pathway and the findings have not been demonstrated in vivo.

P-glycoprotein

Romiti et al. (2008) report that the hexane root extracts of all three Echinacea

species inhibited Pgp, with E. pallida, a little used species, having an effect at 3 μg/mL.

The E. angustifolia and E. purpurea extracts were inhibitory at 30 μg/mL (Romiti et al.,

2008). The ketone compounds of E. pallida were found to be inhibitory as low as 3 μg/mL.

The following experiments were designed to investigate the generation of

metabolites of the predominant alkylamides, the tetraenes, in Echinacea spp. by human

liver microsomes and investigate which isoforms of CYP450 are responsible for

metabolism of the tetraenes in hopes of contributing to the scant data thus far available.


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Methods

Extract preparation

The echinacea extract used in these experiments was supplied by a previous

graduate student at the University of North Carolina, Greensboro and was prepared as

follows: E. purpurea was cultivated at Horizon Herbs Inc. (Williams, OR, USA) and the

species was verified by Richard Cech (Owner, Horizon Herb, LLC). A voucher specimen

was collected and submitted to the University of North Carolina Herbarium in Chapel Hill

accession number 583422. Glassware and utensils for the preparation of the extract were

soaked overnight in Minntech cold sterilant (Minneapolis, MN, USA) to control for

bacterial contamination. After scrubbing the roots of E. purpurea and rinsing with

deionized water, the roots were soaked for 20 minutes in 95% ethanol, which was then

discarded. The roots were blended in ethanol (ratio 1g:2 mL ethanol), pressed and allowed

to macerate for 7 days at 4 ºC. Extracts were then filter sterilized and stored a -70 ºC until

the time of experiments. Extracts were assayed utilizing the Limulus Amoebacyte Lysate

(LAL) assay (Cambrex Bioscience, Rockland, ME, USA) for endotoxins which proved to

be negative.

Liver microsome and CYP450 assays

Dodeca-2E,4E,8Z,10Z-tetranoic acid isobutylamide (Z-tetraene, Chromadex, Santa

Ana, CA, USA) and E. purpurea radix extract were prepared at a concentration of 10 and

100 μg/mL in ethanol, respectively and added to the assay tubes. Assay tubes also included


126

pooled human liver microsomes or CYP450 isoforms (40 µL, Moltox, Boone, NC, USA),

NADPH (1 mM) or cell culture grade water, in addition to sterile phosphate buffer (pH

7.4, 0.1 M) bringing volume to a total of 1 mL. Assays were terminated by the additions of

400 μL of reagent grade ethanol to precipitate proteins. Samples were then centrifuged for

5 minutes at 12,000 rpm and supernatant was filter sterilized through a 0.2 μM cellulose

acetate membrane filter for preparation of LC-MS analysis.

HPLC-ESI-MS Analysis

An ion trap mass spectrometer with electrospray ionization source (LCQ

Advantage, ThermoFisher, San Jose, CA) was employed. The solvent gradient, has been

previously described (Spelman et al., 2009c), as follows, where solvent A is aqueous acetic

acid (17mM, original pH 2.74) and solvent B is neat HPLC grade acetonitrile. For t = 0 to

4 min, a constant composition of A-B (90:10 v/v); for t = 4 to 15 min, a linear gradient

from A-B (90:10, v/v) to A-B (60:40, v/v); for t = 15 to 30 min, a linear gradient from A-B

(60:40,v/v) to A-B (40:60, v/v); for t = 30.1 to 35 min, a constant composition of A-B

(0:100,v/v); for t = 35.1 to 43 min, a constant composition of A-B (90:10, v/v). The mass

spectrometer was operated in the positive ion mode with a scan range of 50.00-2000.00.

Spray, capillary, and tube lens offset voltages were 4.5 kV, 3 V and -60V, respectively.

Quantitative analysis of the Z-tetraene was carried out for triplicate supernatants

from the liver microsome assays. Quantitation was accomplished using the previously

published methods (Cech et al., 2006a; Sasagawa et al., 2006). Briefly, calibration curves

were plotted for the Z-tetraene as the peak area of the selected ion chromatogram for the


127

protonated molecular ion versus concentration. A concentration range of 1.0 to 50 µM was

used.

The concentrations of metabolites (Figure 4.2) were calculated via the calibration

curve for Z-tetraene. These were calculated as µg/mL eq to signify a concentration in units

equivalent to µg/mL of the Z-tetraene. This approach has been previously utilized for the

quantification of alkylamides in echinacea (Matthias et al., 2005). It should be noted that

this method results in estimates of metabolite concentration, due to the likely differing

sensitivity of the metabolites within the mass spectrometer, which likely differ from that of

the standard. This does, however, allow for concentrations to compare metabolites

between samples.


The mean and the standard error of the mean (SEM) were determined for each set

of concentrations. Comparison of means was conducted using a two tailed t-test for paired

data when differences were observed. The mean values were considered significantly

different if p < 0.05. Where appropriate, outlying data points were rejected on the basis of

the Q-test. Statistical analyses were performed with Microsoft Excel (2003).


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Results

Metabolites of dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide

Figure 4.1 illustrates the selected ion chromatograms of the Z-tetraene sample after

exposure to human liver microsomes for 120 minutes. The m/z of the compound in Figure

4.1B increased by 16 amu compared to the parent compound, suggesting that it is

hydroxylated or epoxidised. A shorter retention time in the reverse phase chromatography

was also observed as would be expected due to increased polarity. A second metabolite

was identified in negative and positive ion mode (Figure 4.1 C and D) having an m/z of

276 and 278 respectively. The increase of 30 amu can be explained by addition of two

oxygen atoms and loss of two hydrogens, suggesting the introduction of a carboxyl (COO)

group. The proposed structures of these metabolites are shown in Table 4.1.

Previous reports have demonstrated that cytochrome P450 isoforms hydroxylate

and epoxidate alkylamides (Cech et al., 2006b; Matthias et al., 2005). Matthias has

persuasively argued, through H NMR, that the epoxide addition is at either C8-C9 or C11-

C12, with a preference for C11-C12 (Matthias et al., 2005). Hydroxylation can take place

on C4-C12 and it is uncertain which product would dominant. For the Z-tetraene it can be

observed (Figure 4.3) that a few possible structural variations are present for m/z


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Figure 4.1 Selected ion chromatograms of the Z-tetraene and its metabolites

After incubation of the Z-tetraene (chromatogram A-248) with human liver microsomes for 120 minutes

(positive mode), the hydroxylated metabolite is observed in chromatogram B (positive mode-264).

Chromatogram C displays the base peak of the anionic COO-tetraene in negative ionization mode (276).

Chromatogram D illustrates the cationic COO-tetraene in positive ionization mode (278).

264, which could correspond to both hydroxylated and epoxidized products. The

carboxylated species (Table 4.1 structure D), was a previously unreported product of

microsomal redox of alkylamides (Cech et al., 2006b). Cech et al. (2005) point out that

there are 3 primary carbons that are available for oxidation to carboxylate on the tetraenoic

isobutylamide (Table 4.1 structure A), the C12 of the fatty acid group and C3 or C4 on the

isobutylamide group. The elucidation of the carboxyl group on the C12 derives from the


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Table 4.1. Parent compound & metabolite structural assignment after oxidation by human liver microsomes

Structure MW

O

NH

A. Dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (Z-tetraene)

247.3

O

NH

OH

B. dodeca-2E,4E,8Z,10Z-tetraene-7-hydroxl acid isobutylamide (OH-tetraene)

263.3

O

NH

O

C. dodeca-2E,4E,8Z,10Z-tetraene-10,11-epoxy acid isobutylamide

263.3

O

NH

O

HO

D. dodeca-2E,4E,8Z,10Z-tetraene-12-oic acid isobutylamide (COO-tetraene)

277.3

Compound A, the Z-tetraene, is the most abundant alkylamide in Echinacea purpurea and parent compound

for the metabolites. After oxidation by human liver microsomes, the hydroxylated (B), epoxidated (C) and

carboxylated (D) metabolites shown are the postulated metabolites. LC/MS in positive ion mode shows

above structures [M +1 (MH+)]. The carboxylated metabolites were detectable for both as M +1 (MH

+) and as

M-1 (M-H-) ions (negative ion mode). The metabolites shown here for the Z-tetraene were observed in the

Echinacea purpurea extract as well.

candidates MS/MS studies. A fragment at m/z 74 which corresponds to the isobutylamide

fragment of the COO-tetraene has previously been demonstrated to correspond to a typical

isobutylamide fragment (Spelman et al., 2009c). Consequently, this rules out the carboxyl


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group location on any carbon but C12 of the fatty acid moiety. MS/MS fragmentation in

past studies has been demonstrated to be effective for identification of alkylamides and is

discussed at length in chapter 2 (Spelman et al., 2009c).

Besides MS/MS fragmentation, the identity of the carboxylated metabolite was

deduced from the increase of mass of 30 amu, characteristic of the addition of a carboxyl

group and the ability to detect the metabolite in question in the negative ion mode of the

MS, which indicates the presence of an acidic moiety (Figure 4.1C). Finally, Cech et al.

(2006b) report undeca-2E-ene-8-10-diynoic acid isobutylamide, which has a terminal

ending of a hydrogen, makes terminal carboxylation impossible. Thus, this alkylamide

forms hydroxylated but not carboxylated metabolites. Consequently, this rules out the

carboxyl group location on any carbon but C12 of the fatty acid moiety.

Other studies have also shown that the extract of E. purpurea radix also contains a

peak at 278 after exposure to liver microsomes, corresponding to the 278 peak found in the

MS studies of isolated tetraenoic acid (data not shown) (Cech et al., 2006b).

Metabolite formation as a function of incubation time

Figure 4.2 exhibits the appearance of metabolites as a consequence of the redox

reactions over 120 minutes that the Z-tetraene undergoes. Figure 4.3 shows the

chromatogram of the unmetabolized the Z-tetraene and as such, serves as a control. Figure

4.3B illustrates the chromatogram of the metabolized the Z-tetraene at 20 minutes. The


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Figure 4.2. Time dependent metabolism of the Z-tetraene.

Concentrations of the metabolites, hydroxylated (OH-tetraene) and carboxylated (COO-tetraene), after

incubation with human liver microsomes. The error bars represent the standard deviation of the

concentrations determined for triplicate assay tubes.

OH-tetraene is formed and as illustrated by the ions at m/z 264 peaks. As seen, it

appears that there may be several possible structures forming such as the epoxide and OH-

tetraene metabolites (Table 4.1, structure C). By 60 minutes it appears that the various ions

at m/z 264 have been reduced to a few dominant structures (Figure 4.3C). Figure 4.2

Shows that the formation of the OH-metabolite increases over 60 minutes and then is

reduced at 120 minutes. The longer incubation times (120 min) favor the production of the

COO-tetraene over the hydroxylated molecular species (Figure 4.2; Figure 4.3D). In

addition, it is apparent that the parent compound decreases in intensity (Figure 4.3D) and

decreases in concentration (Figure 4.2) over time.


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Figure 4.3. Selected ion chromatograms of the Z-tetraene and metabolites as a function of time.

The increase in peak areas of the metabolites as a function of time, as the parent compound, a standard,

decreases in peak area as displayed in the chromatograms. All chromatograms are selected for 248, 264, 278

ions. A. The control at time 0. B. At time 20 minutes the largest peak area in the chromatogram is the parent

compound the Z-tetraene. The development of several metabolites with a weight 264 ± 0.15 amu is seen,

likely representing the addition of a hydroxyl group on various locations on the carbon chain of the parent

compound. C. At time 60 minutes a clear development of the preferred OH-tetraene is seen. In addition, the

carboxylated parent compound is seen increasing in peak area. D. At time 120 minutes, the COO-tetraene is

seen to be the dominant metabolite while the peak area of the tetraene is greatly reduced.

Previous reports suggest that the formation of the tetraene metabolites in E.

purpurea radix extracts is less complete than for the Z-tetraene standard (Cech et al.,

2006b). Matthias et al. (2005) suggest that the isobutylamides with a terminal alkyne group

may inhibit particular CYP450 isoforms. This may be at least partially responsible for this

effect. It is likely that the polyphenols, with their known affect on CYP450 activity, may

also play a role in the inhibition of the oxidation of the tetraenes.


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CYP450 1A1 is responsible for the formation of dodeca-2E,4E,8Z,10Z-tetraene-7-

hydroxl acid isobutylamide

Figure 4.4 shows the mass spectrometry results of an experiment analyzed after the

Z-tetraene was incubated with CYP1A1 without NADPH (Figure 4.4A) and with

Figure 4.4 Metabolism of dodecatetraenoic acid isobutylamide by CYP450 1A1

A. Incubation of the tetraenoic isobutylamide with CYP1A1 without NADPH serves as a control. As

demonstrated by the selected ion (248, 264) chromatogram there is no OH-tetraene present.

B. With the addition of NADPH to CYP1A1 and the tetraenoic isobutylamide the formation of the OH-

tetraene ion is present at 264 (MH+).


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NADPH (Figure 4.4B). In the chromatogram in Figure 4.4A the absence of NADPH

serves as a control. It can be seen that there are no other peaks present except for the parent

compound. The appearance of the m/z 264 peak in Figure 4.4B after 60 minutes incubation

demonstrates that CYP1A1 has metabolized the OH-tetraene to a compound with a weight

of 263 (MH+ = 264). The base peak at m/z 264 elutes at the same time as the compound at

m/z 264 when the Z-tetraene is incubated with the human liver microsomes, suggesting

that this is the same compound.

Figure 4.5. Incubation of the Z-tetraene with CYP450 isoforms 1A2, 1B1, 2A6, 3A4.

Incubation of the Z-tetraene with various CYP450 isoforms shows no apparent metabolite formation. A-D

represent experiments with different isoforms of CYP450, 1A2, 1B1, 2A6, 3A4, respectively. All

chromatograms displayed are experiments conducted with NADPH. The controls (without NADPH) are not

shown. As illustrated there are no detectable metabolites present as can be seen by the absence of peaks other

than 248. Drift of the 248 peak is seen due to an extended run of the LC-MS.


136

Figure 4.5 depicts the results of experiments conducted with other isoforms of the

CYP450 enzyme family. As is depicted by these chromatograms, there appear to be no

other metabolites generated under these experimental conditions. Under these conditions

the only CYP450 isoform that generates detectable metabolites of The Z-tetraene are

CYP1A1.

Discussion

Three possible structural assignments have been proposed for the metabolites of the

Z-tetraene after the incubation with human liver microsomes. Whereas a role for enzymes

such as flavin-containing monooxygenases or peroxidases cannot be excluded, the

NADPH dependence exhibited for the formation of these metabolites argues for a role of

the CYP450 families. The presented data suggest that CYP1A1 is involved in the

generation of the OH-tetraene. The CYP1 family contains both CYP1A1, CYP1A2 and

CYP1B1. The isoforms CYP1A1 and CYP1A2, are substrate-inducible cytochrome P450s

with 70% homology, that are responsible for the metabolism of numerous xenobiotics, but

contain very different patterns of tissue expression. While CYP1A2 is expressed primarily

in the liver, CYP1A1 is expressed predominantly in extrahepatic tissues such as the lungs,

lymphocytes and placenta (Raunio et al., 1998; Shah et al., 2009). CYP1A1 is also known

to have a rapid transcript turnover. This has been postulated to be an evolutionary

advantage and appears to be conserved across species (Lekas et al., 2000).

Of pertinence to the metabolism of the tetraenes, 1A1 is known to metabolize

arachidonic acid and eicosapentaenoic fatty acids which are both highly unsaturated as are

the tetraene isomers, which are fatty acid derivatives (Schwarz et al., 2004). As can be seen


137

in Figure 4.4B, the hydroxylated metabolite (m/z 264) is seen only after the Z-tetraene and

CYP1A1 is incubated in the presence of NADPH. While the identity is not certain, it is

highly likely that the structural assignment given to this metabolite (Table 4.1B) is correct

due to the identification of this metabolite by other researchers (Cech et al. 2006; Matthias

et al. 2005) and the subsequent MS/MS studies (Cech et al. 2006).

While the data suggest that CYP1A2 and CYP3A4 are not involved in the

metabolism of the alkylamides, some investigations suggest that extracts of E. purpurea

root may mildly reduce clearance of substrates of CYP1A2 and CYP3A4. Human trials by

Gorski et al. (2004) and Gurley et al. (2004) demonstrate wide inter-individual variability

of metabolism of various echinacea preparations. Gurley et al. (2004) conclude that there

is a minimal risk of drug interaction from echinacea preparations. These data suggest that,

as is the case with pharmaceutical drugs, while the general effect on drug metabolism by

echinacea products on consumers may be insignificant, rare individuals within the group

could possibly experience a more significant induction or inhibition of these enzyme

systems or drug transporters. In general, further study of inter-individual variability is

needed.

CYP1A2, which metabolizes theophylline and other pharmaceuticals, may be

particularly relevant for asthma patients. It must be mentioned, however, that clinicians

have observed an improvement of asthma and bronchitis with echinacea use, suggesting

that echinacea may be useful to asthmatics (Stansbury, 1993). Supporting these

observations, Sharma et al. (2006) found that preparations of root and aerial portions of

echinacea reversed the inflammatory response of human bronchial epithelial cells to

rhinovirus. Furthermore, particular alkylamides of Echinacea spp. are cyclooxygenase

(Hinz et al., 2007; Muller-Jakic et al., 1994) and lipoxygenase (Merali et al., 2003; Muller-


138

Jakic et al., 1994) inhibitors, as well as antiviral (Hudson et al., 2005; Vimalanathan et al.,

2005) and cannabionomimetic (Gertsch et al., 2006; Gertsch et al., 2004; Matovic et al.,

2007; Raduner et al., 2006; Woelkart et al., 2005). Considering that such activity may offer

a significant reduction of spasm and the inflammatory processes of asthma (Lunn et al.,

2006; Pertwee and Ross, 2002; Singh and Budhiraya, 2006), possibly protect against upper

respiratory tract infections (Schoop et al., 2006) and that echinacea inhibition of CYP1A2

is insignificant to mild as found in the human trials reviewed here, recommendations for

individuals with asthma to halt echinacea should be reserved for exceptional

circumstances.

Echinacea may mildly inhibit the cytochrome P450 3A4 enzyme system; this

inhibition may be tissue specific. For example, Mouly et al. (2005) report that selected

substrates can increase hepatic CYP3A activity, yet have no effect on intestinal CYP3A4

or, conversely, substrates may generate CYP intestinal activity without inducing hepatic

effects. Gorski et al. (2004) also report tissue specific activity, finding that E. purpurea

root extracts demonstrate inhibition of intestinal, and induction of hepatic, CYP3A

activity. The molecular rationale behind these differential effects is still unclear. Gorski’s

group (2004) suggests that echinacea root preparations taken with high oral

availability/low clearance drugs (e.g. alprazolam) may induce hepatic CYP3A4 resulting in

decreased serum concentrations of CYP3A4 substrates. Conversely, they suggest that low

oral clearance/first-pass metabolism drugs (e.g. buspirone) taken with echinacea might

result in increased drug serum concentrations. More data is needed to support this

contention.

It has been suggested that CYP3A4 is the most important enzyme for the

metabolism of antineoplastic drugs (Meijerman et al., 2006). Thus the information that


139

echinacea may modulate, even weakly, the activity of 3A4 may encourage healthcare

providers to warn against the use of echinacea with patients undergoing chemotherapy.

However, such warnings could be disadvantageous to the patient; in vivo studies of E.

purpurea aerial parts demonstrate a reduction in chemotherapy induced leucopenia (Bauer,

1996; Melchart et al., 1995; Roesler et al., 1991; See et al., 1997; Steinmuller et al., 1993),

while E. purpurea root has demonstrated stimulation of natural killer cells (Delorme and

Miller, 2005; Gan et al., 2003; Lersch et al., 1990) and prolongation of life in leukemic

mice (Currier and Miller, 2001). Additionally, a number of Echinacea species and

constituents have demonstrated direct antineoplastic activity in vitro (Chicca et al., 2007;

Currier and Miller, 2000; Melchart et al., 2002). Thus again, case by case

recommendations are needed for cancer patients rather than a generalized recommendation

to halt echinacea use.

Another isoform of the CYP 450 system, 2D6, is known to play a primary role in

the metabolism of pharmaceuticals used to treat psychiatric disorders (attention

deficit/hyperactivity disorders, bipolar disorder, depression, schizophrenia) as well as

cardiovascular disorders (β-blockers). This potential of 2D6 metabolism of the alkylamides

was not tested in these studies. However, Echinacea purpurea products containing root or

aerial parts in both human (Gurley et al., 2004) and in vitro models (Yale and Glurich,

2005) has shown no activity on 2D6 substrate metabolism. The available research suggests

that no interactions are expected between Echinacea purpurea products and substrates of

CYP2D6.

Other possible enzymes that deserve investigation for the metabolism of

alkylamides include CYP1B1 and CYP2E1which are native to leukocytes, as well as the

CYP2 CYP4, CYP5 and CYP8 families, many of which are expressed in the liver.


140

CYP4A, CYP5 and CYP8A are known to metabolize eicosanoids. CYP2A6 was

investigated, however, CYP2C9 was not investigated and is known to metabolize

arachidonic acid. The CYP4A isoforms (primarily kidney expression, secondarily hepatic

tissue), such as CYP4A1, CYP4A2, CYP4A3, CYP4A8, are also known to metabolize

arachidonic acid and other unsaturated fatty acid derivatives (Cowart et al., 2002). All of

the isoforms previously mentioned were, unfortunately, unavailable for investigation.

In reference to the effects of the alkylamides on drug metabolism, no case reports

were found documenting an echinacea-drug interaction, a finding supported by other

reviewers (Basch et al., 2005; Dergal et al., 2002; Fugh-Berman and Ernst, 2001; Izzo and

Ernst, 2001; Messina, 2006). The fact that echinacea products rank second in sales of

herbal remedies in the U.S. (Blumenthal et al., 2006) and the number of people using

multiple pharmaceuticals (>75 million) (Slone Epidemiology Center), suggest that drug-

herb interactions with echinacea products are uncommon events. Barrett (2003a) concludes

that less than 100 serious adverse events have been reported involving echinacea for an

estimated >10 million courses of treatment, leaving the risk estimate of less than 1 in

100,000.

While there are scant data on the metabolism of echinacea constituents and their

effects on metabolism of exogenous chemicals, there is even less data on the influence of

the metabolites of Echinacea spp. on immunomodulatory activity. The only paper thus far

found is by Cech et al. (2006) that alkylamide metabolism caused a decrease in

immunomodulatory effects as compared to the native alkylamides. Specifically the

carboxylated metabolite (Table 4.1, structure D) of the Z-tetraene loses its IL-2 inhibitory

effect (Cech et al. 2006). However, it is possible that the novel metabolites reported here


141

could also influence other immune parameters, such as monocyte/macrophage and

neutrophil activity.

Medicinal plants, while necessarily part of healthcare in developing regions, are

becoming increasingly popular in the industrial nations. Estimates suggest that up to 30%

of the North American population use herbal supplements (Nelson and Perrone, 2000).

Given that nearly 3.6 billion prescriptions were purchased in 2005 (Kaiser Family

Foundation, 2006) and 30% of Americans report using five or more drugs in the same

week (Slone Epidemiology Center) it comes as no surprise that the incidence of consumers

taking herbs in conjunction with prescription medications is quite high. Eisenberg et al.

(1998) found that up to 15 million adults were concurrently using prescription medications

and herbal supplements. A nationwide telephone survey by Kaufman et al. (2002) supports

this, finding that 16% of respondents had taken both prescribed medications and

supplements in the week preceding the interview. Consequently, the study of the

metabolism of phytochemicals and these phytochemicals effect on metabolism are crucial.

Even though 80% of the echinacea products sold to consumers are made from

purpurea (Li, 1998), the current body of scientific literature on echinacea can be confusing

due to the multiple species in use – namely E. purpurea, E. pallida and E. angustifolia -

which have phytochemical similarities, but also have notable differences, particularly

around the identity and concentration of alkylamides and other key constituents (Bauer,

1996; Chen et al., 2005; Hudson et al., 2005). In addition, all three species are often used

interchangeably for the treatment of cold, flu, respiratory infection, and inflammation

(Bauer and Foster, 1991). Multiple species, plant parts, and preparations are also used,

each of which may have a different constituent profile (Spelman et al., 2009c; Williamson,

2006). “Echinacea” may refer to the roots, aerial parts, whole plant or a combination of the


142

above; echinacea products can be made from fresh or dried plant parts, and may be

prepared by juicing, alcohol extraction, infusion, decoction, or consumed as tablets or

capsules (Mills and Bone, 2000).

Currently, it has not been clearly elucidated as to how constituents of echinacea are

metabolized by the body or which of those constituents affect the metabolism of other

exogenous chemical inputs. Preliminary studies suggest that extraction conditions, the

various species and botanical part of echinacea have differing effects on the cytochrome

P450 enzyme system (Raner et al., 2007). For example, Raner et al. (2007) found that the

hydrophobic constituents of E. purpurea (e.g. alkylamides) are much more inhibitory to

CYP450 than its hydrophilic constituents (e.g. phenylpropanoids) and this work is in

agreement with other CYP450 evaluations (Matthias et al., 2005). Modari et al. (2007)

found that higher alkylamide content of echinacea preparations was associated with greater

CYP3A4 inhibitory activity, although the inhibitory activity was overall weak in effect.

Recent work by Spelman et al. (2009c) demonstrated quantitative and qualitative

differences in alkylamide concentrations, including the alkylamides reported to modulate

CYP activity, depending on the extraction technique and the use of fresh or dry plant

material in ethanolic preparations of Echinacea purpurea radix (see chapter 2). Thus,

ethanolic extracts as well as other echinacea preparations may differ in their activity on the

CYP 450 system and therefore generalizations about metabolism of echinacea products

and their possible influence on the metabolism of drugs may be misleading (Freeman and

Spelman, 2008).

The search for and appraisal of information relating to the metabolism of

phytochemicals and the phytochemical influence of drug metabolism has thus far been a

challenge for researchers and educators, and readily accessible information among the


143

scientific and medical community is lacking. Metabolic studies on phytochemicals have

only recently been published in the scientific literature. Unfortunately, it appears that in

regard to phytochemical influence on the metabolism of pharmaceuticals, much of the

literature does not evaluate the quality of evidence from which conclusions are drawn.

Of particular concern for healthcare providers, Freeman and Spelman (2008) report

that drug-herb interactions related to echinacea products were cited in some 49 articles,

only 16% (8) of these 49 papers contained primary data relevant to interactions between

echinacea products and pharmaceuticals. Two studies were clinical trials (Gorski et al.,

2004; Gurley et al., 2004) and the remaining were in vitro assays, three of which did not

contain complete information about the concentration of extract used (Budzinski et al.,

2000; Foster et al., 2003; Yale and Glurich, 2005); only half of the studies verified the

authenticity of the echinacea (Gorski et al., 2004; Moulick and Raner, 2003; Raner et al.,

2007; Yale and Glurich, 2005). Yet, there are very few investigations investigating the

metabolites that result from echinacea phytochemicals.

Previous researchers have demonstrated that the Z-tetraene is a CB2 ligand

(Gertsch et al., 2004; Matovic et al., 2007; Raduner et al., 2006). Hence, it has been

suggested that considering the strict structural requirements for ligands of the cannabinoid

2 (CB2) receptors, that the COO-tetraene likely has a greatly reduced affinity for CB2

(Cech et al. 2006). However, recent evidence suggests that the IL-2 effect is due, at least in

part, to PPARγ activity (Spelman et al., 2009b). Consequently, it is also possible,

especially considering the considerable promiscuity of the PPARγ receptor to divergent

phytochemical structural motifs (Spelman and Burns, 2009), that tetraenoic isobutylamide

is converted from an agonist to an antagonist with the carboxylation of this alkylamide. In

addition, the ligand binding domain of PPARα PPARβ/δ PPARγ is particularly


144

homologous (Shearer and Billin, 2007). Considering that epoxyeicosatrienoic acids,

carboxylated fatty acid derivatives, are known to activate human and mouse peroxisome

proliferator-activated receptor-alpha (Cowart et al., 2002), the possibility of the tetraenoic

isobutylamide metabolites activating isoforms of PPAR needs to be explored.

Summary

The generation of metabolites from alkylamides and the effect on alkylamides and

Echinacea spp. on metabolic processes is poorly represented in the literature at this point

in time. The presented data suggest that metabolites from the alkylamides are generated by

the cytochrome P450 system. The above data suggest that there are metabolites formed

from the predominant alkylamide dodeca-2,4,8,10-tetraenoic acid isobutylamide that

possible include; epoxidizee, hydroxylated and carboxylated metabolites. These data

suggest that CYP450 1A1 is responsible for the formation of the hydroxylated metabolite.

In addition Echinacea purpurea root appears to have a relatively low potential to

produce CYP-mediated effects on drug metabolism. However, further pharmacokinetic

testing is required before conclusive statements can be made.

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Electron Microscopy of Jurkat Cells

Chapter 5 Alkylamide Activation of PPARγ

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Chapter 5

The activation of PPARγ by undeca-2E-ene-8,10-diynoic acid isobutylamide

Introduction

Recent investigations utilizing alkylamides from the genus Echinacea demonstrates

that dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (Z-tetraene) and dodeca-2E,4E-

dienoic acid isobutylamide have a reasonable affinity for the cannabinoid receptors,

binding CB2 (Ki ~ 50 nM) with ca. 30 to 50 fold selectivity over CB1 (Raduner et al.,

2006). Broad-based receptor screening demonstrates that binding to CB2 is highly

selective (Gertsch et al., 2006). Accordingly, this has provided an explanation, although

limited, to the basis for the immunomodulation of Echinacea spp. (Woelkart et al., 2005).

There have been frequent reports in the primary literature that suggest cannabinoid

ligands may have CB independent activity. For example, 2-arachidonoylglycerol (2-AG) is

known to modulate a number of immune processes, including cytokine secretion via CB2.

Supporting this is the significant up-regulation of 2-AG production by a number of WBC

species upon activation (Berdyshev, 2000). Derocq et al. (1998) showed that the

cannabinoid ligands anandamide, as well as (R)-methanandamide and

palmitylethanolamide, potentiated the growth of the murine interleukin (IL)-6 dependent

lymphoid cells and IL-3 dependent myeloblastic cells. They believe that this effect was a

noncannabinoid receptor-mediated process: CP 55940 which is a highly potent synthetic

cannabinoid with affinity for both CB1 and CB2 was inactive; the CB1 and CB2

antagonist SR 141716A and SR 144528 alone or in combination could not block the

cannabinoid activity; inactivation of both CBs by pertussis toxin did not change the


153

activity of anandamide; and the fatty acid, arachidonic acid, which has no affinity for CB

receptors, was able to trigger comparable activity to the structurally similar anandamide.

Although cytokine inhibition by leukocytes is speculated to be transduced by CB2, non-

cannabinoid modes of activity appear to be involved as well (Klein, 2005; Pertwee and

Ross, 2002).

Other receptor systems are known to respond to fatty acids and their derivatives.

For example, the peroxisome proliferator-activated receptors (PPARs) consist of a set of

three receptor sub-types which are members of the nuclear receptor superfamily of ligand-

activated transcription factors. They function as lipid sensors, regulating gene expression

in a variety of metabolically active tissues and have a significant role in cellular energy

balance, fuel utilization, the metabolism of fatty acids and other lipids. They also play a

role in the fibrotic and hypertrophic responses in the heart and vascular wall and the

generation and remodeling of adipose tissue (Bishop-Bailey and Wray, 2003).

Recently PPARγ, PPARα and PPARβ/δ agonists have been demonstrated to exhibit

anti-inflammatory and immunomodulatory properties thus opening up new avenues for

research. For example, B and T lymphocytes and monocytes are known to express PPARγ.

Both anandamide and 2-AG, in a number of models, demonstrate concentration dependent

inhibition of IL-2 production, a classical sign of T cell activation (Rockwell et al., 2006).

Moreover, anandamide induces a concentration-dependent inhibition of interleukin-2 in

primary splenocytes which is unaffected by SR141716A and SR144528. In the same

model, arachidonic acid shows similar activity as anandamide which is attenuated by

pretreatment with nonspecific cyclooxygenase (COX) inhibitors as well as COX-2 specific

inhibitors. Pretreatment with the PPARγ antagonist T0070907 also attenuated anandamide

mediated suppression of IL-2 secretion (Rockwell and Kaminski, 2004). Yang et al. (2000)


154

observed that PPARγ free Jurkat cells did not respond to PPARγ ligands troglitazone and

15d-PgJ2 (an endogenous ligand), but PPARγ expressing Jurkats did exhibit IL-2

inhibition in response to PPARγ agonists.

IL-2, an autocrine/paracrine factor, plays a key role in multiple processes of T cell

biology. Initially, activation of the IL-2 receptor (IL-2R) was found to be crucial for

inducing proliferation of T cells in vitro (Hatakeyama et al., 1989). Subsequent

investigations revealed that IL-2R signaling activates genes such as c-myc and c-fos, which

are crucial to proliferation (Miyazaki et al., 1995; Nelson and Willerford, 1998). Later

investigations demonstrated that IL-2 plays an important role in sensitizing T cells for

activation-induced cell death (Lenardo, 1991) and plays a key role in autoimmunity

(Suzuki et al., 1995; Willerford et al., 1995). Currently it is known that IL-2 dependent

signals are required for T regulatory development, homeostasis and function (Burchill et

al., 2007). Opportune for laboratory investigations, IL-2 production is a characteristic

response of T cell activation, exhibiting virtually no basal level expression, but is rapidly

secreted upon T cell activation (Rockwell et al., 2006). For full activation of the IL-2

response, multiple transcription factors are involved including NFκB and cooperative

binding of NFAT and AP-1 for transcriptional activity of the IL-2 gene (Jan et al., 2002).

AP-1 is generated by dimerization of c-jun and c-fos for the transcription of IL-2.

Helper T-cells produce IL-2 in response to antigen recognition by signaling through

the T-cell receptor. In particular, the E6.1 cell line (Jurkats), established from a 14 year old

boy with T cell leukemia, is a widely used model for studying IL-2 regulation due to their

basal levels of IL-2 being nearly absent (<50 pg/mL). Upon dual stimulation, phorbol

esters and either lectins or monoclonal antibodies against the T3 antigen, large amounts of

IL-2 are produced by Jurkats (Weiss et al., 1984).


155

Jurkat T cells exhibit low levels of surface CB2 receptors (Bouaboula et al., 1993).

And while, Raduner et al. (2006) found high affinity of 2E,4E,8Z,10E/Z-tetraenoic acid

isobutylamide (tetraenes) and dodeca-2E,4E-dienoic acid isobutylamide for CB2 they also

suggested the possibility of the involvement of a second receptor due to the fact that

SR144528 is incapable of fully inhibiting influx of Ca2+

in HL60 cells induced by 2-AG

and alkylamides. Consequently, although alkylamide binding to CB2 is established, there

may be other targets beyond the CB2 for specific alkylamides. Consequently, the focus of

the present studies was to investigate the effects of an alkylamide that has been previously

established to have negligible CB2 affinity on IL-2 production using the Jurkat cell line.

Hence, the hypothesis that signal transduction by undeca-2E-ene-8,10-diynoic acid

isobutylamide (UDA) inhibits the release of IL-2 in Jurkat cells by PPARγ was tested.

Methods

Reagents

All reagents were from Sigma-Aldrich (St. Louis, MO) except the following:

undeca-2E-ene-8,10-diynoic acid isobutylamide (UDA, MW 231.34; certificate of analysis

verified identity by NMR and HPLC, and purity of ≥99% by HPLC lot #21235-501;

(Chromadex Inc., Santa Anna, CA); troglitazone (MW 441.5; Rezulin, abbreviated as

TZD, gift from Ron Morrison, UNCG, Department of Nutrition); T0070907 (MW 277.7;

Cayman Chemicals, Ann Arbor, MI); ethanol (AAPER, Shelbyville, KY); nanopure water

(Nanopure Diamond D11931, Barnstead International, Thermolyne, Dubuque, IA); calf

serum (Colorado Serum Co., Denver, CO); human IL-2 Duo Set ELISA Kit-DY202 (R&D


156

Systems, Minneapolis, MN); Jurkat E6.1 cells (ATCC, Manassas, VA); 3T3-L1

preadipocytes (a gift from Ron Morrison); nitro-cellulose membranes (Bio-Rad, Hercules,

CA); anti-human PPAR γ primary antibody (Aviva Systems Biology, San Diego, CA); and

goat anti-rabbit conjugated to horse radish peroxidase (US Biological, Swampscott, MA).

Immunodectection of PPARγ protein in Jurkat cells by Western blotting

Cells were harvested (2 x 106 cells) and lysed by boiling for 5 minutes. Cell

extracts were analyzed in a denaturing 10% polyacrylamide gel, electrotransferred to a

supported nitro-cellulose membrane, and immunoblotted with the PPARγ primary

antibody (Aviva Systems ARP32880_T100). The membranes were soaked in blocking

buffer [5% nonfat dry milk diluted in Tris-buffered saline-0.1% Tween-20 (TBS-T)] for 1

h at room temperature with the indicated primary antibody (1:10,000). After washing,

membranes were developed with horseradish peroxidase-conjugated secondary antibodies

and visualized with a chemiluminescent detection system (GE Healthcare/Amersham

Biosciences, Buckinghamshire, England). A double band is the expected image resulting

from this antibody due to the non-specific binding to PPARγ1 and PPARγ2 which have

slight weight differences.

Mitogenic stimulation

Optimal stimulant conditions to elicit sub-maximal IL-2 production by Human E6.1

Jurkat T cells were determined using phorbol 12-myristate 13-acetate (PMA, Sigma-

Aldrich) and phytohemagglutinin (PHA, Sigma-Aldrich, Milwaukee, WI). Jurkat cells


157

(2×106 cells/mL, ATCC, Manassas, VA) were treated with 0.125 – 4 μg/mL PHA with 1, 2

or 3 ng/mL PMA; or individually with PMA (1 and 3 ng/mL) and PHA (0.25 and 2.0

μg/mL) at various concentrations and incubated at 37 °C, 5% CO2, and 95% relative

humidity. Supernatants were harvested after 24 h and immediately assayed for IL-2 with an

ELISA kit (IL-2 DuoSet, R&D Systems Minneapolis, MN). Secreted IL-2 concentration

vs. PMA/PHA concentrations were plotted and IL-2 concentration determined by the

association curve produced using Excel (2007).

Jurkat cell culture and IL-2 ELISA

Jurkat T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640

medium with 10% FBS, 2 mM L-glutamine and 1 mM Na pyruvate. After serum starvation

for 7 hours, Jurkat cells were plated in 96 well culture plates at 1.25 x 105 cells/mL in

RPMI 1640 (without phenol red) with 10% FBS. Cells were treated with PMA (1.25

ng/mL) and PHA (0.25 μg/mL) and the selective antagonist T0070907 or DMSO vehicle

was added to appropriate wells and incubated at room temperature for 15 minutes after

which alkylamides and thiazolidine (TZD) were added. Plates were then incubated for 18

hours. Cell supernatants were collected (100 µL) and assayed for IL-2 by ELISA (Human

IL-2 Duo Set ELISA Kit). All test conditions were assayed in triplicate and verified with

repeated experiments three times. The IL-2 calibration curve determined by ELISA was

R2= 0.9996.


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Cell Survival by XTT assay

The cytotoxicity of UDA was measured by the XTT colorimetric assay (Scudiero et

al., 1988), which was performed with the same plated cells cultured for the IL-2 cytokine

testing in dual ELISA/XTT assays. In the dual assay, cells are harvested for XTT assays

and the supernatant is harvested for the ELISA assay. For the cell viability standard curve,

unstimulated cells were plated in triplicate at 2.5 x 104, 2 x 10

4, 1.5 x 10

4, 7.5 x 10

3, 5.0 x

103, and 2.5 x 10

3 cells/well. After removal of 100 µL supernatant from all wells, 100 μL

of 1 mg/mL of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

(XTT) plus 0.02 mM phenazine methosulfate (PMS), in medium was added. After 5 h

incubation, ODs at 450 nm (Teacan Sunrise plate reader, Grödig, Austria) were measured

and cell concentrations extrapolated from known cell concentrations in standard curve.

Test conditions were assayed in triplicate and repeated in two experiments.

Reverse Transcription PCR Analysis

Cell extracts were prepared from the Jurkats cells run under described conditions

and total RNA was isolated using the Qiagen (Valencia, CA) Minikit 50. Primer and probe

sequences used were sense 5V-ggatagcctctcttactaccac-3V and antisense 5V-

tcctgtcatggtcttcacaacg-3V for c-fos (280 base pairs); sense 5V-caggtggcacagcttaaaca-3V

and antisense 5V- tttttctctccgtcgcaact-3V for c- jun (180 base pairs). First strand cDNA

was synthesized from mRNA with Qiagen Onestep RT-PCR kit. The cDNA was reverse

transcribed (RT) and amplified. Distilled water was substituted for cDNA template as a

negative control. The resultant cDNA samples were subjected to 35 cycles of PCR


159

amplification in the presence of specific sense and antisense primers. The RT denaturing,

annealing and extension temperatures are below.

1. Reverse transcriptase 50 ○C x 30’

2. Denaturing of reverse transcriptase/DNA 95 ○C x 15’

3. Denaturing 94 ○C x 1’

4. Annealing 59 ○C x 1’

5. Extension 72 ○C x 1’

6. Go to 3

7. Extension 72 ○C x 1’

The amplified PCR products were resolved on a 1.5% agarose gel visualized with

ethidium bromide under UV light illumination. Gel imaging and densitometry was aquired

with Αlpha Innotech Corporation (Alexandria, VA) utilizing UV transillumination.

Relative quantitation was made spectrophotometrically by densitometry.

Fibroblast cell culture and differentiation

3T3-L1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) plus

10% calf serum, 4 mM L-glutamine and 1 mM Na pyruvate. Cells were plated in 6 well

plates in a total of 1.5 mL of medium and grown to confluence over 4-5 days. Forty-eight

hours after confluence was achieved (Day 0), insulin (10 μg/mL) was added to all wells

except the insulin free control. On day 2, media was changed to contain FBS and the

following treatment conditions, which were added to separate triplicate wells: UDA (5.0

μg/mL, 7.5 μg/mL and 10 μg/mL); TZD positive control (10 μM); insulin negative control


160

(10 μg/mL); and vehicle control (EtOH:DMSO 0.4%:0.1%) were added. Every 2 days,

the media and all reagents, including treatments, were replenished after a PBS wash. The

experiment was halted on day 5, at which time adipogenesis was determined by

photomicroscopy by morphology and the presence of the obvious prominent lipid

vacuoles. Images were taken with a SPOT digital camera mounted on an Olympus BX60

fluorescence microscope.


All data are expressed as means ± SE of experiments conducted in triplicate.

Statistical analysis was performed using Student’s t-test and analysis of variance (one-way

ANOVA). The accepted level of significance was p < 0.05.

O

N

Figure 5.1. Undeca-2E-ene-8,10-diynoic acid isobutylamide (UDA).

Results

PHA titration to determine submaximal IL-2 production

Immune stimulants for Jurkats lymphocytes were selected based on confirmatory

experiments as well as previous studies (Sasagawa et al., 2006). Previous work has

determined that in order to demonstrate IL-2 inhibition in response to echinacea and its


161

Figure 5.2. PMA/PHA optimization for IL-2 response.

Cell concentration 2 x 104 were incubated for 24 h under standard conditions. Insignificant differences are

seen in PMA concentrations of 1,2 or 3 ng/mL with titration of PHA from 0.125 to 4.0 μg/mL. Error bars

indicate standard error of the means.

alkylamides, that suboptimal IL-2 response was necessary (Sasagawa et al., 2006).

Through a few experiments, this was confirmed (data not shown).

PHA or PMA, when tested in isolation with Jurkats, do not induce an IL-2 response

over 24 h, but the combination is well established to induce IL-2 production. Figure 5.2

demonstrates the optimal mitogenic stimulation conditions of Jurkats for PMA/PHA.

Concentrations tested for PHA were 0.125, 0.25, 0.5, 1.0, 2.0, 4.0 μg/mL. This in turn, was

assayed against PMA at 1, 2, 3 ng/mL. PHA between 0.5 and 1.0 μg/mL was observed to

give the optimal IL-2 response. At a cell concentration of 2 x 104 cells the calculated PHA

concentration necessary to stimulant ≈70% of the maximal IL-2 concentration (150 pg/mL)

was 0.22 μg/mL. An IL-2 ELISA assay resulted in a 0.25 μg/mL for ≈70% of the maximal

IL-2 concentration. This varied insignificantly at different PMA concentrations of 1, 2, or


162

3 ng/mL. Higher concentrations of PMA exhibited the same PHA pattern in that the PHA

concentration of 0.5 μg/mL provided optimal IL-2 response.

Figure 5.3. IL-2 response between 3 to 18 hours.

A rapid increase of IL-2 is seen from 3 to 18 h after stimulation with PMA 1.0 ng/mL and PHA 0.25 μg/mL.

Error bars indicate standard error of the means.

Figure 5.4. IL-2 response between 18 and 48 hours.

IL-2 levels are seen to fluctuate in response to PMA 1.0 ng/mL and PHA 0.25 μg/mL from 18 to 48 hour

time interval. Statistical significance is for comparison of 18h vs. all other time points on the PMA/PHA

curve only. * p<0.05; ** p<0.005


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Determining optimal timing for IL-2 production

Figures 5.3 and 5.4 illustrate the IL-2 response as a function of time as assessed by

ELISA. Jurkat cells were stimulated with suboptimal PMA/PHA (1.0 ng/mL / 0.25 μg/mL)

and allowed to incubate for 24 hours, with samples taken at 3, 9 and 18 hours (Figure 5.3).

The experiment was repeated and samples of the supernatant were taken at 18, 24, 30, 36,

42 and 48 hours (Figure 5.4). The IL-2 calibration curve determined by ELISA was 0.9999

and 1.000 for Figures 5.3 and 5.4 respectively.

The maxium secretion found for IL-2 was previously stated to be 24 hours in E6.1

Jurkats (Sasagawa et al., 2006). Figure 5.3 and 5.4 exhibits the results of Jurkats incubated

with suboptimal PMA/PHA immune stimulation as determined by the experiments

illustrated in Figure 5.2. Maximal concentrations of IL-2 were determined to occur at 18

and 30 hours, with a significant drop off starting at 36 hours. Comparison of the results of

the 18 and 30 hour time intervals were not statistically significant.

Figure 5.5. Immunodetection of PPAR in Jurkat cells by Western blotting.

Nuclear extracts of untreated Jurkat E6.1 cells were treated with rabbit polyclonal PPARγ antibody from 1

µg/mL to 4 µg/mL. A band at 55 kDa, which increases in intensity with increasing PPARγ antibody,

confirms the expression of the PPAR-γ nuclear protein in these cells. Mass (kDa) of the marker proteins are

shown on the right hand side of the blot.


164

Presence of PPARγ in Jurkat cells

The Western blot technique was utilized to verify the presence of the PPARγ

protein in Jurkats, although Jurkat cells have previously been demonstrated to contain the

PPARγ nuclear hormone receptor (Clark et al., 2000). Nuclear extracts of untreated Jurkats

were subjected to polyclonal PPARγ antibodies from 1 μg/mL to 4 μg/mL following

standard Western blot procedures. Figure 5.5 confirms the presence of PPARγ receptor in

Jurkat E6.1 cells. A faint signal is seen at 1 μg/mL that grows in intensity with increasing

concentrations of antibody. These results demonstrate that while the PPAR protein is not

highly expressed in this strain of Jurkats, it is present. The manufacturer suggest a double

band is the expected result with the use of this particular PPARγ antibody as seen on their

website and confirmed by Aviva’s technical support. The antibody binds both PPARγ1 and

PPARγ2 which have a slight mass difference and result in the dual bands.

The PPARγ selective antagonist T0070907 attenuates the IL-2 inhibition of

undeca-2E-ene-8,10-diynoic acid (UDA) at suboptimal Jurkat stimulation

To assess the response of T cells to UDA (Figure 5.1), suboptimal IL-2 stimulation

of Jurkats by PMA/PHA (PMA 1.25 ng/mL & PHA 0.25 μg/mL) was used. Previous work

has utilized this model system to demonstrate that PPARγ mediates IL-2 inhibition in T

cells (Clark et al., 2000; Rockwell and Kaminski, 2004; Rockwell et al., 2006). Cells were

serum starved for 7 hours, plated and then exposed to vehicle control (EtOH/DMSO),

positive control (TZD-a selective PPARγ agonist), positive control with antagonist

(TZD/T0070907), UDA or UDA with antagonist (UDA/T0070907) at increasing


165

Figure 5.6. PPARγ antagonist T0070907 attenuates the UDA induced inhibition of

IL-2 secretion by suboptimally stimulated Jurkat E6.1 cells.

Cells were subjected to suboptimal mitogenic stimulation (PMA 1.25 ng/mL, PHA 0.25 μg/mL) after serum

starvation, for induction of IL-2 in all cases except in the no treatment group (No Tx). The selective agonist

troglitazone (TZD) represents a positive control (black bar). Stripped bars represents the combination of 2.5

μM of T0070907 with the matched treatments (vehicle-VC, UDA, TZD) shown in the left bar of each pair.

Results show a dose dependent response of IL-2 by UDA and the positive control TZD. The T0070907

treatments dose-dependently attenuate the IL-2 inhibition (up to 1 μg/mL UDA) that occurs upon treatment

of Jurkat cells with increasing doses of UDA (grey bars). The positive control (TZD black bar), also shows

significant inhibition of IL-2 which is blocked by T0070907. Values are mean ± S.E. of experiments

performed in triplicate. One-way ANOVA was used to determine statistical significance between the vehicle

(VC) versus the various treatments (UDA or TZD) treated groups (* p < 0.05) and the UDA versus the

UDA/T0070907 treated groups († p<0.05).

concentrations. T0070907 was held at constant concentration (2.5 μM). After 18 h

incubation, ELISA of the supernatants collected from wells treated with the various

conditions was performed. The results demonstrate that UDA dose-dependently inhibits

IL-2 production beginning at 0.33 μg/mL (1.4 μM) (Figure 5.6). When the selective

PPARγ antagonist T0070907 is added with UDA, a dose-dependent attenuation of the IL-2

inhibition is seen starting at 0.33 μg/mL. At this concentration, the IL-2 level returns to

baseline in the wells treated with T0070907. However, at higher levels of UDA treatment,


166

0

5000

10000

15000

20000

25000

30000

VC TZD

4.42

0.033 0.1 0.33 1 3.3

μg/mL

Ce

ll co

nce

ntr

atio

n (

cells

/mL)

UDA UDA/T0070907

* * † * †*

Figure 5.7. Cell survival assay (XTT assay) of Jurkat E6.1 cells treated with UDA and T0070907.

Cells were subjected to identical conditions as the IL-2 assays. The selective agonist troglitazone (TZD)

represents a positive control (black bar). Stripped bars represent the combination of 2.5 μM of T0070907

with the matched treatments (vehicle-VC; UDA; or TZD) shown in the left bar of each pair. A proliferative

effect by UDA (grey bars) on Jurkat cells is observed. A trend showing inhibition of proliferative effects is

observed with the addition of the selective PPARγ antagonist T0070907. The positive control, selective

PPARγ agonist TZD combined with T0070907, shows a statistically significant inhibition of proliferation as

compared to TZD alone (black bar). Values are mean ± S.E. of experiments performed in triplicate. One-way

ANOVA was used to determine statistical significance between the vehicle (VC) versus the UDA- or TZD-

treated groups (* p < 0.05), and the UDA versus the UDA/T0070907 treated groups († p<0.05).

the antagonistic effect of T0070907 on IL-2 secretion is lost, as would be expected in

increasing concentrations of a competitive agonist and a fixed concentration of antagonist.

Cell survival of Jurkats after treatment with UDA and UDA/T0070907 does not

account for IL-2 modulation

To confirm that the IL-2 changes seen in Jurkats treated in the experimental

conditions were due to UDA and UDA/T0070907 and not due to decreases of cell number,


167

the same experimental conditions as run in the experiments illustrated by Figure 5.6 were

assayed with XTT reagent. As Figure 5.7 demonstrates, there is a statistically significant

(p<0.05) proliferative effect of UDA. Thus the IL-2 inhibition induced by UDA is not due

to cell death. In addition the data demonstrate that the increase of the IL-2 by those cells

treated with the PPARγ selective antagonist T0070907 are not due to an increase in cell

number.

Effect of PPARγ activation by UDA on transcriptional activation of c-jun and c-fos

The IL-2 promoter contains several regulatory elements that can bind different

transcription factors, including NFAT, AP-1, and NFκB (Crabtree, 1989). Recent

investigations have demonstrated that UDA inhibits NFκB expression in Jurkats (Matthias

et al., 2008). AP-1, a collaborator of NFAT, has also shown to be inhibited by 15d-PGJ2

and ciglitazone, established ligands of PPARγ (Chung et al., 2003). C-fos protein and it

heterodimeric partner, c-jun, generate the AP-1 complex (with other basic leucine-zipper

proteins). Consequently, the role of PPARγ in Jurkat cells was investigated by measuring

the transcript levels of c-jun and c-fos by RT-PCR. Under experimental conditions as

previously described with the exception of using 1 and 20 μg/mL of UDA against

PMA/PHA stimulated Jurkats, nuclear extracts were harvested for RT-PCR analysis and

supernatants were collected for a confirmatory IL-2 ELISA. The ELISA confirmed

statistically significant IL-2 inhibition for both concentrations tested (1 and 20 μg/mL) but

unexpectedly, not for TZD (Figure 8A).

Agarose (1.5%) gel electrophoresis analysis of RT-PCR products generated by different

primer pairs (jun, fos) provided relative spectrophotometric quantitation (Figures 5.8B and


168

Figure 5.8. Relative quantitation in IL-2 protein and transcription factors c-fos and c-jun in response

to UDA.

All graphs and gels represent the same experiment with cells harvested for PCR and supernatants collected

for IL-2 ELISA. Cells were exposed to 4.42 μg/mL TZD (black bars), 1.0 μg/mL UDA (light gray bars) and

20 μg/mL UDA (dark grey bars) Expression normalized to the PMA/PHA stimulated groups (Vehicle

Control) which were set at 1. Electrophories agarose gel (1.5%) images are stained with ethidium bromide

and illuminated with UV light. A. IL-2 change in Jurkats as assessed by IL-2 ELISA. B. c-fos change as

assessed by PCR. C. c-jun change as assessed by PCR.


169

5.8C). The various PCR analyses demonstrate effects on the transcription of the assayed

genes by UDA and TZD (4.42 μg/mL positive control). The expected inhibition of c-fos by

TZD was observed (Figure 5.8B) although it is mild, however TZD had a slight

stimulatory effect on c-jun transcription (Figure 5.8C). Conversely, the UDA had a

stimulatory effect on c-fos at both concentrations of 1 and 20 μg/mL (Figure 5.8B). A mild

inhibitory effect was observed on c-jun at 20 μg/mL but not at 1 μg/mL (Figure 5.8C).

Although by visual inspection, the appearance of the 1 μg/mL band appears to be more

intense than the appearance of the vehicle control, the densitometry indicates otherwise.

This was verified repeatedly, including by running a 10 fold dilution of the PCR products

and confirming the number sets.

The involvement of c-jun and c-fos in IL-2 expression has been previously

established. The activity of the AP-1 transcription factor has been utilized to establish the

involvement of the nuclear receptor and upstream transcription factor PPARγ. Modest

decrease of AP-1 is correlated with PPARγ agonism (Rockwell et al., 2006).

Treatment of 3T3-L1 preadipocytes with UDA as an indicator of PPARγ

involvement

The 3T3-L1 cell line has been widely established as a model to indicate PPARγ activity

since Wright et al (2000) produced pharmacological data demonstrating that PPARγ

function is required for hormonally induced differentiation. Considering that PPARγ is not

highly expressed by Jurkats, 3T3-L1 differentiation was utilized to confirm or refute the

hypothesis that UDA induces PPARγ activity.


170

Vehicle Untreated Vehicle Untreated + Insulin TZD 10 μM + Insulin

UDA 5 μg/mL + Insulin UDA 7.5 μg/mL + Insulin UDA 10 μg/mL + Insulin

Figure 5.9. Dose-dependent response of 3T3-L1 cell differentiation by UDA.

Increasing concentrations of UDA enhance 3T3-L1 differentiation, as observed by microscopy. 3T3-L1 cells

were plated in 6 well plates with 1.5 mL of medium and grown to confluence over 5 days. After confluence

was achieved, UDA, insulin positive control (10 μg/mL), TZD (10 μM), or vehicle control (EtOH/DMSO)

were added. Medium and treatment conditions were replenished every 2 days after PBS wash.

Photomicrographs were taken using an Olympus inverted microscope at day 5. Results shown are

representative of triplicate experiments.

3T3-L1 cells were grown to confluence and then exposed to experimental

conditions including a positive control (TZD 10 μM), a negative control (insulin-10 μg/mL

and vehicle controls), and treatment with UDA (+ insulin 10 μg/mL) at increasing

concentrations (5.0 μg/mL, 7.5 μg/mL and 10 μg/mL). It was been previously observed

that because 3T3-L1 fibroblast adipogenesis typically requires approximately 7 days

coupled with the efficient processing of lipids by 3T3-L1 cells, higher concentrations of


171

PPARγ agonists are generally required as compared to other short-term assays such as IL-2

secretion in Jurkats (Rockwell et al., 2006).

Differentiation of 3T3-L1s was assessed morphologically by the relatively round

shape of the adipocytes and by the presence of the obvious prominent lipid vacuoles seen

by microscopy. Figure 5.9 shows that the vehicle control and the insulin control exhibit no

differentiation, while UDA induced a dose dependent adipogenesis in 3T3-L1 cells that

was morphologically indistinguishable from that induced by the selective PPARγ agonist

positive control (TZD).

Discussion

PPARγ plays a role in a variety of diseases including diabetes, atherosclerosis,

inflammation, cancer and autoimmune disorders (Desvergne et al., 2004; Khan and

Vanden Heuvel, 2003; Rees et al., 2008; Vamecq and Latruffe, 1999; Yuan et al., 2004).

Thus, novel PPARγ ligands may offer further therapeutic options in a wide array of

diseases. PPARγ ligands include fatty acid derivatives, as well as the selective PPARγ

agonists known as the thiazolidinediones (TZDs). Both class of compounds have been

shown to reduce IL-2 levels in T cells (Clark et al., 2000; Cunard et al., 2004; Itoh et al.,

2008; Yang et al., 2000; Yang et al., 2002). This effect has been shown to be due to

binding of the PPARγ nuclear receptor by reporter assays (Rockwell et al., 2006).

Moreover, endocannabinoids, which share common structure with some of the unsaturated

alkylamides, have been shown to reduce IL-2 levels via PPARγ (Rockwell and Kaminski,

2004; Rockwell et al., 2006).


172

Previous work has demonstrated ambiguous results on the effects of alkylamides on

IL-2. A number of isolated alkylamides from Echinacea spp., the 2,4-diene alkylamides,

have shown IL-2 inhibition in a model utilizing PMA/PHA and Jurkat cells (Sasagawa et

al., 2006). However, a model system using PMA/ionomycin along with purified 2,4-diene

alkylamides showed no IL-2 effects on rat splenocytes (Goel et al., 2002). Extracts of

echinacea, rather than isolated alkylamides, have also shown ambiguous results. Extracts

void of alkylamides have no effect on IL-2 levels (Luettig et al., 1989), while 50%

ethanolic extracts of the most common commercial Echinacea spp. also showed no effect

on IL-2 in an ex vivo model of human T lymphocytes infected with influenza virus

(McCann et al., 2007). In older subjects (> 64 y/o) IL-2 was reduced by the commercial

species E. pallida and E. angustifolia, but not E. purpurea (50% ethanol extracts-ex vivo).

In the same model system young subjects (age not specified) lymphocytes were isolated

and showed no IL-2 response to any echinacea species (Senchina et al., 2006). Finally, an

in vivo murine model using continuous dosing of aerial parts (unidentified echinacea

species) demonstrated dramatic increases in IL-2 levels from weeks 4 to 6. This effect

declined, but were still above the norm, at weeks 7 and 8 (Cundell et al., 2003). Thus, type

of extraction, type of immune stimulation, subject age, dose duration and concentration all

appear to be important factors in the IL-2 response of T cells to echinacea.

The current trend in assigning a target for the immunomodulation induced by the

alkylamides of Echinacea spp. has been to invoke the CB2 receptor. And while much of

the evidence is compelling, it should be noted that the CB2 receptor has variable

expression levels in white blood cell types. For example, B cells are reported to have the

highest expression levels of CB2 followed by natural killer cells which have similar levels

as neutrophils and CD8 T cells. Monocytes have less CB2 expression than CD8 T cells but


173

this is considerably greater than CD4 T cells, which are reported to have the lowest level of

CB2 expression. The Jurkat cell line has been reported to not express CB2 (Bouaboula et

al., 1993).

CB-2 activity has been shown for the 2,4-diene olefinic alkylamides (Raduner et

al., 2006), the alkylamide UDA (Figure 5.1) has shown negligible affinity for CB2

(Kliewer et al., 1994). Raduner et al. (2006) demonstrated that inhibition of TNF-α

expression in macrophages is partially due to CB2 activity. Nevertheless, they also found

that the CB2 protein could not fully account for the TNF-α inhibition due to the activity of

UDA. Moreover, UDA has shown activity in Jurkat and macrophage assays, in some cases

effects different than the high affinity CB2 ligand the Z-tetraene (Matthias et al., 2008;

Matthias et al., 2007). For example, in Jurkats stimulated with PMA, UDA preexposure

caused a profound decrease in NFκB at 200 ng/mL while the same dose of Z-tetraene had

no effect. Conversely, in Jurkats stimulated with LPS, UDA preexposure had no effect on

NFκB, while Z-tetraene increases levels of NFκB.

IL-2 secretion is a characteristic response of T cell activation. This cytokine is vital

in adaptive immune response due to essential roles in T cell proliferation, differentiation

and cell survival. Central to our discussion, both anandamide and 2-AG, in a number of

models, demonstrate concentration dependent inhibition of IL-2 (Rockwell et al., 2006).

Moreover, anandamide and 2-arachidonylglycerol induce a concentration-dependent

inhibition of interleukin-2 in primary splenocytes which is unaffected by the CB1 and CB2

antagonist, SR141716A and SR144528 respectively (Rockwell and Kaminski, 2004;

Rockwell et al., 2006). In the same model arachidonic acid shows similar activity as

anandamide which is attenuated by pretreatment with nonspecific COX inhibitors as well

as COX-2 specific inhibitors (Rockwell and Kaminski, 2004). Pretreatment with the


174

PPAR-γ antagonist T0070907 also attenuated anandamide and 2-arachidonylglycerol

mediated suppression of IL-2 secretion (Rockwell and Kaminski, 2004; Rockwell et al.,

2006).

Yang et al. (2000) observed that PPARγ free Jurkat cells do not have an IL-2

response to PPARγ ligands troglitazone and 15d-PgJ2, but PPARγ expressing Jurkats did

exhibit IL-2 inhibition in response to the those PPARγ agonists. Clark et al. (2000) and

Rockwell et al. (2006) also found PPARγ inhibitory activity on IL-2 secretion by the

PPARγ agonists ciglitazone and 15d-PgJ2. Rockwell et al. also ruled out CB1 and CB2

activity on inhibition of IL-2 in their model of Jurkat cells and 2-AG ligand by

demonstrating the inhibition of transcriptional activity of the crucial IL-2 transcriptional

factors, NFAT and NFκB. Thus, in the absence, but not in the presence, of T0070907 IL-2

secretion was inhibited. Finally, Rockwell et al. (2006) also found that 2-AG treatment

doubled the PPARγ/ PPAR response elements (PPRE) and suppressed mRNA levels for

IFN-γ and IL-4 in Jurkats. As has been previously stated, PPARs are believed to be lipid

sensors. Thus, select fatty acids and their derivatives are recognized as PPAR ligands

(Nakamura et al., 2004; Rodriguez-Cruz et al., 2005). Notably, the alkylamides in

echinacea are fatty acid derivatives.

Using two well established PPARγ mediated biological responses, IL-2 response

and PPARγ-dependent adipogenesis in 3T3-L1 cells, the activity of UDA was probed. As

shown in Figure 5.6, the selective PPARγ antagonist T0070907 at 2.5 μM is effective in

attenuating the IL-2 inhibitory effects of UDA at alkylamide concentrations between 330

ng/mL to 1 μg/mL. This is in range of relevant physiological alkylamide concentrations

based on in vivo human pharmacokinetic data (Matthias et al., 2005a). T0070907 appears

to lose its ability to attenuate the IL-2 inhibition at higher concentrations of UDA. This


175

apparent loss of antagonist activity is an expected observation of competitive agonism.

The fixed dose of T0070907 (2.5 μM) appears to be overwhelmed by increasing

concentrations of UDA ranging over two orders of magnitude. It is also possible that at

high UDA concentrations, IL-2 inhibition occurs independently of PPARγ activity.

Alternatively, recent data suggests that the PPARγ receptor, which presents a particularly

large binding cavity as compared to other nuclear receptors, may bind more than one

ligand at a time (Itoh et al., 2008). For example, rosiglitazone, a selective PPARγ agonist

structurally similar to the agonist used in these studies, occupies only about 40% of the

ligand-binding site in the ternary complex of PPARγ, leaving adequate room for other

ligands (Nolte et al., 1998). Crystal structures of PPARγ demonstrate that PPARγ can bind

two 9-(S)-hydroxyoctadecadienoic acid molecules concurrently (Itoh et al., 2008). Thus a

more involved explanation of the loss of the antagonism by T0070907 at higher UDA

concentrations may be that PPARγ is binding both T0070907 and UDA at the same time

and this may result in an overall IL-2 inhibitory effect.

Past evidence demonstrates that PPARγ's ability to repress the activities of

numerous transcription factors, including NF-κB, NFAT and AP-1 has diverse activity on

immune and inflammatory responses (Jiang et al., 1998; Park et al., 2003; Ricote et al.,

1998; Shipley and Waxman, 2003). For example, PPARγ is known to be up-regulated

during the activation of mouse and human T cells (Cunard et al., 2002; Tautenhahn et al.,

2003; Wang et al., 2002) and this has been found to be time and concentration dependant

(Cunard et al., 2002; Tautenhahn et al., 2003). Intriguingly, up-regulation of PPARγ has

been reported to be in response to the anti-inflammatory cytokine, IL-4, in murine T cells

(Cunard et al., 2002). In turn, the PPARγ ligand, rosiglitazone has been observed to up-

regulate IL-4 production (but is down-regulated by 2-AG) (Efrati et al., 2009).


176

The IL-2 promoter is reported to increase in activity after T cells are treated with

PMA/PHA (Yang et al., 2000). The IL-2 promoter contains several regulatory elements

that can bind different transcription factors, including NFAT, AP-1, and NFκB (Crabtree,

1989). AP-1, a dimer consisting of c-jun and c-fos, results in a cooperative composite

enhancer (NFAT∙AP-1) that regulates expression of many cytokine genes including the

upregulation of IL-2 (Macian et al., 2001). Without AP-1, NFAT cannot bind to DNA-

NFAT sites. However, on activation, PPARγ physically associates with the NFAT to block

NFAT binding to DNA and thus, transcriptional activity. This ultimately blocks the

production of IL-2 (Macian et al., 2001).

Recent evidence has shown that anandamide and lipoxygenase products, previously

discussed as agonists of PPARγ, also have an inhibitory role on IL-2 promoters (Sancho et

al., 2003; Yang et al., 2002). NFκB inhibition has been found to be independent of CB1,

CB2 and the vanilloid receptor (an alternative site for cannabinoid binding) (Sancho et al.,

2003). In addition, AP-1 is reported to be inhibited by known PPARγ ligands (Chung et al.,

2003). Specific to c-fos expression in murine mesangial cells exposed to ciglitazone, there

is a marked down-regulation of c-fos (Ghosh et al., 2003).

It is also possible that the IL-2 inhibition could be explained by UDA acting as a

partial agonist. The prevailing dogma, suggest that ligands for the nuclear receptor

superfamily are classified as either ‘agonists’ or ‘antagonists’. Sporn et al. (2001) suggest

that this is misleading, pointing out that tamoxifen, classified as an estrogen antagonist,

acts on bone and uterine tissue as an agonist. Moreover, activity is dependent on the

cellular milieu; tamoxifen can act as an agonist in the absence of estrogen and as an

antagonist in the presence of estrogen in MCF-7 cells. Alkylamides have recently been


177

suggested to act as partial agonists in a fibroblast/adipocyte model (Christensen et al.,

2009).

Christensen et al. (2009) showed that hexadeca-2E,9Z,12Z,14E-tetraenoic acid

isobutylamide, up to 200 μM, stimulated glucose uptake but did not induce adipocyte

differentiation as compared to control. However, they did find induction of adipogenesis in

a PPARγ activation dependent model (DEX) and inhibition of adipogenesis in a PPARγ

independent model (MDI) for an undisclosed concentration of dodeca-2E,4E-dienoic acid

isobutylamide, an alkylamide found in both main commercial species of echinacea

(Christensen et al., 2009). However this alkylamide did not exhibit PPARγ activation

except at high concentrations (80 μM).

UDA, is considered a 2-ene alkylamide, which commonly contain diacetylinic tails.

Levels of 2E-monoene diacetylenic alkylamides, like undeca-2E-ene-8,10-diynoic acid

isobutylamide, appears to be dependent on the age of the echinacea plant. In the six species

in which UDA has been identified, E. angustifolia, E. sanguinea, E. simulata, E.

tennesseensis, E. atrorubens, E. laevigata, it is significantly higher in concentration in the

older plants (Wu et al., 2004). Interestingly, previous work has suggested the highest

concentration of UDA is found in E. sanguinea (Wu et al., 2004). E. simulata is also

known to contain relatively large concentrations of UDA (Bauer and Foster, 1991).

Of the two main commercial species that constitute echinacea extract preparations,

E. angustifolia and E. purpurea, the 2-ene alkylamides are more characteristic of the

alkylamides found in E. angustifolia. This is opposed to the 2,4-diene alkylamides, more

characteristically found in E. purpurea, which are more commonly olefinic alkylamides.

However, both echinacea species contain olefinic and acetylinic classes of alkylamides

(Spelman et al., 2009). The olefinic 2,4-dienes have demonstrated moderate to high CB2


178

affinity, while the diacetylinic 2-enes have thus far, with few exceptions, demonstrated

negligible CB2 affinity (Raduner et al., 2006). Considering Christensen et al. (2009) recent

work demonstrating activation of PPARγ in fibroblasts with olefinic alkylamides at high

concentrations (40-100 μM) and our results showing PPARγ activation with a diacetylinic

alkylamide at much lower concentrations (22-44 μM), it is possible that other diacetylinic

alkylamides which have lower affinities for CB2 than the olefinic alkylamides, may

activate PPARγ.

Accordingly, it may be that E. angustifolia and E. purpurea differ in the degree of

PPARγ and CB2 mediated immunomodulation. Previous studies report that the alkylamide

investigated here, UDA, comprises 5% of the alkylamide content of E. angustifolia root

(Matthias et al., 2005b). Furthermore, up to 55% of E. angustifolia alkylamides possess a

diacetylenic tail like that of UDA, and a large percentage of these are also 2-ene

alkylamides (Wills and Stuart, 1999). This is in contrast to E. purpurea root, which does

not contain UDA in detectable concentrations (Spelman et al., 2009), and contains less

than 45% diacetylenic alkylamides overall (Matthias et al., 2005b).

A series of experiments comparing the ethanol extracts of E. angustifolia and E.

purpurea on a series of cytokines, suggested that E. angustifolia had more immune activity

than E. purpurea. Of interest, previous reports by the eclectic physicians of the early-mid

19th

to mid 20th

century, who brought echinacea into clinical practice, suggested that the

two species differed in effect (King et al., 1898). Further research is needed to evaluate

how E. angustifolia and E. purpurea vary in their effects, possibly due to differential

actions on pathways such as PPARγ and CB2. Future investigations should explore if the

echinacea alkylamides subtly shift endocannabinoid and PPARγ tone without the profound

side effects observed with the synthetic cannabinoids and the PPARγ TZD ligands.


179

These results should be interpreted carefully. There are other proteins known to

directly influence adipocyte differentiation besides PPARγ. This includes

CCAAT/enhancer binding proteins (C/EBP), and the basic helix-loop-helix-leucine zipper

transcription factor sterol regulator element-binding-protein-1c (Yeh et al., 1995).

Synthetic and endogenous PPARγ agonists have been demonstrated to block C/EBP

transcriptional activity by forming PPARγ∙C/EBPβ complexes. Activation of C/EBP is

known to engage the PPARγ pathways in both 3T3-L1 cells (Lefterova et al., 2009) and T

cells (Blanquart et al., 2003). Of particular relevance, a recent gene microarray suggested

C/EBPβ is an upstream activation node for many of the pathways activated by echinacea

(Altamirano-Dimas et al., 2007). Further investigations are needed to more clearly

elucidate the role of PPARγ in the presented results.

Summary

These experiments illustrate the involvement of PPARγ in the inhibition of IL-2

secretion by T cells in response to undeca-2-ene-8,10-diynoic acid isobutylamide. The data

indicate a decrease in IL-2 levels in this model system starting at 330 ng/mL of undeca-2E-

ene-8,10-diynoic acid isobutylamide, which is reversed by the addition of a PPARγ

selective antagonist. In addition, preliminary PCR studies suggest that c-fos and c-jun

expression is altered supporting the hypothesis of PPARγ involvement and the possibility

that UDA function as a partial agonist to PPARγ. In addition, the dose-dependent

differentiation of 3T3-L1 fibroblast in response to undeca-2E-ene-8,10-diynoic acid

isobutylamide also is indicative of induction of PPARγ activity.


180

However, the possibility that other targets are involved in this effect is not ruled out

by these data. Consequently, it is possible that there is a combination of effects due to

PPARγ and other targets that inhibit IL-2 production and induce adipocyte differentiation.

These results, coupled with previous results demonstrating cannabinoid activity of the

alkylamides, suggest that the immunomodulatory potential of the echinacea alkylamides is

likely due to polyvalent activity. Further investigations are needed to elucidate the role of

PPARγ and other potential targets in the IL-2 inhibitory response of T cells to alkylamides,

including undeca-2E-ene-8,10-diynoic acid isobutylamide.

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Chapter 6 Discussion

187

Chapter 6

Discussion

Natural products have been called “a population of privileged structures selected by

evolutionary pressures to interact with a wide variety of proteins and biological targets for

specific purposes”(Koehn and Carter, 2005). The short of this is that food and medicinal

plants may often lead to broad spectrum effects, that over an evolutionary time scale have

become conditionally essential for human health.

In regard to echinacea, a past prominent remedy for U.S. physicians, one of the top

sellers in the natural products industry and currently a frequently physician prescribed

remedy in Germany, the hunt for bioactive constituents has lead investigators into the

elucidation of a number of constituent groups. The phenylpropanoids, such as cichoric acid

and echinacoside, the polysaccharides and glycoproteins, the alkylamides and, most

recently, the lipoproteins (Pugh et al., 2008) have all been considered “actives” of

echinacea. It seems likely, through the examples of other heavily researched medicinal

plants, that the pharmacological effects of echinacea are best studied under a network

pharmacology model which allow for the recognition of multiple actives and effects on

diverse biochemical pathways (Spelman, 2009).

Nonetheless, in the series of investigations presented here, it has been necessary to

narrow the focus to one group of constituents, or at times, even one constituent. As a

result this discussion will focus on the alkylamides, their extraction, metabolism and

stability, as well as the PPARγ activity of undeca-2E-ene-8,10-diynoic acid isobutylamide

(UDA).


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Extraction

The preliminary studies presented here suggest that the alkylamides are sufficiently

concentrated in extracts of E. purpurea to provide biological activity. Lower

concentrations of alkylamides than those found in the ethanolic extracts highlighted in

Chapter 2, have proven in pharmacokinetic tests to result in blood levels in humans of

alkylamides ranging from 17.4 ng/mL after a single oral dose of E. angustifolia tincture

(Dietz et al., 2001) and 44 ng/mL after a single dose of E. purpurea tincture (Matthias et

al., 2005a) to 336±131 ng/mL after ingestion of an ethanolic extract of E. purpurea and E.

angustifolia in tablet form (Matthias et al., 2007a). Consequently, the ethanolic extracts

were used in all steps in these series of experiments, generating data that is relevant to

researchers, clinicians and consumers. Of interest to all of the before mentioned groups is

that, depending on the ratio of plant to solvent and whether the extraction is fresh or dry,

alkylamide groups are extracted differentially. For example, the acetylene alkylamides

under different conditions appear to extract at different concentrations than the olefinic

alkylamides (Spelman et al., 2009b). Due to the fact that there has been different

bioactivity shown for the acetylene versus the olefinc alkylamide (Chicca et al., 2009;

Gertsch et al., 2004; Matthias et al., 2008; Matthias et al., 2007b; Matthias et al., 2005b;

Raduner et al., 2007; Spelman et al., 2009a), this is of significance to many potential

applications of echinacea products.


189

Metabolism

The metabolism of the alkylamides has predominantly focused on one alkylamide,

dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (Z-tetraene). This alkylamide is of

significance to due to the elucidation of its CB2 activity (Matovic et al., 2007), but also

because of the potential of drug-herb interactions (Freeman and Spelman, 2008). It is

present in all 3 commercial species of echinacea (and most other Echinacea spp.) and

occurs as a series of three isomers (tetraenes). The CB2 affinity is highest for the Z-

tetraene (≈57 nM), but the other isomers, dodeca-2E,4E,8E,10Z-tetraenoic acid

isobutylamide and dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide, also show CB2

affinities that could be physiologically relevant (≈4.5 & 9.1 μM, respectively).

Thus far, two independent labs have reported the hydroxylated metabolite of the

tetraenes reported in chapter 3 of this document (Cech et al., 2006; Matthias et al., 2005b).

In chapter 4 a carboxylated tetraene metabolite is reported. The carboxylated metabolite

has, thus far, shown different immune activity than the native tetraenes. Specifically, the

carboxylated metabolite appears to have diminished IL-2 inhibitory activity. This may

prove to be of significance as the pharmacokinetics of these compounds becomes

understood further.

There has been data generated on other alkylamide metabolites, namely the

alkylamide studied in the chapter 5 on PPARγ activity of undeca-2E-ene-8,10-diynoic acid

isobutylamide (UDA). Cech et al. (2006) have shown that after exposure to human liver

microsomes, UDA is metabolized to a hydroxylated structure (but not carboxylated).

While this is interesting, there is still no data available for the physiological and


190

pharmacological effects of the hydroxylated metabolites. However, a potential target for

hydroxylated metabolites will be suggested below.

Degradation

Considering that the extraction process was found to generate differential

concentrations of alkylamide types, it should be no surprise that the degradation of

alkylamides is also variable over time. The data presented here show that the tetraenes are

stable over one year. In comparison, the acetylenic alkylamides appear to degrade at a

much higher rate (over 50% loss) than the olefinic tetraenes in ethanolic extracts in the

same time period. In addition, the alkylamides are found to degrade significantly in both

cut/sift and powdered forms of echinacea root.

This finding is key to researchers, clinicians and consumers. For researchers it

suggests that the age of the plant material purchased and the processing of the plant

material are key to generating full spectrum alkylamide extracts. Even if isolation of

alkylamides is the goal, the quantity of alkylamides makes a difference on expense, time

investment, energy investment and quantity of solvents. For clinicians and consumers, it

suggest that all echinacea products are not created equal and that besides the expected

expiration date, products should ideally have a harvest date, and a processing date. If the

alkylamides (or the phenylpropanoids) are the dominant active constituents in echinacea,

and this is still debatable, then the age of the material will potentially have a significant

impact on efficacy. Moreover, due to differential effects of the alkylamides, an aged

echinacea remedy may have different bioactivity than an extract made with newly

harvested plant material. This is of course applicable to medicinal plant extracts generally


191

and is an impediment to rational clinical use and any regulatory efforts. There are many

issues to be worked out regarding this issue and they include not only the data generated

by analytical techniques, but also include politics, economics and special interest groups

who have considerable resources to propose or oppose regulations.

Bioactivity: alkylamides as PPARγ activators

A number of fatty acid amides, a structurally diverse endogenous congener of

molecules, are known to be bioactive (Table 6.1). Due to their role in cell signaling, these

molecules, and their analogs, may prove to have diverse activity due to their interface with

a number of receptor systems, including, but not limited to CB2 and PPARγ. Table 6.2

Table 6.1. Bioactive Fatty Acid Amides

Receptor Type Ligands

CB1 Anandamide; N-Dihomo-γ-linolenoylethanolamine;

5Z,8 Z,11 Z –eicosatrienoylethanolamine; N-Acyldopamines; N-Arachidonoyldopamine

CB2 Anandamide; N-Dihomo-γ-linolenoylethanolamine;

5Z,8 Z,11 Z -eicosatrienoylethanolamine

GABAA Primary fatty acid amides; Oleamide

Non-CB1/CB2 GPCR (aorta)

N-Acyldopamines; N-Arachidonoyldopamine;

GPR18 N-arachidonoylglycine

GPR55 N-Palmitoylethanolamine

GPR119 N-oleoylethanolamine

PPARα Anandamide; N-oleoylethanolaminel; N-Palmitoylethanolamine;

N-oleoyldopamine

PPARγ Anandamide; N-oleoylethanolamine; ; N-oleoyldopamine

TRPM8 Anandamide

TRPV1 Anandamide; N-oleoylethanolamine; N-linolenoylethanolamine; N-

linoleoylethanolamine; N-Acyldopamines; N-arachidonoyldopamine; N-acylamino acids; N-arachidonoyltaurine

TRPV4 N-acylamino acids; N-arachidonoyltaurine

5HT2A, 5HT2C, 5HT7 Primary fatty acid amides; Oleamide

From (Farrell and Merkler, 2008)


192

illustrates the endogenous ligands of CB2 and PPARγ and key alkylamides of echinacea

that have shown PPARγ activity or CB2 activity. Table 6.2 includes UDA, the alkylamide

found to activate PPARγ in Chapter 5.

Besides the N-alkylamides found in a variety of plant families, the N-

acylethanolamines with their ethanolamide head group, are also worthy of mention. In

addition, enzymes involved in these fatty acid derivatives biosynthesis and degradation,

may also be a source of further insights in the activity of this class of molecules in

mammalian systems. In particular, COX and LOX are relevant. Considering the broad

activity exhibited by the fatty acid amides, there is a wide range of activities including

cancer, cardiovascular disease, inflammation, pain, drug addition, eating disorders, anxiety

and depression (Farrell and Merkler, 2008).

PPARγ plays a profound role in metabolic and immunological processes and as a

result may play a role in many disease processes. Thus far this includes inflammation,

cancer and autoimmune disorders and the list is growing (Desvergne et al., 2004; Khan and

Vanden Heuvel, 2003; Rees et al., 2008; Vamecq and Latruffe, 1999; Yuan et al., 2004).

PPAR ligands inhibit the expression of proinflammatory cytokines such as IL-1ß, IL-2,

IL-6, IL-8, MCP-1, TNF- , and MMPs in various cell types. This includes cell types such

as monocyte/macrophages, endothelial cells, smooth muscle cells, and adipocytes. These

activities have been reported to be due to transcriptional regulation and nontranscriptional

interference with signaling pathways such as NFκB (p65, p50), NFAT, activator protein-1

(AP-1 = fos·jun), MAPK cascade, and STAT-1/STAT-3 (Chinetti et al., 2000; Dubuquoy

et al., 2002).


193

Table 6.2. Ligands of CB2 and PPARγ

CB2 ligands PPARγ ligands

1. O

NH

OH

Anandamide MW 347.3 (PPARγ)

6.

O

OH

O 15-deoxy-Δ

12,14-prostaglandin J2

MW 316.4 (fibroblasts activity 7 μM)

2.

O

O

OH

OH

2-arachidonylglycerol MW 378.3 (PPARγ)

7. O

OH

OH 13-hydroxyoctadecadienoic acid MW 296.4

3.

NH2

O

Dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide MW 247.3 (5 μM monocytes isomeric mix; PPARγ activation fibroblasts at 80 μM))

8. O

OH

O 13- oxooctadecadienoic acid MW 294.4

4.

NH2

O

Dodeca-2E,4E-dienoic acid isobutylamide MW 251.3 (PPARγ activation fibroblasts at 80 μM)

9.

NH2

O

Undeca-2E-ene-8,10-diynoic acid isobutylamide MW 231.3 (fibroblast activity 22-44) μM;T cell activity 1.4 μM)

5.

NH2

O

Dodeca-2E,4E,8Z-trienoic acid isobutylamide MW 249.3 (PPARγ activation fibroblasts at 80 μM)

10. O

NH2

Hexadeca-2E,9Z,12Z,14E-tetraenoic acid

isobutylamide MW 303.3(fibroblast activity 30 μM)

a. When information was available concentrations found to be active are stated.

b. Compounds 3 & 4 are also PPARγ activators.


194

As discussed in chapter 5, the endogenous cannabinoid ligands anandamide and 2-

AG, bind the PPARγ nuclear receptor. This has brought pause to cannabinoid researchers

as the resolution of the signal transduction by CB2 has been obscured. For example, IL-2

inhibition was thought to be a classic CB2 response (Kaplan et al., 2005). Most recently

however, two independent groups have shown that IL-2 inhibition is due to endogenous

and some synthetic cannabinoids binding to PPARγ (Clark et al., 2000; Kaplan et al.,

2005; Ouyang et al., 1998; Rockwell and Kaminski, 2004; Rockwell et al., 2006). In

regard to echinacea, these results suggest the possibility of metabolic and immunogenic

effects mediated by PPARγ. While the immunogenic effects of the alkylamides of

echinacea are already well established, little to no work has been done on metabolic

activity of echinacea. Thus far, two papers have suggested that the alkylamides of

echinacea may have an influence on the physiological metabolism of fatty acids and blood

glucose via PPARγ (Christensen et al., 2009; Spelman et al., 2009a). The Spelman (2009a)

paper is a summation of Chapter 5. Christensen et al. (2009) suggested that the lipophilic

fractions of E. purpurea could be useful in the management of insulin resistance and type 2

diabetes. While this is possible, it is intriguing that one of the traditional uses of echinacea

is listed as diabetes (Duke and Duke, 2002; Mills and Bone, 2000).

Are there previous investigations suggesting PPAR activity?

Gertsch et al. (2006) argues for the alkylamides being classified as

cannabinomimetics due to an overall effect similar to cannabinoids; IL-2 inhibition and a

Th biasing effect towards an increase of Th2 activity (Klein, 2005). However, the same


195

argument can be made for PPARγ which modulates immune function in a Th2 direction

(Saubermann et al., 2002; Yang et al., 2002). Hence, in consideration of the results

presented in chapter 5, in addition to CB2 activity, at least some of the alkylamides may

mediate their immunomodulatory effects through PPARγ activity.

Besides the investigations that show direct PPARγ involvement for the alkylamides

of echinacea (Christensen et al., 2009; Spelman et al., 2009a), many of the previous

investigations into echinacea and the alkylamides of echinacea can be extrapolated to

suggest PPARγ activity. This includes the IL-2 activity, anti-neoplastic and life extension

effects of echinacea extracts (Brousseau and Miller, 2005; Currier and Miller, 2001;

Sasagawa et al., 2006).

The IL-2 inhibition, previously discussed as due to PPARγ signaling (Rockwell and

Kaminski, 2004; Rockwell et al., 2006), of other alkylamides besides UDA has been

demonstrated (Sasagawa et al., 2006). Sasagawa et al. (2006) showed that dodeca-

2E,4E,8Z,10Z-tetraenoic acid and dodeca-2E,4E-dienoic acid isobutylamides also inhibited

IL-2 activity. While both of these alkylamides have been reported to be CB2 ligands, this

suggest the possibility of PPARγ activity as well. In unreported experiments, I found that

both of these alkylamides at moderate to high concentrations (≈43 μM) induced 3T3-L1

differentiation. Christensen et al. (2009) also reported that these alkylamides induced

PPARγ activation although only weakly at high concentrations (40-100 μM).

Besides these observations, more indirect extrapolations can be made. For example,

a series of experiments have shown that the ingestion of a commercially prepared E.

purpurea extract extends life in murine models. In one experiment, healthy mice were

dosed daily (0.45 mg/mouse) from 7 weeks of age to 13 months. By 13 months, 46% of the

control group was alive as opposed to 74% of the experimental group. In addition, NK cell


196

counts were elevated and stayed elevated throughout the experimental groups lifetime as

compared to the controls (Brousseau and Miller, 2005). In another investigation, mice were

injected with leukemic cells and started on the same dose and preparation of E. purpurea.

By day 9, an elevation of NK cells was seen in the experimental group. By day 27 the

untreated mice had died and by 3 months, 1/3 of the echinacea treated group were still

alive and went on to live full life spans with 2 x the normal NK cell count as normal mice

(Currier and Miller, 2001). Finally, mice that were immunized against leukemia 5 weeks

prior to injection with live leukemic cells, exhibited a survival rate of about 30%. When

echinacea was combined with the immunization, the survival rate went up to 60% group

(same dose and preparation). In this case the echinacea treated group had NK levels 3 x the

immunized mice. Intriguingly, past studies on structure activity relationships of

alkylamides have suggested that some of these compounds promote terminal

differentiation of leukemia to a benign state (Harpalani et al., 1993). However, these

previously mentioned effects have not been shown for an echinacea preparation containing

only alkylamides or isolated alkylamides.

What is interesting about the results suggesting PPARγ activation by alkylamides is

that activation of PPARγ is known to increase life span (Chung et al., 2008; Erol, 2007;

Martin et al., 2006; Yamagishi et al., 2009; Zhang and Zheng, 2008) and have a

modulatory role on NK cells (Zhang et al., 2004; Zhang and Young, 2002). Notably,

PPARγ ligands have a positive influence on leukemia models (Abe et al., 2002; Emi and

Maeyama, 2004; Liu et al., 2009) and synergize with known leukemic cancer drugs (Bertz

et al., 2009).

Considering that select alkylamides may have PPARγ activity and CB2 activity, E.

angustifolia and E. purpurea may differ in the degree of PPARγ and CB2 mediated


197

immunomodulation as suggested in Chapter 5. If the olefinic alkylamides do activate

PPARγ, the fact that E. angustifolia has less of these olefins (< 45%), which are the

primary alkylamides shown to target human CB2 (Raduner et al., 2006; Woelkart et al.,

2008), suggest that there may be differences in the physiological activity of echinacea

species based on the concentration of the various alkylamide species.

Other possible targets; C/EBP, RXR, TRPV1

The C/EBP protein

There is also the possibility that CCAAT/enhancer binding proteins (C/EBP), and

retinoic X receptor (RXR) proteins are involved in the immunomodulatory activity of

echinacea alkylamides. The C/EBP protein (like PPAR) are synthesized in multiple

isoforms and are involved in a broad range of physiological processes including as a

mediator of IL-6 transcription and signaling (Poli et al., 1990). Due to their leucine zipper

structure, they are able to form both homo- and heterodimers with other proteins.

In the case of 3T3-L1 differentiation there has been controversy over which

receptor is the “master” regulator. C/EBPα, which induces its own expression as well as

the transactivation of PPARγ, is reported as the transcriptional master regulator of late

adipocyte differentiation (Camp et al., 2002). Though, without C/EBPβ early stage

differentiation would not proceed either (Lefterova et al., 2008).

Besides a prominent role in adipocyte differentiation, C/EBP is known to play a

role in IL-2 inhibition. C/EBPβ has been shown to suppress IL-2 and IFN expression and

accordingly, plays a role in the commitment of Th0 cells to differentiate towards Th2 cells


198

(Berberich-Siebelt et al., 2000). Synthetic and endogenous PPARγ agonists have been

demonstrated to block C/EBP transcriptional activity by forming PPARγ∙C/EBPβ

complexes.

Further support for the alkylamides of echinacea influencing C/EBP activity comes

from gene microarrays. Altamirano-Dimas et al. (2007) reported that C/EBPβ was a likely

upstream activation node for many of the pathways activated by echinacea after running

their data through Ingenuity Pathways Analysis software.

The RXR protein

RXRs, occurring in 3 isoforms α, β, γ, are known as the major heterodimer partners

of several nuclear receptors including the PPAR isoforms. The endogenous ligand is 9E

retinoic acid, yet another fatty acid like molecule. RXR heterodimers bind to a variety of

ligands derived for cholesterol, fatty acids, and glucose to regulated target genes that

mediate metabolic homeostasis (Nohara et al., 2009).

PPARγ is an obligate heterodimer with the RXR to transduce signals. As with other

PPAR nuclear receptors, a ligand binding to PPARγ or RXR promotes dimerization,

interaction with DNA and modulates transcription of several genes (Sun et al., 2007). The

PPAR ∙RXR heterodimer is known to play a key role in mediating the inflammatory

process and therapeutic applications

in inflammation-related diseases are underway

(Desreumaux et al., 2001; Dubuquoy et al., 2002).

Of interest, the PPAR ∙RXR heterodimer responds to ligands for either receptor

and binding of both receptors is a synergic integration of two ligand-dependent pathways.

Although the lipophiles that bind RXR appear to be more complex than the N-alkylamides,


199

the metabolized versions of alkylamides are remotely similar to the retinoids. Moreover, it

may be possible that PPARγ binding of alkylamides may recruit endogenous retinoic acid

to bind RXR, thereby synergizing the effect of the alkylamides.

The Vanilloid Receptors

The vanilloid family of receptors also known as the transient receptor potential

cation channels are currently known to have 6 distinct receptor members in humans

(Vennekens et al., 2008). The transient receptor potential cation channel, member 1

(TRPV1), was the first vanilloid receptor identified and is also called the capsaicin receptor

as it binds the alkylamide capsaicin from Capsicum annum (chili peppers). The TRPV1

receptor is known to play a role in nociception resulting from inflammation and

neuropathic pain (Tobin et al., 2002). Central to this discussion, the endogenous

cannabinoids are known to bind this receptor.

While Gertsch’s (2006) investigations have suggested that the alkylamides from

echinacea did not bind TRPV1, others have shown that hydroxylated alkylamides may

bind TRPV1 and another vanilloid receptor member, TRPA1 (Riera et al., 2009). The

metabolites of the alkylamides need to be investigated for possible vanilloid receptor

activation.

Synergic activity?

Synergy is often cited as a potential basis of action for many medicinal plant

species (Kirakosyan et al., 2009; Spelman et al., 2006; Williamson, 2001). Considering the


200

number of compounds found in a medicinal plant extract, it seems highly likely that either

pharmacokinetic potentiation or pharmacodynamic enhancement is likely for certain

medicinal plant extract. For example, an Actea racemosa (black cohosh) product has been

shown to modulate over 400 genes in MCF-7 cells (Gaube et al., 2007). In the case of

echinacea, considering the activities that have been suggested, including antimicrobial,

antioxidant, anti-inflammatory and immune upregulation, it is possible that synergic

activity plays a role in echinacea’s activity on the immune system.

Recent work by Chicca et al. (2009) has shown pharmacodynamic synergic activity

by the alkylamides on the cannabinoid system. This includes inhibition of fatty acid amide

hydrolase (FAAH), the enzyme the controls the half-life of the endogenous cannabinoids,

direct binding to CB2 and facilitated transport of the endocannabinoids (Chicca et al.,

2009).

Given that PPARγ binds the endogenous cannabinoids, it is also possible that an

extended half-life of the endocannabinoids, a pharmacokinetic potentiation, prolongs

signaling of the PPARγ and CB2 systems. In effect, this may lead to pharmacodynamic

synergic activity due to alkylamide activation and extended half-life of the endogenous

agonists of PPARγ and CB2. In addition, considering that the RXR∙PPARγ heterodimer

responds to ligands for either receptor and binding of both receptors represents an

integration of two ligand-dependent pathways, this generates further potential

pharmacodynamic enhancement. Furthermore, due to the possibility of PPARγ receptor

binding two ligands simultaneously, the likelihood of additive, synergic or antagonistic

activity just within the PPARγ LBD domain is also possible.

Besides the alkylamides there are other compounds that have recently shown

PPARγ and CB2 activity in echinacea species. For example, kaempferol, a ubiquitous


201

flavonoid also found in echinacea, exhibits ligand and allosteric effector properties on

PPARγ (Fang et al., 2008; Liang et al., 2001; Spelman and Burns, 2009). β-caryophyllene,

occurring in echinacea and many other medicinal plant species is also a CB2 agonist

(Gertsch et al., 2008). Such multi-pathway activation by diverse constituents as well as

polyvalent activity by just the alkylamides generates a plethora of complexities that

obscure the role of any one constituent in signaling pathways. With the existing data it

does appear that multiple pathways, at least PPARγ and CB2 signal transduction pathways,

converge for an immunomodulatory effect by the alkylamides, and possibly other

constituents.

Future work

Considering the presented data on PPARγ activity by UDA, and the previous work

by Christensen et al. (2009) and Spelman et al. (2009a), there is building evidence that

PPARγ may be a key site in the alkylamide induced immunomodulation of echinacea. In

light of the previous discussion, there are also other key sites to consider besides PPARγ

and CB2 as discussed above.

As we gain further insights into the interaction between cellular

signaling/regulation and botanical compounds or food constituents, Shay et al. (2005)

suggest a few principles are crystallizing. First, as with the alkylamides, it is increasingly

obvious that one ingested dietary compound may interact with a number of systems such

as PPAR regulation, antioxidative protection, and effects on cell signaling processes.

Second, the inherent promiscuity of nuclear receptors permits the interface of multiple


202

phytochemicals with the lipid and immune regulating systems of the body (Shay and Banz,

2005).

The complexity of studying multicomponent remedies requires models that can

assess multiple pathways simultaneously, since ingestion of a medicinal plant product (or

food plant) likely activates multiple pathways. While the polyvalent nature of medicinal

plant extracts adds to the potential of complex signal transduction, which is difficult to

fully comprehend in current in vitro and in vivo models, recent technological

breakthroughs such as gene microarrays and the “omics” platforms, in combination with

new constructs such as network pharmacology, offer more realistic maps of real-time

physiology.

A common argument about medicinal plants is that they must be “assessed” in

similar models as single chemical entities. While safety is of the utmost of importance and

a rational requirement for the multicomponent products originating from medicinal plants,

the current safety (and efficacy) model is based on the assessment of single molecules with

steric factors and electronic forces that differ from the majority of the 1biosynthesized

molecules. An evaluation of molecules generated from natural products shows that many

do not conform to Lipinski’s rule of 5, a foundational principle used in drug development

to guide the structural development of new chemical entities. This leads to the question, is

it rational to evaluate the less complex new to nature molecules synthesized in laboratories

by the same criteria as a series of molecules selected, through hundreds of millions of years

of evolutionary high throughput screening, for different structural foundations?

Intriguingly, the alkylamides do not conform to Lipinski’s rule of 5 (Basso et al., 2005;

Gertsch et al., 2006) yet appear to offer a therapeutic option to modulate immune function.

1 Biologically active allelochemicals/secondary metabolites from plants.


203

Future work may show that a diet rich in phytochemical content may result in

complex signaling patterns, similar to what early humans were accustomed to, via a variety

of promiscuous nuclear receptors, as well as other enzymes and receptors systems, that

cannot be observed by following only one food or botanical constituent (Shay and Banz,

2005).


204

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1Mol. Nutr. Food Res. 2008, 52, 000 –000 DOI 10.1002/mnfr.200700113

Review

A critical evaluation of drug interactions withEchinacea spp.

Camille Freeman1, 3 and Kevin Spelman 2, 3

1 Department of Physiology and Biophysics (MS candidate), Georgetown University, Washington, DC, USA2 Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, NC, USA3 Department of Herbal Medicine, Tai Sophia Institute, Laurel, MD, USA

Accurate information concerning drug–herb interactions is vital for both healthcare providers andpatients. Unfortunately, many of the reviews on drug–herb interactions contain overstated or inaccu-rate information. To provide accurate information on drug–herb interactions healthcare providersmust account for product verification, dosage, medicinal plant species, and plant part used. This crit-ical review assessed the occurrence of drug interactions with one of the top selling botanical rem-edies, echinacea including Echinacea angustifolia, E. pallida, and E. purpurea. Only eight paperscontaining primary data relating to drug interactions were identified. Herbal remedies made fromE. purpurea appear to have a low potential to generate cytochrome P450 (CYP 450) drug–herb inter-actions including CYP 450 1A2 (CYP1A2) and CYP 450 3A4 (CYP3A4). Currently there are no ver-ifiable reports of drug–herb interactions with any echinacea product. However, further pharmacoki-netic testing is necessary before conclusive statements can be made about echinacea drug–herb inter-actions. Given our findings, the estimated risk of taking echinacea products (1 in 100 000), the num-ber of echinacea doses consumed yearly (A10 million), the number of adverse events (a100) and thatthe majority of use is short term, E. purpurea products (roots and/or aerial parts) do not appear to bea risk to consumers.

Keywords: Angustifolia / CYP 450 / Echinacea / Pallida / Purpurea /

Received: March 20, 2007; revised: November 21, 2007; accepted: January 1, 2008

1 Introduction

Medicinal plants, while necessarily part of healthcare indeveloping regions, are becoming increasingly popular inthe industrial nations. Estimates suggest that up to 30% ofthe North American population use herbal supplements [1].Given that nearly 3.6 billion prescriptions were purchasedin 2005 [2] and 30% of Americans report using five ormore drugs in the same week [3] it comes as no surprise thatthe incidence of consumers taking herbs in conjunctionwith prescription medications is quite high. Eisenberg et al.

[4] found that up to 15 million adults were concurrentlyusing prescription medications and herbal supplements. Anationwide telephone survey by Kaufman et al. [5] supportsthis, finding that 16% of respondents had taken both pre-scribed medications and supplements in the week precedingthe interview.

Echinacea is one of the most popular herbal supplements,with a sales ranking of #2 in the mass market [6] and totalsales in 2006 (excluding Walmart sales), reaching morethan $36 million in the US [7]. Native Americans intro-duced echinacea to European settlers, after which it becameone of the most popular remedies used by the Eclecticphysicians for a variety of conditions including septicemia,typhoid fever, and catarrhal conditions of the respiratoryand reproductive tracts [8, 9]. Additionally, physicians prac-ticing from the 19th century through the mid 20th centuryused echinacea to treat other infections and counteract ven-omous snakebites [10]. Echinacea was included in theNational Formulary from 1906 until 1950, eventually fall-ing out of favor as pharmaceuticals came to dominate clini-

Correspondence: Kevin Spelman, Department of Chemistry and Bio-chemistry, 435 Science Bldg, UNCG, Greensboro, NC, USAE-mail: [email protected]: +1-336-334-5402

Abbreviations: CYP 450, cytochrome P450; CYP1A2, cytochromeP450 1A2; CYP3A4, cytochrome P450 3A4; CYP2D6, cytochromeP450 2D6; OATP-B, organic anion-transporting polypeptide; ROS, re-active oxygen species

i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mnf-journal.com

C. Freeman and K. Spelman Mol. Nutr. Food Res. 2008, 52, 000 –000

cal therapeutics. Currently, the aerial portion of Echinaceapurpurea and the roots of E. pallida are approved for coldsand infection of the respiratory and urinary tracts by theGerman Commission E [11].

The current body of scientific literature on echinacea canbe confusing not only due to the multiple species in use –namely E. purpurea, E. pallida, and E. angustifolia – whichhave phytochemical similarities, but also have notable dif-ferences, particularly around the identity and concentrationof alkylamides and other key constituents [12–14]. Eventhough 80% of the echinacea products sold to consumersare made from purpurea [15], all three species are oftenused interchangeably for the treatment of cold, flu, respira-tory infection, and inflammation [16]. Multiple species,plant parts, and preparations are also used, each of whichmay have a different constituent profile [17]. “Echinacea”may refer to the roots, aerial parts, whole plant, or a combi-nation of the above; echinacea products can be made fromfresh or dried plant parts, and may be prepared by juicing,alcohol extraction, infusion, decoction, or consumed as tab-lets or capsules [18].

Many authors suggest that taking herbs such as echinaceais potentially unsafe for those taking prescription medica-tions, and statements of warnings/cautions about drug–herb interactions are frequently found in the scientific andmedical literature [19–21]. In discussing ranking schemesof medicinal plant efficacy and safety, Mills (Mills, S., sub-mitted pers. comm.) points out that if safety statementswere graded to the same rigor as efficacy statements, mostof the papers citing poor safety records of herbal remedieswould fail the burden of proof. For example, Messina [19],as well as Vickers and Zollman [21], citing an inaccuratereport that echinacea is hepatoxic due to pyrrolizidine alka-loid content [22], contend that echinacea should not be usedwith known hepatotoxic drugs. Other authors have notedthat the echinacea poses no risk of hepatoxicity [23–25].The saturated pyrrolizidines found in echinacea lack anytoxicity, unlike unsaturated pyrrolizidines which are highlysusceptible to nucleophilic reactions [26].

To the detriment of the healthcare field, such misleadinginformation appears to be common in the literature, misin-forming healthcare providers and unnecessarily frighteningpatients. Brazier and Levine [27] found that of 240 articlesthat pertained to drug–herb interactions, the majority of thereports were anecdotal – less than 10% reported primarydata. Coon [28] reported that of 165 possible drug–herbinteractions, only 13% were supported by clinical data. Ofthe 352 papers we found as relevant to drug interactionswith medicinal plant preparations in a MEDLINE searchlimited to 2006, only 4.2% were actual case reports and4.0% were clinical trials. It appears that the vast majority ofthe data on drug–herb interactions are not based on primaryreports. Hence, the aim of this review is to provide a criticalevaluation of the primary literature on the evidence of druginteractions with Echinacea spp.

2 Methods

We searched the MEDLINE, SciFinder Scholar, Web ofScience, Cochrane Library, CAPLUS, and EBSCO data-bases using the terms echinacea, and “drug interaction,”“herb–drug interaction,” or “P-glycoprotein” with a cutoffdate of 12-15-2006. A manual search of bibliographiesfrom published articles was also performed. Inclusion crite-ria included the following research models: clinical trials,in vivo, ex vivo, and in vitro investigations. The search waslimited to English-language papers; otherwise no limitswere placed on the search.

The authors independently assessed abstracts and titlesof each publication for evidence pertaining to drug–herbinteractions, and acquired full articles that made commentson any Echinacea species and drug–herb interactions. Pub-lications that referenced an interaction but did not containan original report were excluded; only primary literaturewas assessed. Each interaction reported in the primary liter-ature was evaluated by both authors and later reconciled.Disagreements were resolved by discussion and consensus.

3 Results

Of the 49 articles located, only 8 contained primary evi-dence of potential interactions between echinacea productsand drugs. All other articles either referenced these eightprimary studies or were based upon hypothetical interac-tions (Table 1). Three of these primary articles did not con-tain complete information about the dosing used [29–31];only four indicated that the product used was verifiedbefore the research was undertaken [31–34]. One of theeight papers tested echinacea in combination with otherherbs [30], and therefore yielded little useful informationconcerning interactions due to echinacea alone and waseliminated from the review.

No documented case studies of echinacea–drug interac-tions were found. Of the eight primary papers, only twowere conducted in a human model [32, 35]. No clinicalpharmacokinetic studies that tested coadministration ofechinacea products and pharmaceuticals were available.Table 2 summarizes the cytochrome P450 (CYP 450) mod-ulation reported in the eight primary papers.

3.1 Cytochrome P450 interactions

3.1.1 CYP 450 1A2 (CYP1A2)Both Gurley et al. [35] and Gorski et al. [32] report humanpharmacokinetic results involving CYP1A2. In a study of12 volunteers who took 1600 mg/day E. purpurea root for8 day, Gorski et al. [32] found significant reduction in oralcaffeine clearance by 27%. The authors postulated thatinhibition at this level could affect CYP1A2 substrates withnarrow therapeutic index such as theophylline and cloza-

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pine. Gurley et al. [35] found no significant inhibition ofCYP1A2 in healthy volunteers after 28 day of 1600 mg/dayof whole-plant extract of E. purpurea, although a 13%decrease in 6 h paraxanthine/caffeine values suggested apossible inhibitory effect of E. purpurea whole-plantextract on CYP1A2. This effect, however, was not statisti-

cally significant and was not considered clinically signifi-cant by the authors.

3.1.2 CYP2C9Yale and Glurich [31] using an unrevealed concentration ofa remarkably concentrated 50:1 E. purpurea aerial extract,

3


Table 1. Primary research on drug interactions with Echinacea spp.

Author System tested Species and part used Model Comments

Budzinski et al.,2000

CYP3A4 Tinctures of (ratio unknown),55% EtOH of E. angustifolia+ E. purpurea (1:1, part unknown);E. angustifolia roots; E. purpurea roots;E. purpurea tops

In vitro – fluorometric microtiterplate assay

Ratio of plant material: menstruum wasnot available. Thus dosing unclear the 1:1ratio refers to proportion of E. angustifoliato E. purpurea. Products used were not au-thenticated.

Foster et al.,2003

CYP3A4,CYP2C9,CYP2C19,CYP2D6

Echinacea Plus – blend includingE. purpurea and angustifolia (plantpart not specified), extract of E.purpurea root, and assorted otherherbs; Echinacea special – 210 mg$triple Echinacea$ root per cup, 18 mgstandardized E. purpurea root extractper cup

In vitro – aliquots screened forability to affect in vitro metabolismof marker substances

Teas were mixtures, dosing of echinaceaunclear, parts used unclear in some cases,mixed teas were not inspected visually orauthenticated

Fuchikami et al.,2006

OATP-B E. purpurea, aerial parts In vitro – uptake of estrone-3-sulfate (an OATP-B substrate)measured in human embryonickidney 293 cells

No human studies contextualize clinicalrelevance of this info; paper does not indi-cate if product was verified to actually beE. purpurea

Gorski et al.,2004

CYP1A2,CYP2C9,CYP2D6,CYP3A

E. purpurea, root In vivo – 12 human volunteersunderwent a control phase totest for metabolism of substratesof these 4 enzymes; then theytook 400 mg E. purpurea root QIDfor 8 day. Substrates were readmi-nistered and blood/urine samplestaken

Formulation used was previously tested byConsumer Labs and shown to have >1%phenols. No adverse events reported byvolunteers or observed by researchers

Gurley, 2004 CYP3A4,CYP1A2,CYP2E1,CYP2D6

Echinacea purpurea,whole-plant extract

In vivo – 12 young adults partici-pated in an open-label studyrandomized for supplementationsequence (three other herbs weretested). Took 800 mg BID for28 day. Phenotypic metabolicratios were used to determineprobe drug clearance before andafter supplementation periods

While mean levels were not affected, therewas interindividual varaion – some indi-viduals appear to have significant inhibi-tion or induction. One person had a minorrash while taking Ech. Product used wastested for marker compounds to confirmidentity of E. purpurea

Moulick andRaner, 2003

CYP2E1,CYP1A

E. purpurea, root In vitro – substrate p-nitrophenolconcentration monitored usinghuman liver microsomes andexpressed CYP450 2E1 and 1A2

Only abstract available. Direct communi-cation with author provided information onspecies, plant part, dose, etc.

Raner et al.,2007

CYP2E1 E. purpurea, root; isolatedalkylamides; cichoric acid

In vitro – substrate p-nitrophenolconcentration monitored usinghuman liver microsomes andexpressed CYP450 2E1

Extract characterized by MS, individual al-kylamides and cichoric acid also tested

Yale and Glurich,2005

CYP2D6, 3A4, 2C9 E. purpurea, aerial portion, used capwith 50 mg of 50:1 extract, 15%pressed juice

In vitro – well plates with cDNA-derived enzymes in microsomesprepared from baculovirus-in-fected insect cells

Product tested by Consumer Lab to ensureidentity of product and minimal lot-to-lotvariation



Table 2. The Effect of Echinacea spp. on CYP 450

CYP 450isoform

Species Plant part Preparation Concentration(dose)

Model Results Author

1A purpurea Root 95% EtOH, 1:2fresh

0.2 – 2% In vitro No effect on ethoxycoumarinmetabolism

(Moulick and Raner,2003)

1A2 purpurea Root Capsule 1600 mg/day68 day

In vivo 27% z in oral caffeine clear-ance

(Gorski et al., 2004)

1A2 purpurea Whole plant Capsule 1600 mg/day628 day

Clinical trial 13% z in paraxanthine/caffeineserum ratios, not statisticallysignificant

(Gurley et al., 2004)

2C9 purpurea Aerial 50:1 extract (15%pressed juice)

Unspecified In vitro Inhibition of 7-methoxy-4-tri-fluoromethyl coumarin

[31] (Yale and Glurich,2005)

2C9 purpurea Root Capsule 1600 mg/day68 day

In vivo 11% – in oral tolbutamideclearance


2D6 purpurea Aerial 50 mg of 50:1 ex-tract (15% pressedjuice)

Unspecified In vitro No effect on AMMC metabo-lism

(Yale and Glurich,2005)

2D6 purpurea Root Capsule 1600 mg/day68 day

In vivo No effect on metabolism ofdextromethorphan


2D6 purpurea Whole plant Capsule 1600 mg/day628 day

Clinical trial No statistical significance in 4-hydroxydebrisoquin/[debriso-quin 4-hydroxydebrisoquin])urinary ratios


2E1 purpurea Root 95% EtOH 1:2fresh

0.4% In vitro 29% inhibition of p-nitrophenolmetabolism

(Raner et al., 2007)

2E1 purpurea Whole plant Capsule 1600 mg/day628 day

Clinical trial No statistical significance in 6-hydroxychlorzoxazone/chlor-zoxazone serum ratios


3A4 angustifolia Root 55% EtOH ratiounspecified

Unspecified IC501 – 2%

In vitro IC50 extrapolated, inhibition of7-benzyloxyresorufin metabo-lism

(Budzinski et al.,2000)

3A4 angustifolia + -purpurea 1:1

Part unknown 55% EtOH ext, ra-tio unspecified

Unspecified IC50A5% and a10%

In vitro Inhibition of 7-benzyloxyresor-ufin metabolism


3A4 purpurea Aerial 55% EtOH ratiounspecified


In vitro \)Moderately inhibitory\) (IC50A5% and a10%), concentrationunlikely to be physiologicallyrelevant


3A4 purpurea Aerial 50 mg of 50:1 ex-tract, 15% pressedjuice

Unspecified In vitro No inhibition of resorufin benzylether and $mild inhibition$ of7-benzyloxy-4-trifluoromethylcoumarin

(Yale and Glurich,2005)

3A4 purpurea Root 55% EtOH ratiounspecified


In vitro Inhibition of 7-benzyloxyresor-ufin metabolism


3A4 purpurea Root Capsule 1600 mg/day68 day

Clinical trial No change in oral clearance ofmidazolam; though FH and FG

affected differently volume ofdistribution (AUC) not signifi-cantly affected


42% – systemic clearance;18% z FH; FG – two-fold; 50%– Foral of midazolam

3A4 purpurea Whole plant Capsule 1600 mg/day628 day

Clinical trial No statistical significant differ-ence in 1-hydroxymidazolam/midazolam serum ratios


HLM purpurea Root 95% EtOH 1:2fresh

0.4% In vitro 27% inhibition of p-nitrophenolmetabolism

(Raner et al., 2007)

AMCC 3-[2-(N,N-diethyl-N methyl amino) ethyl]-7-methoxy-4-methylcoumarin; HLM, human liver microsomes; FH, hepatic availability;FG, GI availability.

Mol. Nutr. Food Res. 2008, 52, 000 –000

reported mild in vitro inhibition. Gorski et al. [32] reportedan 11% reduction in clearance of tolbutamide, a substrateof CPY2C9, after 8 day of E. purpurea root supplementa-tion. Gorski et al. [32] suggest this interaction is not clini-cally significant.

3.1.3 CYP 450 2D6 (CYP2D6)Evidence from the Gurley et al. [35] and Gorski et al. [32]studies presented above, as well as in vitro evidence fromYale and Gurlich [31] have shown no interaction between E.purpurea (aerial parts or root) and CYP2D6. Both humanstudies concluded that no interactions were expectedbetween E. purpurea and substrates of CYP2D6.

3.1.4 CYP2E1Gurley et al. [35] found no significant effects on CYP2E1after 28 day of 1600 mg/day E. purpurea whole-plantextract. In vitro data from Raner et al. [34], suggest inhibi-tion of CYP2E1 by an ethanolic extract of E. purpurea root,reporting a concentration of 2 lL/mL of a 95% EtOHextract producing a 27–29% inhibition. Of particular inter-est was the finding that the same concentration of a 33%EtOH extract of E. purpurea root did not demonstrateCYP2E1 inhibition.

3.1.5 CYP3A4Conflicting data were reported for CYP3A4; in vitroresearch, some at concentrations not obtainable with oraldosing, suggested mild to strong inhibition on CYP3A4while human trials show no overall CYP3A4 activity. Bud-zinski et al., using benzyloxyresorufin as a substrate andextract of (i) the E. purpurea aerial portion or (ii) a mixtureof E. purpurea aerial portions and E. angustifolia root (1:1),observed moderate inhibition of CYP3A4 (unspecifiedextract ratio, IC50 A5% and a10%). Using solely the rootsof both angustifolia and purpurea resulted in higher inhibi-tion of CYP3A4 (unspecified extract ratio, IC50 A1% anda5%) [29]. The concentrations of extracts used in theseassays, if typical of commercial extracts, are unobtainableby oral dosing.

Further in vitro research by Budzinski et al. [29]reported a significant difference in the effects of E. pur-purea versus E. angustifolia roots on CYP3A4. WhileE. angustifolia demonstrated a strong inhibitory effect onCYP3A4 activity, E. purpurea was found to be a mildinhibitor of CYP3A4 for 7-benzyloxy-4-trifluoromethyl-coumarin metabolism and had no activity on resorufinbenzyl ether metabolism, except at nonrelevant physiolog-ical concentrations (resulting in mild inhibition) [35].Another in vitro investigation [29] demonstrated hydroe-thanolic extracts of E. purpurea aerial portion and a com-bination of E. purpurea and E. angustifolia (plant partsunspecified) moderately inhibited CYP3A4-mediated7-benzyloxyresorufin metabolism while root extracts ofE. purpurea and E. angustifolia demonstrated strong inhib-

ition. Unfortunately, not enough data were provided to cal-culate the concentration of plant material that generatedthese results.

Human investigations by Gorski et al. [32], found nochanges in the metabolism of midazolam, a CYP3A4 sub-strate, after participants ingested 1600 mg of E. purpurearoot daily for 8 day. While the authors observed an 85%increase in intestinal availability of midazolam, a 15%reduction of hepatic availability (p a 0.003) was also noted.The authors postulated that the induction of hepaticCYP3A4 counteracted inhibition of intestinal CYP3A4,leading to little to no effect in midazolam metabolism over-all [32]. Using E. purpurea whole-plant extract (aerial androot combined) in a human trial Gurley et al. [35] found nostatistically significant differences in CYP3A4 phenotypicratios [35]. CYP 450 phenotypic ratios have been shown toprovide a practical method for predicting CYP-mediateddrug interactions [35].

3.2 Organic anion-transporting polypeptide(OATP-B)

Fuchikami et al. [36] found inhibition of OATP-B by the aer-ial parts of E. purpurea in vitro. The clinical significance ofthis finding is unclear, as few drugs are metabolized via thispathway and the findings have not been demonstrated invivo.

3.3 P-glycoprotein

No primary reports involving the constituents of any Echi-nacea species could be found.

4 Discussion

The search for and appraisal of information relating todrug–herb interactions are a challenge for researchers andeducators; accurate, readily accessible information for themedical community is lacking. Unfortunately, reports ondrug–herb interactions have only recently been publishedin the scientific literature. Perhaps due to the void in pri-mary data, it appears that the majority of authors make rec-ommendations without evaluating the quality of evidencefrom which their conclusions are drawn.

A common shortcoming of many drug–herb interactionreports is that investigators fail to report species, plant partused, preparation type and concentrations assayed, and/ordose, each of which may affect the accuracy of reporting andthe potential for drug–herb interactions. Additionally, arecent study of echinacea products found that the relation-ship between labeled milligrams and measured milligramswas poor, suggesting that clinical and research studies maybe difficult to correlate with the products taken by consum-ers [37].

5



Our results demonstrate that although drug–herb inter-actions related to echinacea products were cited in some 49articles, only 16% (8) of these 49 papers contained primarydata relevant to interactions between echinacea productsand pharmaceuticals. Two studies were clinical trials [32,35] and the remaining were in vitro assays, three of whichdid not contain complete information about the concentra-tion of extract used [29–31]; only half of the studies veri-fied the authenticity of the echinacea [31–34]. One paperwas dropped because echinacea was used in combinationwith other herbs [30].

The bulk of the available data on possible drug–echina-cea interactions concerns the CYP 450s, a large family ofhepatic phase I detoxification enzymes that also are essen-tial in a myriad of enzymatic reactions implicated in tissuesother than the liver [38]. CYP 450 genes code for enzymesthat play a role in the metabolism of drugs and foreignchemicals as well as arachidonic acid metabolism and for-mation of eicosanoids, cholesterol metabolism, bile acidsynthesis, steroid metabolism, vitamin D and vitamin Ametabolism [38]. CYP450s are crucial in understanding theclinical pharmacology of drug interactions in addition tointerindividual variability in drug metabolism.

In vitro studies suggest that E. purpurea root may mildlyreduce clearance of substrates of CYP1A2 and CYP3A4.Human trials by Gorski et al. [32] and Gurley et al. [35]demonstrate wide interindividual variability of metabolismof various echinacea preparations. Gurley et al. [35] con-cludes that there is a minimal risk of drug interaction fromechinacea preparations. These data suggest that, as is thecase with pharmaceutical drugs, while the general effect ofechinacea products on consumers may be insignificant, rareindividuals within the group could possibly experience amore significant induction or inhibition of these enzymesystems or drug transporters. In general, further study ofinterindividual variability is needed.

CYP1A2, which metabolizes theophylline and other phar-maceuticals, may be particularly relevant for asthmapatients. It must be mentioned, however, that clinicians haveobserved an improvement of asthma and bronchitis withechinacea use, suggesting that echinacea may be useful toasthmatics [39]. Supporting these observations, Sharma etal. [40] found that preparations of root and aerial portions ofechinacea reversed the inflammatory response of humanbronchial epithelial cells to rhinovirus. Furthermore, partic-ular alkylamides of Echinacea spp. are cyclooxygenase [41,42] and lipoxygenase [41, 43] inhibitors, as well as antiviral[14, 44] and cannabionomimetic [45–49]. Considering thatsuch activity may offer a significant reduction of spasm andthe inflammatory processes of asthma [50–52], possiblyprotect against upper respiratory tract infections [53] andthat echinacea inhibition of CYP1A2 is insignificant to mildas found in the human trials reviewed here, recommenda-tions for individuals with asthma to halt echinacea should bereserved for exceptional circumstances.

Echinacea may mildly inhibit the CYP 450 3A4 enzymesystem; this inhibition may be tissue specific. For example,Mouly et al. [54] report that selected substrates can increasehepatic CYP3A activity, yet have no effect on intestinalCYP3A4 or, conversely, substrates may generate CYPintestinal activity without inducing hepatic effects. Gorskiet al. [32] also report tissue specific activity, finding that E.purpurea root extracts demonstrate inhibition of intestinal,and induction of hepatic, CYP3A activity. The molecularrationale behind these differential effects is still unclear.Gorski's group suggests that echinacea root preparationstaken with high oral availability/low clearance drugs (e.g.,alprazolam) may induce hepatic CYP3A4 resulting indecreased serum concentrations. Conversely, they suggestthat low oral clearance/first-pass metabolism drugs (e.g.,buspirone) taken with echinacea might result in increaseddrug serum concentrations. More data are needed to supportthis contention [32].

It has been suggested that CYP3A4 is the most importantenzyme for the metabolism of antineoplastic drugs [55].Thus the information that echinacea may modulate, evenweakly, the activity of 3A4 may encourage healthcare pro-viders to warn against the use of echinacea with patientsundergoing chemotherapy. However, such warnings couldbe disadvantageous to the patient; in vivo studies of E. pur-purea aerial parts demonstrate a reduction in chemotherapyinduced leucopenia [12, 56–59], while E. purpurea roothas demonstrated stimulation of natural killer cells [60–62]and prolongation of life in leukemic mice [63]. Addition-ally, a number of Echinacea species and constituents havedemonstrated direct antineoplastic activity in vitro [64–66]. Thus again, case by case recommendations are neededfor cancer patients rather than a generalized recommenda-tion to halt echinacea use.

Another isoform of the CYP 450 system, CYP2D6, isknown to play a primary role in the metabolism of pharma-ceuticals used to treat psychiatric disorders (attention defi-cit/hyperactivity disorders, bipolar disorder, depression,schizophrenia) as well as cardiovascular disorders (b-block-ers). E. purpurea products containing root or aerial parts inboth human [35] and in vitro models [31] has shown noactivity on CYP2D6. The current research suggests that nointeractions are expected between E. purpurea products andsubstrates of CYP2D6.

The available data also suggest that extraction condi-tions, the various species and botanical part of echinaceahave differing effects on the CYP 450 enzyme system [34].For example, Raner et al. [34] found that the hydrophobicconstituents of E. purpurea (e.g., alkylamides) are muchmore inhibitory to CYP450 than its hydrophilic constituents(e.g., phenylpropanoids) and this work is in agreement withother CYP 450 evaluations [67]. Recent work by Spelmanet al. (Spelman, K., Wetschler, M. H., Cech, N. B., submit-ted.) demonstrated quantitative and qualitative differencesin alkylamide concentrations, including the alkylamides

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Mol. Nutr. Food Res. 2008, 52, 000 –000

reported to modulate CYP activity, depending on theextraction technique and the use of fresh or dry plant mate-rial in ethanolic preparations of E. purpurea radix. Thus,ethanolic extracts as well as other echinacea preparationsmay differ in their activity on the CYP 450 system andtherefore generalizations about echinacea products anddrug–herb interactions may be misleading (Spelman, K.,Wetschler, M. H., Cech, N. B., submitted.).

Although there are two human trials in this collection ofdata, the remaining five studies attempt to extrapolate datafrom in vitro models. The fact that replicating relevant mod-els of human physiology are difficult with in vitro methodsis widely acknowledged. Researchers are globally awarethat in vitro experimental models are often poorly represen-tative of in vivo models, especially in regard to gene expres-sion, enzyme levels and metabolism [68]. One constraintincludes the absence of digestive and metabolic processingof compounds within an extract by in vitro models(although one study, Fuchikami et al. [36], attempted a“digestion” in their model). Cech et al. [69] has demon-strated that alkylamide metabolites of human liver micro-somes have different immunological activity as comparedto their nonmetabolized counterparts, yet such significantfindings are frequently missed in vitro. Another limitationconcerns the experimental concentrations of echinaceaextracts utilized for in vitro models. Two of the studiesappear to have used concentrations of extract that areimpossible to achieve by oral dosing of echinacea extracts[29, 31]. Central to the evaluation of these studies are theprevious investigations that have demonstrated that 0.4 ng/mL (l0.31 ng/mL) are the maximum serum levels reachedby the dominant alkylamide, dodecatetraenoic acid isobuty-lamide, in echinacea [70]. This particular alkylamide hasbeen previously shown, in vitro, to play a role in inhibitionof CYP 450 3A4 [67]. A more recent study has suggestedthat the lowest obtainable IC50 for CYP 3A4 of dodecate-traenoic acid isobutylamide to be 1.96 mg/mL, almost5000-fold higher than the predicted concentration withinhepatocytes [71]. Thus, within the restraint of the availabledata, it seems unlikely, even with chemical potentiation byother echinacea constituents, that echinacea will generatedrug interactions, via CYP 450, of any consequence.

A more obscure and less considered complication of invitro research is the oxidative stress inherently generated byin vitro models. The increased exposure to oxygen due to invitro conditions leads to significant reactive oxygen species(ROS) generation and may produce results that are notreflective of in vivo conditions [68]. Key to this issue is thatmany CYP 450 mechanisms are based in redox chemistry.In considering in vitro research on the CYP 450 system,increased ROS generation due to in vitro conditions mayskew the results of these assays. In the case of echinaceaextracts, increased ROS generation is particularly likelydue to the ease of oxidation of the polyphenols of echina-cea. Accordingly, there is an obligatory caution that must

be observed in extrapolating data derived from in vitro workto clinical realities, especially in regard to CYP 450 assays.

A missing yet, crucial point in much of the drug metabo-lism literature is that fruits, vegetables, grains, and legumes,with their inherent phytochemical content, modulateCYP450 enzyme expression, and activity [72]. Yet, exceptfor grapefruit, and tyramine-containing foods, the majorityof discussion on drug–herb interactions involves herbalmedicine and neglects the more frequently consumed plant-based foods. Humans are routinely exposed to foods such asthe cruciferious vegetables with their indole-3-carbinolsthat modulate phase I and phase II detoxification [38]. Theubiquitous flavonoids found in most plant-based foods arealso known to modulate a number of isoforms of CYP 450[73]. Other commonly consumed phytochemicals such ascaffeine are known to modulate CYP 450 function [73].Hence, it is crucial that pharmacokinetic studies investigat-ing the possibility of drug–herb interactions control fordiet. Fortunately, Gorski et al. [32], requested CYP 450appropriate dietary restrictions of their subjects, as did Gur-ley et al. [35]. However, in both trials dietary restrictionsdepended on voluntary compliance as there is limitedopportunity for such trials to impose dietary restrictionswithout significant burden. Nonetheless, to avoid ambigu-ity and to provide optimal evaluation of drug–herb interac-tions, it is crucial that clinical trials on drug–herb interac-tions include measures to rule out dietary effects on drugmetabolism.

Ultimately, to generate clinically relevant data on theCYP 450 enzyme system, multiple substrate concentrationsas well as multiple test inhibitor concentrations are required[27, 28, 74]. Unfortunately, this is commonly neglected inboth in vitro and in vivo research and leaves much to theunknown. In the meantime, it appears that many warningsabout the effects on Echinacea spp. on drug–herb interac-tions and the CYP 450 system have been based on inad-equate evidence leading to inaccuracies in the literature.

We suggest more objective and detailed methods forreporting drug–herb interactions. Since many consumersdo not realize that herbs qualify as supplements, investiga-tors, and clinicians must enquire about both “supplement”and “herb” use when recording a proper medical history[29]. Additionally, product verification, dosage, species,and plant part used need to be documented. Occurrences ofpotential drug–herb interactions need to be investigatedwith greater accuracy and detail to identify what is clini-cally relevant and what is not. Currently, 68–90% of dataon drug–herb interactions is based on unsubstantiated orpoor quality information [28] resulting in anecdotal report-ing.

Of the three Echinacea species typically used in herbalmedicine, the available research predominantly focused onE. purpurea, which accounts for 80% of commercial pro-duction [15]. No case reports were found documenting anechinacea–drug interaction, a finding supported by other

7



reviewers [19, 74–77]. The fact that echinacea productsrank second in sales of herbal remedies in the US [6] andthe number of people using multiple pharmaceuticals (A75million) [3], suggest that drug–herb interactions with echi-nacea products are uncommon events. Barrett [78] con-cludes that less than 100 serious adverse events have beenreported involving echinacea for an estimated A10 millioncourses of treatment, leaving the risk estimate of less than 1in 100000.

5 Conclusion

Currently there are no verifiable reports of actual drug–herb interactions with any echinacea product. Both E. pur-purea aerial parts and root appear to have a relatively lowpotential to produce CYP-mediated drug–herb interac-tions. However, further pharmacokinetic testing is requiredbefore conclusive statements can be made. Further researchis also needed to confirm the potential for herb–drug inter-actions involving other species of Echinacea. In order tofurther clarify drug–herb interactions, healthcare providersand researchers must account for product verification, dos-age, species, and part used. Additionally, data obtainedfrom one product should not be extrapolated to other prod-ucts, species, plant parts, or dosages.

Although there is not enough evidence to support theneed for precautions at this time, a prudent clinicalapproach is to monitor patients taking echinacea productsconcurrently with substrates of CYP3A4 or CYP1A2.Given the data in this review, the number of echinacea dosesconsumed yearly and that the majority of use is short termfor the treatment of upper respiratory tract infections,E. purpurea products, consisting of roots and aerial parts,do not appear to be a risk to consumers provided that thepreparations are authentic. Lastly, for proper public safetymeasures, it is imperative that drug–herb reporting is donemethodically.

K. S. expresses appreciation to Tai Sophia Institute, Univer-sity of North Carolina and Nadja Cech, Ph.D. for support inwriting this manuscript. C. F. wishes to thank AdrianeFugh-Berman, MD for her guidance and support whileworking on this project.

The authors have declared no conflict of interest.

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8


Kevin Spelman is a researchscientist currently using proteo-mics methodology at the Uni-versity of North Carolina atGreensboro to investigate bio-active compounds from medici-nal plants. Mr. Spelman was afounding faculty member of thefirst BS degree in botanicalmedicine in the US and mostrecently of the first MS degreein the clinical herbal medicine inthe US. He is also a member ofthe College of Practitioners of

Phytotherapy in the UK and a doctoral student at the Univer-sity of Exeter in the UK.

Camille Freeman received a Master of Science in HerbalMedicine from the Tai Sophia Institute in Laurel, MD. She is alicensed nutritionist and a certified Nutrition Specialistthrough the American College of Nutrition. At the time thismanuscript was written, Camille was a graduate student atGeorgetown University in the Department of Physiology andBiophysics. She maintains a private practice serving the DCarea, with a focus on female reproductive health and educa-tion.

Mol. Nutr. Food Res. 2008, 52, 000 –000

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9



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10


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Journal of Pharmaceutical and Biomedical Analysis 49 (2009) 1141–1149

Contents lists available at ScienceDirect

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journa l homepage: www.e lsev ier .com/ locate / jpba

Comparison of alkylamide yield in ethanolic extracts prepared from fresh versusdry Echinacea purpurea utilizing HPLC–ESI-MS

Kevin Spelman, Matthew H. Wetschler, Nadja B. Cech ∗

Department of Chemistry and Biochemistry, The University of North Carolina Greensboro, P.O. Box 26170, Greensboro, NC 27402, United States

a r t i c l e i n f o

Article history:Received 13 November 2008Received in revised form 30 January 2009Accepted 10 February 2009Available online 20 February 2009

Keywords:HPLC–ESI-MSElectrosprayEchinaceaAlkylamideIsobutylamide2-MethylbutylamideQuality assessmentEthanolic extractionTincture

a b s t r a c t

Echinacea purpurea (L.) Moench, a top selling botanical medicine, is currently of considerable interest dueto immunomodulatory, anti-inflammatory, antiviral and cannabinoid receptor 2 (CB2) binding activitiesof its alkylamide constituents. The purpose of these studies was to comprehensively profile the alky-lamide (alkamide) content of E. purpurea root, and to compare yields of alkylamide constituents resultingfrom various ethanolic extraction procedures commonly employed by the dietary supplements indus-try. To accomplish this goal, a high performance liquid chromatography–electrospray ionization massspectrometry (HPLC–ESI-MS) method was validated for quantitative analysis of several E. purpurea alky-lamides. Using this method, at least 15 alkylamides were identified and it was shown that fresh and dryE. purpurea extracts prepared from equivalent amounts (dry weight) of roots, with exceptions, exhibitedsimilar yield of specific alkylamides. However, the amount of total dissolved solids in the dry extractwas higher (by 38%) than the fresh extract. Two extracts prepared from dried roots at different ratiosof root:solvent (1:5, w:v and 1:11, w:v) were similar in yield of total dissolved solids, but, there weredifferences in quantities of specific alkylamides extracted using these two root:solvent ratios. In addition,the important bioactive dodecatetraenoic acid isobutylamides are fully extracted from dry E. purpurearoot in 2 days, suggesting that the manufacturing practice of macerating Echinacea extracts for weeksmay be unnecessary for optimal alkylamide extraction. Finally, the identification of a new alkylamidehas been proposed. These results demonstrate the differences of the described extractions and utility ofthe analytical methods used to determine the wide-ranging individual alkylamide content of commonlyconsumed Echinacea extracts.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

The investigations described herein employ high performanceliquid chromatography coupled to electrospray ionization massspectrometry (HPLC–ESI-MS) for the comprehensive characteriza-tion of several extracts from the medicinal plant Echinacea purpurea(L.) Moench. Echinacea is widely used for the treatment of upperrespiratory infections, and is a global top seller. Three main speciesof echinacea are used clinically and available to consumers, Echi-nacea pallida, E. purpurea and E. angustifolia. Of these, E. purpurearepresents 80% of commercial production [1]. E. purpurea prod-ucts range from the injectables prepared to rigorous Europeanpharmaceutical manufacturing standards, to the low tech ethano-

Abbreviations: CB2, cannabinoid 2 receptor; HPLC–MS, high performance liquidchromatography–mass spectrometry; ESI-MS, electrospray ionization mass spec-trometry; MW, molecular weight; Tetraenes, isomers of dodeca-2,4,8,10-tetraenoicacid isobutylamide.

∗ Corresponding author. Tel.: +1 336 334 3017; fax: +1 336 334 5402.E-mail address: nadja [email protected] (N.B. Cech).

lic extractions or “tinctures” that follow general manufacturingpractices (GMPs) of the United States dietary supplements indus-try. Although in Germany the aerial parts are preferred, ethanolicextracts of echinacea root make up a large source of sales andclinical use in the United States. Manufacturing practices gener-ally dictate whether the starting plant material should be fresh ordry, but in the case of echinacea species, both fresh and dry rootextracts are commercially available. To further complicate matters,these extracts are prepared with varying ratios of plant:solventdepending on the manufacturer. Currently, there are few investi-gations comparing the efficiency of extracting active constituentsunder these various extraction conditions. The few studies investi-gating the extraction of alkylamides have generally utilized driedroot, while alkylamide extraction of fresh roots has scarcely beenstudied [2]. There is currently a lack of information regarding differ-ences in chemical composition among extracts prepared using freshversus dried Echinacea. One of the goals of the studies conductedherein was to provide such information.

Four constituent groups are currently believed to be the sourceof activity in the echinacea genus; alkylamides (alkamides),phenylpropanoids (caffeic acid derivatives), polysaccharides, and

0731-7085/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.jpba.2009.02.011


1142 K. Spelman et al. / Journal of Pharmaceutical and Biomedical Analysis 49 (2009) 1141–1149

glycoproteins [3]. However, in extractions with ethanol concen-trations above 40%, only very low levels of polysaccharides areleft in suspension, and denaturing of proteins is expected [4,5].Thus, the major constituents of ethanolic echinacea extracts arephenylpropanoids and alkylamides. To date, human pharmacoki-netic studies of Echinacea spp. suggest that the alkylamides are themajor constituent group circulated in plasma [6].

Alkylamides have been of pharmacological interest since the tin-gling paresthesia from chewing plants rich in these compoundswere noted [7]. This anesthetic property was utilized by nativeAmericans [8] and eventually by physicians in the early 20th cen-tury for a variety of purposes including as a sialogogue, antitussiveand for toothache. Alkylamides were later recognized as insectici-dal [9] and oncolytic [10]. Recent investigations have demonstratedimmunomodulatory activity of alkylamides in vitro [11] and in vivo[12], as well as direct antiviral activity [13]. Most recently, thesecompounds have become a subject of interest due to their elucida-tion as agonists of the cannabinoid receptor 2 (CB2) receptor [14].

Given that the alkylamides appear to be one of the key con-stituent groups responsible for pharmacological activity of E.purpurea, the studies described herein focused on this class of con-stituents. A number of analytical studies, the bulk by Bauer et al.,have relied on liquid chromatography with UV detection (LC-UV) toanalyze echinacea alkylamides [15–18]. However, comprehensiveprofiling of echinacea alkylamide content using LC-UV alone hasbeen challenging. These compounds are present at widely differ-ent concentrations, and many of them are isomeric. Consequently,co-elution of structurally similar alkylamides is common, and UVdetectors may not detect minor alkylamide constituents because oflow concentrations and/or co-elution with other compounds. Massspectrometry (MS) provides a distinct advantage over UV detec-tors due to its sensitivity and the ability to select by mass the ionscorresponding to the compounds of interest [19]. As the data pre-sented here will demonstrate, this advantage makes HPLC–ESI-MSan ideal technique for the comprehensive analysis of the isomericalkylamide content in E. purpurea. Although several investigatorshave previously employed HPLC–ESI-MS to the analysis of alky-lamides in Echinacea [20–22], none of the previous methods havebeen validated for quantitative purposes. Furthermore, because ofthe abundance of isomeric alkylamides in E. purpurea, even withthe use of MS detectors, misidentification or incomplete identifi-cation of alkylamides has been common [22–24]. With this study,we present the first validated HPLC–ESI-MS method for the analy-sis of alkylamides in E. purpurea. This method enables quantitativecomparison of alkylamide content in various E. purpurea extracts.In addition, by relying on MS–MS fragmentation patterns to dis-tinguish isomeric alkylamides, we report a more comprehensiveprofile of alkylamide content in E. purpurea than has previouslybeen published.

2. Experimental

2.1. Reagents

The following chemicals and reagents were used:Acetonitrile (high performance liquid chromatography (HPLC)

grade) (Honeywell Burdick and Jackson, Muskegon, MI), acetic acid(Fisher Chemical, Fairlawn, NJ), alkylamide standards (ChromadexInc., Santa Anna, CA), ethanol (AAPER, Shelbyville, KY), nanopurewater (Nanopure Diamond D11931, Barnstead International, Ther-molyne, Dubuque, IA).

2.2. Plant material

Cultivation of E. purpurea took place in Grants Pass, OR at PacificBotanicals. Fresh, dormant roots of E. purpurea were harvested in

March 2007. Species was verified by Richard Cech (Horizon Herbs,Williams, OR) and voucher specimens were submitted to the Uni-versity of North Carolina Herbarium in Chapel Hill, NC (accessionnumbers 583416 and 583417). The roots were two years-old at timeof harvest.

2.3. Plant extractions

A typical protocol [25] for the manufacture of ethanolic extractswas followed in all extractions, except that post washing, the rootswere briefly soaked (5 min) in 70% ethanol as a disinfectant, andblown partially dry with compressed air. A loss of the isomericdodeca-2,4,8,10-tetraenoic acid isobutylamides (tetraenes) of 1.4%(against final fresh root concentrations) was calculated from theinitial rinse. The roots were then cut into small pieces (≤1 cm wide)and extracted using three different extraction techniques, fresh rootextraction (1:2, w:v) and dry root extraction at two different root tosolvent ratios, 1:11 and 1:5 (w:v). All ratios are expressed as massraw plant material (E. purpurea roots) in weight (g) per volume (mL)of extraction solvent.

To prepare fresh root extracts, samples of the cut roots (65 g)were blended using a Waring Blender (Tarrington, CT) in a solventof 95% ethanol (AAPER, Shelbyville, KY) at a ratio of 1 g roots:2 mLsolvent. Samples of root from the same batch were dried in an ovenat 50 ◦C and water content was determined to be 74.5%. Dry rootextractions were carried out with the same method as the freshexcept that the solvent consisted of 74.5% ethanol and 25.5% water(to account for the plant water removed upon drying). To makethese dry root extracts, 16.6 g of dried root was added to 179 mL ofsolvent (74.5% ethanol) for a ratio of 1:11 and 16.6 g of dried rootwas added to 83 mL of solvent (74.5% ethanol) for a ratio of 1:5. Fourreplicate extracts were prepared at each extraction ratio (fresh 1:2,dry 1:11 and dry 1:5).

Aliquots (dry root 1:11, 200 �L) for the extraction as a functionof time study were taken on a daily basis (days 2–33) during theprocess of maceration and stored at −70 ◦C until the time of analysisfor alkylamide content. After maceration for one month, the solventwas removed from all of the extracts using a hydraulic press. Theextracts were then aliquoted into 1 mL portions in polypropylenemicrocentrifuge tubes and kept in the dark at room temperatureuntil needed for analysis. Previous investigations have establishedstability of alkylamides under these conditions [26]. All extractionswere macerated at 24 ◦C.

2.4. Preparation of samples and standards

Prior to analysis, samples were removed from storage andallowed to reach room temperature. Aliquots (500 �L) from allextractions were centrifuged at 14,000 rpm (Savant Speedvac Sc110,Farmingdale, NY) for 5 min. Supernatant was then diluted in thesame solvent used for extraction (70% ethanol), and pipetted(300 �L) into autosampler vials (Agilent Technologies, Santa Clara,CA) for LC-MS analysis. Several dilutions were prepared from eachextract to adjust alkylamide content to within the linear dynamicrange of the method. Neat samples were used for determinationof dodeca-2E-ene-8,10-diynoic acid and1000-fold dilutions wereused for analysis of the isomers of dodeda-2,4,8,10-tetraenoic acidisobutylamide.

Alkylamide primary standards were purchased from Chro-madex (Santa Anna, CA) with certificates of analysis verifyingidentity by NMR and HPLC, and purity of ≥99% by HPLC.Concentrated stock solutions of dodeca-2E-ene-8,10-diynoic acidisobutylamide (molecular weight 245.37, lot # 04950-601) anddodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (molecularweight 247.38, lot # 04953-102) were prepared at 5 mg/mL inethanol and stored at 4 ◦C. The stock solutions were diluted in


K. Spelman et al. / Journal of Pharmaceutical and Biomedical Analysis 49 (2009) 1141–1149 1143

ethanol to produce final concentrations of 0.1, 10, 50, 100 and500 �M.

2.5. HPLC–ESI-MS analysis

An ion trap mass spectrometer with electrospray ionizationsource (LCQ Advantage, ThermoFisher, San Jose, CA) was employed.The solvent gradient, which was a minor variation on that previ-ously published [21], was as follows, where solvent A is aqueousacetic acid (17 mM, original pH 2.74) and solvent B is neat HPLCgrade acetonitrile. For t = 0–4 min, a constant composition of A–B(90:10, v/v); for t = 4–15 min, a linear gradient from A–B (90:10,v/v) to A–B (60:40, v/v); for t = 15–30 min, a linear gradient fromA–B (60:40, v/v) to A–B (40:60, v/v); for t = 30.1–35 min, a con-stant composition of A–B (0:100, v/v); for t = 35.1–43 min, a constantcomposition of A–B (90:10, v/v). The mass spectrometer was oper-ated in the positive ion mode with a scan range of 50.00–2000.00.Spray, capillary, and tube lens offset voltages were 4.5 kV, 3 V and−60 V, respectively.

2.6. Quantitative and qualitative analysis of alkylamides

Constituents in the extracts were identified according totheir molecular weights, HPLC retention times, and previouslyestablished MS–MS fragmentation patterns [27]. For quantitativedetermination of alkylamide content, calibration curves were plot-ted as the log of the area of the selected ion chromatogram forthe protonated alkylamide of interest versus the log of concentra-tion. Extract samples were analyzed neat and at 1000-fold dilutionwithin the same run as the calibration standards. The concen-trations of dodeca-2E-ene-8,10-diynoic acid isobutlyamide and ofthe isomers of dodeda-2,4,8,10-tetraenoic acid isobutylamide weredetermined by plugging the relevant peak area into the linearregression equation for the corresponding calibration curve. Allsamples for which quantitative comparisons were made were ana-lyzed within a single run.

2.7. Method validation

Method validation was conducted according to InternationalCommittee of Harmonization (ICH) guideline [28]. Alkylamidestandards prepared as described in Section 2.4 were analyzed intriplicate on three separate days (for a total of 9 analyses of eachstandard). To assess accuracy, a “measured concentration” for eachstandard was back-calculated from the corresponding calibrationcurve. The measured concentration reported was an average ofthe measured concentrations calculated on three separated days.This measured concentration was then compared to the theoret-ical concentration of each standard, and the % relative differencewas reported as the “residual”. Repeatability was determined asthe relative standard deviation among the back-calculated concen-trations for the triplicate analyses of each standard within a singlerun. Intermediate precision was calculated as the relative standarddeviation among the back-calculated concentrations for three runsconducted on three separate days. The limit of detection (a mea-sure of the sensitivity of the method) was determined based on theconcentration required to give a signal to noise ratio (S:N) of 3:1 inthe relevant selected ion chromatogram. Limit of quantitation wasbased on the signal necessary to achieve a S:N of 10:1.

2.8. Statistical analysis

The standard error of the mean (SEM) was determined foreach set of concentrations or peak areas. Data are expressed asthe mean ± SEM and comparison of means was conducted usinga two tailed t-test for paired data when differences were observed.

The mean values were considered significantly different if p < 0.05.Where appropriate, outlying data points were rejected on the basisof the Q-test. Statistical analyses were performed with MicrosoftExcel (2003).

2.9. Determination of yield of dissolved solids

Polypropylene microcentrifuge tubes (1.5 mL) were weighedbefore addition of 500 �L aliquots of centrifuged extracts. Afterdehydration in the speedvac for 39 h at 24 ◦C, the mass of dissolvedsolids for each sample was determined. The ratio of the mass ofdissolved solids to the amount of dry root used in the equivalentvolume of extract was then calculated, providing a measure of thequantity of dissolved solids extracted per mass of Echinacea root(extract yield). For the fresh extract, the dry weight of the root usedfor the extract was calculated by subtracting the mass of the watercontained in the roots from the total mass of the fresh roots.

3. Results and discussion

In this section, a comprehensive profile of alkylamide con-stituents in E. purpurea is listed, with a description of how thesecompounds can be identified using HPLC–ESI-MS. In addition,quantities of dissolved solids and specific alkylamides present invarious E. purpurea extracts are compared. The extracts analyzedhere were prepared using several different procedures commonlyemployed in the dietary supplement industry. Analysis of theseextracts provides insight into the similarities and differences inextract composition that result from these variations in extractiontechnique.

3.1. Identification of alkylamides

Table 1 lists alkylamides identified from E. purpurea, with refer-ences that refer to publications in which these identifications weremade. This is the most comprehensive listing of alkylamides of E.purpurea root to date. Most reports and reviews list some, but notall, of the alkylamides present in this species [16,24,29,30]. Past esti-mates suggest the presence of eleven alkylamides in the roots of E.purpurea [31,32]. Table 1, lists a total of 17 compounds, although,as described later on, some of these identifications are only tenta-tive and one compound (C) was not detected in our samples of E.purpurea.

Many of these compounds were present in the E. purpureaextracts investigated here, as demonstrated in Fig. 1. This figureshows a base peak chromatogram (Fig. 1A) obtained from analy-sis of the 1:5 dry root E. purpurea extract. Peak labels correspondto alkylamide designations in Table 1, and were confirmed basedon comparison of retention time with previous investigations [21]and MS–MS spectra (Table 2, Fig. 2). A few of the minor alky-lamides in the extract were obscured by co-elution with majoralkylamides, but can be visualized through the use of selected ionchromatograms (Fig. 1B–D).

The isomeric compounds D, E, F and G all demonstrate MH+ ionsat m/z 244 and compounds P and Q both have MH+ ions with m/z258. Nonetheless, these alkylamides are distinguishable based onan MS–MS spectra. Table 2 illustrates the primary fragments thatresult from collisionally induced dissociation of several isomericalkylamides. Structurally similar fragments have been groupedwith designations of i, ii, iii, iv and v for ease of reference.

One of the major groups of fragments formed by collision-ally induced dissociation is the acyllium ion (fragment group iin Table 2), as previously reported by Hiserodt et al. [27]. Theseions form due to a charge-remote hemolytic cleavage that yieldsa resonant distonic radical cation, which subsequently undergoeshydrogen rearrangement. Alkylamides F and G (Fig. 2D and E) show



Table 1Alkylamides from Echinacea purpurea.

Designation Alkylamide MW Reference

A Undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide 229.32 [32]B Undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide 229.32 [42]C Undeca-2E-ene-8,10-diynoic acid isobutylamide 231.34 [35]D Undeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide 243.35 [32]E Undeca-2Z,4E-diene-8,10-diynoic acid 2-methylbutylamidea 243.35F Dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide 243.35 [42]G Dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide 243.35 [32]H Dodeca-2E,4E,10E-triene-8-ynoic acid isobutylamide 245.37 [32]J Dodeca-2E-ene-8,10-diynoic acid isobutylamideb 245.37 [35]K Dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide 247.38 [32]L Dodeca-2E,4E, 8Z,10Z-tetraenoic acid isobutylamideb 247.38 [32]M Dodeca-2E,4E, 8E,10Z-tetraenoic acid isobutylamide 247.38 [38]N Dodeca-2E,4E,8Z-trienoic acid isobutylamide 249.40 [32]O Dodeca-2E,4E-dienoic acid isobutylamide 251 41 [31]P Trideca-2E,7Z-diene-8,10-diynoic acid isobutylamide 257.38 [31]Q Dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide 257.38 [40]R Dodeca-2,4,8,10-tetraenoic acid 2-methylbutylamide 261.41 [36]

a Proposed structure of newly identified alkylamide.b Compounds J and L were utilized as standards.

Fig. 1. Characteristic chromatograms obtained by liquid chromatography–massspectrometry analysis of an E. purpurea root extract (1:5). Panel A shows a base peakchromatogram (plot of the most abundant ion in the mass spectrum versus time),with peak labels that correspond to the designations in Table 1. Several minor alky-lamides that coelute with the compounds shown in the base peak chromatogramcan be distinguished with selected ion chromatograms, as shown in panels B (massrange 245.5–246.5), C (mass range 249.5–250.5) and D (mass range 257.5–258.5).

an acyllium ion at m/z 171, while alkylamides D and E (Fig. 2A andB) result in the acyllium ion at m/z 157.

Two additional fragments useful for elucidation of alkylamidestructure are the group ii and group iii fragments (Table 2). Thegroup ii fragments result from the loss of the amide portion alky-lamide, and correspond to the remaining alkyl chain. The group iiifragments are observed at an m/z value 2 amu above the group iifragments. Recent work utilizing deuterated alkylamides suggeststhat in the diene alkylamides, group iii fragments are formed whenan unsaturated bond is lost and the remaining double bond shiftsto the 3 position (in 2,4-dienes), with a subsequent gain of twohydrogens [27].

In combination, the group ii and iii ions can be used to determine(1) whether the alkylamide is a diene; (2) how many carbons arepresent in the alkyl chain; and (3) the identity of the amide moiety(isobutyl versus 2-methylbutyl). Isobutylamides will have two frag-ments corresponding to a loss of 101 (group ii) and 99 (group iii)from the MH+ precursor ion (Fig. 2D–F). For 2-methylbutylamides,the fragments will reflect the additional carbon in the amide moiety,and fragment ions corresponding to a loss of 115 (group ii) and 113(group iii) from the MH+ precursor ion will be observed (Fig. 2A–C).

The group iv fragments (Table 2) correspond to the MH+ ion ofthe protonated alkylamide that remains after loss of the N-alkylgroup. Loss of the N-isobutyl group results in a significant fragment(relative intensity 26–60%) at m/z 188 for compounds F and G and185 for compound Q, while loss of the N-(2-methylbutyl) group

Table 2Fragments formed by collisionally induced dissociation (MS–MS) of the MH+ ion of various E. purpurea alkylamides.

Designation, name, m/z Group id,RIc 30–90%

Group iie,RI 30–90%

Group iiif ,RI 60–100%

Group ivg,RI 26–100%

Group vh,RI < 3–20%

D 244a Undeca-2E,4Z-diene-8,10-diynoic acid 2-metylbutyl amide 157 129 131 174 188, 202, 216E 244 Undeca-2Z,4E-diene-8,10-diynoic acid 2-methylbutylamideb 157 129 131 174 188, 202, 216F 244 Dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide 171 143 145 188 202, 216G 244 Dodeca-2E,4Z-diene-8,10-diynoic acid isobutylamide 171 143 145 188 202, 216P 258 Trideca-2Z,7E-diene-8,10-diynoic acid isobutylamide 185 157 159 202 216,230Q 258 Dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide 171 143 145 188 202, 216

a The number beneath the letter designation indicates the m/z value for the MH+ ion.b Proposed structure for compound E based on retention time and MS/MS fragmentation.c RI corresponds to relative intensity.d The group i fragments correspond to acyllium ions as shown in Fig. 2.e The group ii fragments are carbocations that correspond to the alkyl chain of the alkylamide and are formed by loss of the amide portion (isobutylamide or 2-

methylbutylamide).f The group iii fragments correspond to the alkyl chain of the alkylamide and are formed by the loss of the amide portion of the molecule and saturation of one of the

double bonds on the alkyl chain.g The group iv fragments correspond to the protonated alkylamide minus the N-alkyl group.h The group v fragments correspond to the protonated alkylamide minus various portions of the N-alkyl group (see Fig. 2).



Fig. 2. MS/MS spectra of isomeric alkylamides. This figure compares fragments that result from collisionally induced dissociation precursor ions that represent the protonatedmolecular ion (MH+) of a number of isomeric alkylamides. Even ions with the same MH+ ion have different fragmentation patterns, facilitating structural assignment.

results in a fragment with m/z 174 for compounds D and E (relativeintensity 56–100%) and a fragment with m/z 202 for compoundP. The mass that is lost to form the group iv fragment serves asan additional confirmation to distinguish isobutylamides from 2-methylbutylamides.

The final fragments that result from collisionally induced disso-ciation of alkylamides are group v in Table 2. They are formed bycleavage of various C C bonds on the N-alkyl substituent (Fig. 2).The group v fragments are useful for verifying whether the N-alkylsubstituent is a 2-methylbutylamide or isobutylamide.



Table 3Calibration parameters, limits of quantification (LOQ) and limits of detection (LOD) for E. purpurea alkylamides.

Slope (±SDa) Intercept (±SDa) R2 Linearity (�M) LOD (�M) LOQ (�M)

Dodeca-2E-ene-8,10-diynoic acid isobutylamide (J) 0.757 (±0.013) 8.00 (±0.023) 0.999 1.0–500 0.051 1.7Dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (L) 0.813 (±0.040) 6.95 (±0.070) 0.993 1.0–100 0.99 3.3

a SD: Standard deviation.

An example of the utility of MS–MS for structural elucidationof alkylamides can be demonstrated for the specific case of alky-lamide P, trideca-2E,7Z-diene-8,10-diynoic acid isobutylamide. TheMS–MS spectrum of the precursor ion at m/z 258 for this compoundis shown in Fig. 2F. An acyllium ion (group i) is present at m/z 185.This suggests a thirteen carbon alkyl chain. This chain length is fur-ther confirmed by the group ii fragment at m/z 157. The presenceof the group iii fragment (m/z 159) indicates that the compoundis indeed a diene alkylamide, and, the characteristic loss of 99 toform this fragment (258–159 = 99) suggests that the compound is anisobutylamide rather than a 2-methylbutylamide. The high inten-sity group iv fragment at m/z 202 further confirms that the N-alkylgroup is an isobutylamide. The low intensity group v fragmentsalso confirm an N-isobutyl group rather than an N-2-methylbutylgroup; additional group v fragments would be observed for a 2-methylbutylamide. Thus, the identity of the compound is proposedto be trideca-2E,7Z-diene-8,10-diynoic acid isobutylamide, whichhas previously been shown to occur in E. pallida, but rarely isreported as occurring in E. purpurea [33]. The E/Z assignmentsfor trideca-2E,7Z-diene-8,10-diynoic acid isobutylamide have beenmade based on comparison of the relative retention times observedin this study with those reported previously [33], and are only ten-tative without NMR confirmation.

With HPLC–ESI-MS and MS–MS data such as those shownin Figs. 1 and 2, all of the previously identified alkylamidesfrom E. purpurea in Table 1 except undeca-2E-ene-8,10-diynoicacid isobutylamide (compound C) were identified in the extractsprepared in this study. Although past work cites the presenceof undeca-2E-ene-8,10-diynoic acid isobutylamide in E. purpurearoot [34,35], this compound was not detected in the echinaceaextracts used for these studies. A commercially available standardof this compound was readily detectable with limit of detectionof 0.15 �M, therefore, it can be concluded that undeca-2E-ene-8,10-diynoic acid isobutylamide was not present in the extractsat concentrations above 0.15 �M. Binns et al. [36], using solely UVspectra and retention time, previously identified undeca-2E-ene-8,10-diynoic acid isobutylamide compound in E. purpurea rootsof wild plants but not cultivated germlings. Hence, it is possiblethat some genetic strains of E. purpurea contain undeca-2E-ene-8,10-diynoic acid isobutylamide while others do not. However,our laboratory has investigated over 20 different US sources of E.purpurea (data not included) and thus far not detected this com-pound. Another possibility is that previous reports of the presenceof undeca-2E-ene-8,10-diynoic acid isobutylamide in E. purpureawere due to improperly identified plant material. Another Echinaceaspecies, E. angustifolia, does produce significant levels of undeca-2E-ene-8,10-diynoic acid isobutylamide, and the misidentificationof echinacea species has often been documented [15,37,38].

In addition to aiding in structural elucidation of known alky-lamides, with HPLC–ESI-MS it was possible to tentatively identifya new alkylamide, the structure of which has not been previouslypublished. This compound is undeca-2Z,4E-diene-8,10-diynoic acid2-methylbutylamide (compound E in Table 1). Identification ofthis compound was based on retention time and the correlationbetween MS–MS fragmentation pattern and alkylamide structure.The mass and fragmentation pattern for compound E (Fig. 2) con-firms that it is a 2-methylbutylamide, and indicates the level ofsaturation and length of the carbon chain. The mass spectral data

do not indicate stereochemistry or bond position; however, relativeretention time does suggest that this compound is the 2Z/4E isomerof compound D. For the previously identified alkylamide isomersthat vary by the 2E/4Z and 2Z/4E stereochemistry, such as com-pounds A/B and F/G, we have demonstrated (Fig. 1) that the 2E/4Zisomer elutes before the 2Z/4E isomer. Thus, it is logical to assumea similar relationship in stereochemistry between compounds Dand E. However, as noted earlier, without NMR confirmation, thereported stereochemistry of this new alkylamide is only tentative.

Bauer and Remiger previously demonstrated that with reversedphase HPLC, alkylamides with terminal alkynes elute early in theseparation followed by tetraene alkylamides [33]. For the purposesof this discussion, compounds A–G, J, P and Q are designated aspolyacetylene amides, while the term “tetraenes” refers to isobuty-lamides with four double bonds in the alkyl chain. Consistent withthe Bauer study, our results (Fig. 1) indicate that the polyacetyleneamides (A, B, C, D/E, F/G, H, J, P, Q) elute early in the separation,between 25 and 31 min, followed by the tetraenes and dienes (K, L,M, N and O). These findings are significant in that alkynes, specifi-cally 8,10 terminal alkynes, have been shown to modulate CYP 450function [34], while tetraene isomer L and alkylamides N and Oare ligands of the CB2 receptor [14]. The results in Fig. 1 suggestpreparatory scale HPLC could be used to separate groups of alky-lamides with differing physiological and pharmacological activity.

3.2. Calibration results and method validation

Table 3 illustrates the linear regression equations and statisticaldata for the alkylamide standards. The linear range of the calibrationcurves was from 1 to 500 �M for dodeca-2E-ene-8,10-diynoic acidisobutylamide (J), and from 1 to 100 �M for dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (L). Correlation coefficients (R2) ofthe alkylamide standards were 0.999 (J) and 0.993 (L). The limit ofdetection (concentration required to give a signal to noise ratio,S:N, of 3:1) for isobutylamides dodeca-2E-ene-8,10-diynoic acidand dodeca-2,4,8,10-tetraenoic acid were 0.051 and 0.99, respec-tively. Limits of quantitation (based on S:N of 10:1) were 1.7 and3.3, respectively.

Table 4 shows the results of method validation for the quantita-tive analysis of alkylamide content, which was accomplished usingInternational Committee of Harmonization (ICH) guidelines [28], asdescribed in Section 2.7. The values for the residuals, repeatability,and intermediate precision conform to the parameters for methodvalidation according to the ICH.

3.3. Comparison of alkylamide yield in various E. purpureaextracts

Three extracts were chosen for comparison of alkylamide con-tent. One of these (fresh 1:2) was prepared from fresh E. purpurearoots using 1 g roots for every 2 mL of solvent. The other two wereprepared from dry E. purpurea roots, one using 1 g dried roots per11 mL solvent (1:11) and the other1 g dried roots per 5 mL sol-vent (1:5). All of the three extracts contain the same percentageof ethanol (69%). The extracts differ only in the nature of startingmaterial (fresh or dry root) and the ratio of root:solvent. Once themass of the fresh roots is adjusted to account for water content, thefresh 1:2 and dry 1:11 extract have equivalent ratios of dry weight



Table 4Validation parameters for quantitative analysis of E. purpurea alkylamides.

Dodeca-2E-ene-8,10-diynoic acid isobutylamide (J)

Theoretical concentration (�M)a Measured concentration (�M)b Residues (%)c Repeatability (%)d Intermediate precision (%)e

1.0 1.1 14 3 210 8.8 −12 2 250 44 −11 1 3

100 99.6 −0.3 1 4500 568 13 0.5 0.7

Dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (L)f

1.0 0.99 −.5 3 610 9.7 −3 3 250 49 −3 0.5 4

100 105 5 3 0.6

a Theoretical concentration based on the mass of standard per volume of solution.b Measured concentration for a given standard is the average of the back-calculated concentration (from the calibration curve) for three analyses conducted on three

separate days.c The residuals (Res) are calculated by first determining the difference between the measured concentration (CM) and the theoretical concentration (CT) and then dividing

this value by the measured concentration: Res = (CM − CT)/CM × 100.d Repeatability corresponds to the relative standard deviation of the back-calculated concentration for triplicate analyses of the same standard on the same day.e Intermediate precision corresponds to the relative standard deviation of the back-calculated concentration for triplicate analyses of the same standard on three different

days.f Validation results are reported for dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide up to 100 �M, the upper limit of the linear range.

Table 5Quantities of the isomeric alkylamides K, L and M and alkylamide J in ethanolic extracts of E. purpurea root.

Type of extract Alkylamides K, L and M Alkylamide J

Fresh 1:2 Dry 1:11 Dry 1:5 Fresh 1:2 Dry 1:11 Dry 1:5

Mean concentration (mg/mL)a 1.4 1.6 7.2 0.0047 .0072 0.013Mean concentration (mM)a 5.7 6.4 29 0.019 0.029 0.054SEb 0.5 0.2 3 0.001 0.0004 0.002

a The mean concentration was calculated for four replicate extractions (n = 4).b SE represents the standard error of the concentrations in mM.

plant material:mL solvent. Therefore, by comparing the compositionof the fresh 1:2 and dry 1:11 extracts, it should be possible to determinehow extract composition differs depending on whether fresh or driedroots are used for extraction. The two extracts prepared from driedE. purpurea differ only in the ratio of g root:mL solvent (1:11 versus1:5), therefore, by comparing the composition of the dry 1:11 and dry1:5 extracts, it should be possible to determine whether changing theroot:solvent ratio has an effect on extract composition.

Utilizing available alkylamide standards, the quantities of alky-lamides in the fresh and dry E. purpurea root extracts weredetermined. Table 5 displays these results in terms of concen-trations of the isomeric tetraenes (compounds K, L and M) anddodeca-2E-ene-8,10-diynoic acid isobutylamide (compound J) permillilitre of solvent. The three different Echinacea extracts, fresh 1:2,dry 1:11 and dry 1:5, all contained these alkylamides. However, asshown in Table 5, the dry 1:5 extract contained the greatest amountof these compounds. This is to be expected given the lower ratio ofg roots:mL solvent used in the preparation of the 1:5 extract.

In order to easily compare how efficiently alkylamides wereextracted in the three different E. purpurea extracts, the quantityof each alkylamide (mg) was divided by the dry weight of E. pur-purea root (g) used to prepare an equivalent volume of extract. Theresulting value is referred to as “alkylamide yield” (Fig. 3). The onlydifference in the fresh 1:2 versus the dry 1:11 extracts is whetherfresh or dry root was used in their preparation. Therefore, assum-ing no loss of alkylamide during the drying process, alkylamideyield would be expected to be very similar for these two extracts.Indeed, the alkylamide yield for the fresh 1:2 versus the 1:11 werecomparable for the tetraenes K, L, M (Fig. 3A), 15.3 ± 2.5 versus17.1 ± 1.3 mg/g, respectively. For alkylamide J, the yield was lower inthe 1:2 extract as compared to the 1:11 extract (0.0506 ± 00.0053versus 0.0775 ± 0.0024 mg/g, respectively). This difference could be

Fig. 3. Comparison of alkylamide yield in fresh 1:2, dry 1:11 and dry 1:5 E. purpurearoot ethanolic extracts. All concentrations represent the mean from four replicateextractions at room temperature. Error bars denote standard error of the mean(SEM). Comparisons were made between fresh 1:2, dry 1:11 and 1:5 extracts, with (*)indicating p < 0.005; (**) indicating p < 0.001. Alkylamide yield is similar in the fresh1:2 and dry 1:11 extracts, indicating no loss of alkylamides in the drying process.Better yield of alkylamide J was obtained for the fresh 1:2 versus dry 1:11 extract.



due to differences in particle size between the extracts, which mayhave given rise to more efficient extraction in the dry as comparedto the fresh extract. Importantly, comparison of alkylamide yield forthe 1:2 and 1:11 extracts does not indicate any significant degrada-tion of alkylamides due to drying. The extracts in this study wereprepared from roots immediately after completion of oven drying.Kabganian et al. [39] came to the same conclusion finding no degra-dation of alkylamides in roots that were oven dried. Whether or notthere is a loss in alkylamide content in E. purpurea roots stored forlong periods of time would be a worthy subject of a future investi-gation.

In comparing the extraction yield of the alkylamides betweenthe 1:11 versus 1:5 dry root extracts, a logical prediction, provideda 1:5 extract is not saturated, would be that the alkylamide yieldwould be the same in the two extracts. As can be seen in Fig. 3,however, this is not the case. While alkylamide yield is similar inthe two extracts, there are statistically significant differences. Foralkylamides K, L, M (Fig. 3A), extraction is more efficient in the 1:5as compared to the 1:11 extract. This is the opposite of what wouldbe expected if the solution were saturated in the case of the 1:5extract; therefore, saturation is not the cause of the differences.Conversely, for alkylamide J, extraction is more efficient in the 1:11extract than the 1:5 extract (Fig. 3B). Previous studies have estab-lished that solvent interactions of N-alkylamides differ dependingon molecular structure and the surrounding phytochemical matrix[40]. Therefore, it is plausible that extraction of certain alkylamidesis favored in more dilute extracts, while concentrated extracts favorthe extraction of structurally different species.

3.4. Comparison of yield of dissolved solids in various E. purpureaextracts

The amount of total dissolved solids in the three extractsdescribed in Section 3.3 was compared. Total dissolved solids are ameasure of how much material overall (alkylamides as well as othercompounds) was dissolved in the original extract. When the massof dissolved solids is divided by the dry weight of the plant mate-rial used to produce an equivalent volume of extract, the resultingvalue is referred to as “extract yield,” which is a measure of howmuch of the initial starting material was converted into extract.

Fig. 4 displays the extract yield (mg dissolved solids/g dry root)for the three E. purpurea extracts under investigation. Overall, theextract yield was similar for all three extracts. However, the yieldfor the fresh root extract (196.6 ± 2.7 mg/g) was slightly lower than

Fig. 4. Yield of dissolved solids in ethanolic extracts of Echinacea purpurea roots.Mass of dissolved solids was determined by evaporation of the ethanol/water solventfrom aliquots of extracts, and this value was ratioed to the quantity of root (dryweight) used to prepare an equivalent volume of extract to calculate dissolved solidsyield. Yield of dissolved solids in the 1:11 extraction does not statistically differfrom 1:5 extraction. The fresh root extraction (1:2) differs from the 1:11 and the1:5 extraction by 26.8% and 32.4% respectively. (*) indicates p < 0.005;(**) indicatesp < 0.001.

Fig. 5. Relative concentration of dodecatetraenoic acid isobutylamides (alkylamidesK, L and M) in an E. purpurea root extraction over time. Relative concentrations (CR)were calculated by dividing the concentration of each sample (CS) by the concen-tration at day 2 (Cday2) and converting to percent: CR = CS/Cday2 × 100. Samples weretaken daily over 28 days from dry root (1:11) ethanolic maceration of E. purpurea.Results show that maximal extraction of dodecatetraenoic acid isobutylamide isachieved by day 2.

the two dry root extracts (269 ± 12 and 290 ± 24 mg/g for the 1:11and 1:5 extracts, respectively, p < 0.001). As mentioned previously,a similar effect was observed for alkylamide J. This difference couldpossibly be attributed to differences in particle size in fresh ver-sus dry extractions. Between the two dried extracts, there was nostatistically significant difference in extract yield. This similaritybetween 1:11 and 1:5 extracts indicates that it is possible, by dou-bling the quantity of root used for the extraction, to double theamount of material dissolved in the solvent, at least up to a ratioof 1:5. It should be pointed out that because of the greater amountof root used to prepare the 1:5 extract, this extract does, overall,contain a greater concentration of dissolved solids then the 1:11extract. However, there is no significant difference between the twoextracts when the amount of dissolved solids is expressed relativeto the mass of root used in the extract.

3.5. Extraction of the tetraene isomers as a function ofmaceration time

Lastly, the quantity of the isomeric dodeca-2,4,8,10-tetraenoicacid isobutylamides present in a macerating E. purpurea extractagainst time was measured (Fig. 5). The results are all displayedrelative to that achieved on the first day when concentrationwas measured (day 2). These tetraene isobutylamides are sig-nificant because they typically compose from 30 to 70% of thetotal alkylamides in echinacea products [41]. The results in Fig. 5demonstrate that the extraction of dodeca-2,4,8,10-tetraenoic acidisobutylamides is complete by day 2. Thus, in terms of the extractionof specifically these compounds, maceration beyond day 2 shouldnot be necessary. This finding is particularly interesting given thatmany dietary supplements manufacturers currently suggest thatthe appropriate time for a maceration is 2 weeks or longer, depend-ing on the part of the plant used [25]. While commonly used longmaceration times seem to have little effect on alkylamide con-tent, long maceration times could actually be detrimental if somecompounds (for example caffeic acid derivatives) degrade duringmaceration. Future investigations of the optimal maceration timefor producing an extract with maximal concentrations of all desir-able constituents are warranted.

4. Conclusion

With these investigations, it has been demonstrated thatHPLC–ESI-MS is an excellent technique for comprehensive analysisof the alkylamide content of E. purpurea extracts. Using HPLC–ESI-MS, a more comprehensive alkylamide profile was obtained thanis typically possible with other analytical approaches. By relying



on collisionally induced dissociation, it was possible to distinguishbetween isomeric alkylamides, and to tentatively identify a newE. purpurea alkylamide, undeca-2Z,4E-diene-8,10-diynoic acid 2-methylbutylamide. In addition, the validated method facilitatedquantitative comparison of alkylamide content among extractsprepared with different extraction procedures. All three extrac-tion techniques investigated here (fresh 1:2, dry 1:5 and dry 1:11)resulted in very similar alkylamide profile, and gave similar yieldsof alkylamides and of total dissolved solids. The similarity in alky-lamide content in fresh 1:2 and dry 1:11 extracts indicates thatdrying of root material at 50 ◦C does not result in a loss of alky-lamides. It appears that either fresh or dried roots can be used toprepare extracts with high alkylamide content, although the overallyield was slightly lower for fresh extracts. Lastly, the analysis of a1:2 fresh root ethanolic extract suggest that the maximum concen-tration of the tetraenes (dodecatetraenoic acid isobutylamides) isachieved by day 2 in an ethanolic extraction.

Although alkylamide yields were, overall, similar with thethree extraction techniques, there were some statistically signif-icant differences in quantities of alkylamides extracted. Notablythe isomeric tetraenes were extracted more efficiently with aroot to solvent ratio of 1:5 (w:v) as compared to a ratio of1:11, while dodeca-2E-ene-8,10-diynoic acid isobutylamide wasextracted more efficiently with a ratio of 1:11. Given that the bio-logical activity of alkylamides differs depending on structure, thesesuggest that pharmacological activity of E. purpurea extracts coulddiffer depending on the ratio of root:solvent used in extraction.Ultimately, in vitro and in vivo studies are needed to elucidate thedifferences in pharmacokinetic and pharmacodynamic activity ofextracts of various Echinacea spp. The results presented in this paperdo, however, suggest that it would be erroneous to assume that allethanolic extracts of E. purpurea result in equivalent phytochemicalprofiles.

Acknowledgements

These studies were made possible by financial support ofResearch Corporation (Cottrell College Science Award # CC6595),the National Institutes of Health National Center for Complemen-tary and Alternative Medicine (R15 AT001466-01), the NationalScience Foundation (MRI grant # 0420292) and the UNC ResearchCompetitiveness Fund. We thank James Snow of Tai Sophia Insti-tute for advice on technical aspects of medicinal plant extractions,Jason Reddick for technical advice, Richard Cech of Horizon Herbsfor providing the voucher specimen for this study, and Tai SophiaInstitute for educational support of KS.

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Rapid Report

Role for PPARγ in IL-2 inhibition in T cells by Echinacea-derivedundeca-2E-ene-8,10-diynoic acid isobutylamide

Kevin Spelman a, Katrina Iiams-Hauser b, Nadja B. Cech a, Ethan Will Taylor c,Nicholas Smirnoff d, Cynthia A. Wenner b,⁎a University of North Carolina Greensboro, Department of Chemistry and Biochemistry, P.O. Box 26170, Greensboro, NC 27405, United Statesb Bastyr University, Department of Basic Sciences, School of Natural Health Sciences, Kenmore, WA 98028, United Statesc University of North Carolina Greensboro, Laboratory of Molecular Medicine, Office of Research, University of North Carolina at Greensboro, Greensboro, NC 27402-6170, United Statesd School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK

a b s t r a c ta r t i c l e i n f o

Article history:Received 20 May 2009Received in revised form 16 August 2009Accepted 17 August 2009

Keywords:EchinaceaAlkylamidesCytokinesUndeca-2E-ene-8,10-diynoicacid isobutylamidePPARIL-2

Certain fatty acid amides from Echinacea spp. have demonstrated moderate to high cannabinoid activity. As aresult, CB2 activation is currently hypothesized to be the basis of activity for immunomodulation by Echi-nacea spp. PPARγ, an orphan nuclear receptor and lipid sensor, is known to inhibit IL-2 production and beactivated by fatty acid derivatives such as the endocannabinoids. In these investigations, we demonstratethat undeca-2E-ene-8,10-diynoic acid, an Echinacea angustifolia-derived alkylamide lacking affinity for theCB2 receptor, inhibits IL-2 secretion in Jurkat T cells through PPARγ activity at low micromolarconcentrations (330 ng/mL). The IL-2 inhibition is reversed by the addition of the selective PPARγantagonist T0070907. Additionally, we show that that undeca-2-ene-8,10-diynoic acid stimulates 3T3-L1differentiation, a process dependent on PPARγ activity. These experiments demonstrate that PPARγ isinvolved in T cell IL-2 inhibition by undeca-2-ene-8,10-diynoic acid and suggest that cytokine modulation bythe alkylamides is due to polyvalent activity.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Peroxisome proliferator activated receptor gamma (PPARγ) is anorphan nuclear receptor that regulates transcription of target genes inresponse to the binding of small lipophilic ligands [1]. It was originallydescribed as a compulsory target in differentiating adipocytes [2].Later it was identified in other tissues [3] and a role inmacrophage [4],dendritic cell [5] and T cell [6] activity was observed. Intriguingly,recent results suggest that endogenous cannabinoids such as ananda-mide and 2-arachidonoylglycerol are agonists of PPARγ [7,8].

Recent work has demonstrated that several of the unsaturatedalkylamides of Echinacea spp. exhibit significant affinity for thecannabinoid-2-receptor (CB2) [9] and that in the presence of thesealkylamides, LPS-induced TNF secretion is down-regulated, a functionof alkylamides targeting CB2 in macrophages [10]. Raduner et al., [11]confirmed the high CB2 affinity of the 2,4-diene unsaturatedisobutylamides (olefinic alkylamides). Moreover, this group also

reported that the selective CB2 antagonist SR144528 is incapable offully inhibiting increases in total cellular concentrations of Ca2+ inHL60 cells induced by 2-arachidonoylglycerol (2-AG) and alkylamides,suggesting the possibility of the involvement of a second receptor.Echinacea spp.-derived alkylamides have been shown to inhibit IL-2secretion by humanT cells [12], but the basis of alkylamide-induced IL-2 inhibition has not yet been reported.

PPARγ activation has recently been found to be responsible for IL-2inhibition induced by the endocannabinoids [7,8]. In addition, Chris-tensen et al. [13] recently demonstrated that various fatty acids andalkylamides from the flowers of E. purpurea activated PPARγ at highconcentrations (40–100 μM). Considering the structural similaritybetween 2-AG and alkylamides, we investigated the possibility ofinduction of PPARγ activity by the alkylamide undeca-2E-ene-8,10-diynoic acid isobutylamide (hitherto referred to as undecaenediynoicacid isobutylamide) found in E. angustifolia and Spilanthes acmella(Fig. 1) [14]. This 2-ene alkylamide, containing a diacetylinic tail, haspreviously been found to have negligible affinity for CB2 [11]. Utilizingthe previously established models of IL-2 inhibition and 3T3-L1adipogenesis, we investigated the possibility that PPARγ is a target ofundecaenediynoic acid isobutylamide, and that interaction of thisalkylamide with PPARγ contributes to alkylamide-induced IL-2 inhibi-tion and adipocyte differentiation.

International Immunopharmacology 9 (2009) 1260–1264

⁎ Corresponding author. Tel.:+1 425 602 3163.E-mail address: [email protected] (C.A. Wenner).

1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.intimp.2009.08.009

Contents lists available at ScienceDirect

International Immunopharmacology

j ourna l homepage: www.e lsev ie r.com/ locate / in t imp


2. Materials and methods

2.1. Reagents

The following materials, chemicals and reagents were used. Allreagents were from Sigma-Aldrich (St. Louis, MO) except the follow-ing: undeca-2E-ene-8,10-diynoic acid isobutylamide (MW 231.34;certificate of analysis verified identity by NMR and HPLC, and purityof ≥99% by HPLC lot #21235-501; (Chromadex Inc., Santa Anna,CA); troglitazone (MW 441.5; Rezulin, abbreviated as TZD, gift fromRon Morrison, UNCG, Department of Nutrition); T0070907 (MW277.7; Cayman Chemicals, Ann Arbor, MI); ethanol (AAPER, Shelby-ville, KY); nanopure water (Nanopure Diamond D11931, BarnsteadInternational, Thermolyne, Dubuque, IA); calf serum (ColoradoSerum Co., Denver, CO); human IL-2 Duo Set ELISA Kit-DY202 (R&DSystems, Minneapolis, MN); Jurkat E6.1 cells (ATCC, Manassas, VA);3T3-L1 preadipocytes (a gift from Ron Morrison); nitro-cellulosemembranes (Bio-Rad, Hercules, CA); anti-human PPAR γ primaryantibody (Aviva Systems Biology, San Diego, CA); and goat anti-rabbitconjugated to horse radish peroxidase (US Biological, Swampscott,MA).

2.2. Immunodectection of PPARγ protein in Jurkat cells by Westernblotting

Cells were harvested (2×106 cells) and lysed by boiling for 5min.Nuclear extracts were analyzed in a denaturing 10% polyacrylamidegel, electrotransferred to a supported nitro-cellulose membrane, andimmunoblotted with the PPAR γ primary antibody (Aviva SystemsARP32880_T100). The membranes were soaked in blocking buffer [5%nonfat dry milk diluted in Tris-buffered saline— 0.1% Tween-20 (TBS-T)] for 1 h at room temperature with the indicated primary antibody(1:10,000). After washing, membranes were developed with horse-radish peroxidase-conjugated secondary antibodies and visualizedwith a chemiluminescent detection system (GE Healthcare/Amer-sham Biosciences, Buckinghamshire, England). A double band is theexpected image resulting from this antibody.

2.3. Fibroblast cell culture and differentiation

3T3-L1 cells were cultured in DMEM plus 10% calf serum, 4 mML-glutamine and 1 mM Na pyruvate. Cells were plated in 6 well platesin a total of 1.5 mL of medium and grown to confluence over 4–5 days. 48h after confluence was achieved (day 0), insulin (10 μg/mL)was added to all wells except the insulin free control. On day 2, mediawas changed to contain FBS and the following treatment conditions,which were added to separate triplicate wells: undecaenediynoic acidisobutylamide (5.0 μg/mL, 7.5 μg/mL and 10 μg/mL); TZD positivecontrol (10 μM); insulin negative control (10 μg/mL); and vehiclecontrol (EtOH:DMSO 0.4%:0.1%) were added. Every 2 days, mediumand all reagents, including treatments, were replenished after a PBSwash. Experiment was halted on day 5, at which time adipogenesiswas determined by photomicroscopy by morphology and the pre-sence of the obvious prominent lipid vacuoles. Images were takenwith a SPOT digital camera mounted on an Olympus BX60 fluore-scence microscope.

2.4. Jurkat cell culture and IL-2 ELISA

Human E6.1 Jurkat T cells were cultured in RPMI 1640with 10% FBS,2 mM L-glutamine and 1 mM Na pyruvate. After serum starvation for7h, Jurkat cells were plated in 96 well culture plates at 1.25×105cells/mL in RPMI 1640 (without phenol red)with 10% FBS. Cells were treatedwith PMA (1.25 ng/mL) and PHA (0.25 μg/mL) and the selectiveantagonist T0070907 or DMSO vehicle was added to appropriate wellsand incubated at room temperature for 15min after which alkylamidesand TZD were added. Plates were then incubated for 18 h. Cellsupernatants were collected (100 µL) and assayed for IL-2 by ELISA(Human IL-2 Duo Set ELISA Kit). All test conditions were assayed intriplicate and verified with repeated experiments three times.

2.5. Cell survival by XTT assay

The cytotoxicity of undecaenediynoic acid isobutylamide wasmeasured by the XTT colorimetric assay [15], which was performedon the same plated cells cultured for the IL-2 cytokine testing in dualELISA/XTT assays. For the cell viability standard curve, unstimulatedcells were plated in triplicate at 2.5×104, 2×104, 1.5×104, 7.5×103,5.0×103, and 2.5×103cells/well. After removal of 100 µL supernatantfrom all wells, 100 μL of 1 mg/mL of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) plus 0.02 mMphenazine methosulfate (PMS), in medium was added. After 5 hincubation, ODs at 450 nm (Teacan Sunrise plate reader, Grödig,Austria) were measured and cell concentrations extrapolated fromknown cell concentrations in standard curve. Test conditions wereassayed in triplicate and repeated in two experiments.

2.6. Statistical analysis

All data are expressed as means±SE of experiments conducted intriplicate. Statistical analysis was performed using Student's t-test andanalysis of variance (one-way ANOVA). The accepted level of sig-nificance was p<0.05.

3. Results

3.1. Presence of PPARγ receptor in Jurkat cells

Jurkat cells have previously been demonstrated to express thePPARγ nuclear hormone receptor [16]. The presence of the PPARγprotein in the Jurkat E6.1 strain of cells used for these experimentswasconfirmed by Western blot (see supplemental information). Nuclearextracts of untreated Jurkats were incubated with polycolonal PPARγantibodies from 1 μg/mL to 4 μg/mL following standard Western blotprocedures. Analysis of Jurkat cell nuclear extracts confirmed theexpression of PPARγ receptor in Jurkat E6.1 cells. A signal of 56 kDa isseen at 1 μg/mL that grows in intensity with increasing PPARγantibody concentration. These results demonstrate that PPARγproteinis expressed in the E6.1 strain of Jurkat cells used in this study.

3.2. The PPARγ selective antagonist T0070907 attenuates the IL-2inhibition induced by undecaenediynoic acid isobutylamide atsuboptimal Jurkat stimulation

To assess the response of T cells to undecaenediynoic acidisobutylamide (Fig. 1), suboptimal IL-2 stimulation of Jurkats by PMA/PHA (PMA 1.25 ng/mL & PHA 0.25 μg/mL) was used. Previous work hasused this model system to demonstrate that PPARγ mediates IL-2inhibition in T cells [7,8,16]. Cells were serum starved for 7h, platedand then exposed to vehicle control (EtOH/DMSO), positive control(TZD-a selective PPARγ agonist), positive control with antagonist (TZD/T0070907), undecaenediynoic acid isobutylamide or undecaenediynoicacid isobutylamide with antagonist (UDA/T0070907) at increasing

Fig. 1. Undeca-2E-ene-8,10-diynoic acid isobutylamide (undecaenediynoic acidisobutylamide).

1261K. Spelman et al. / International Immunopharmacology 9 (2009) 1260–1264


concentrations. T0070907 was held at constant concentration (2.5 μM).After 18 h incubation, ELISA of the supernatants collected from wellstreatedwith the various conditionswas performed. Results demonstratethat undecaenediynoic acid isobutylamide dose-dependently inhibits IL-2 production beginning at 0.33 μg/mL (1.4 μM) (Fig. 2). When theselective PPARγ antagonist T0070907 is added with undecaenediynoicacid isobutylamide, a dose-dependent attenuation of the IL-2 inhibitionis seen starting at 0.33 μg/mL. At this concentration, the IL-2 level returnsto baseline in thewells treatedwith T0070907.However, at higher levelsof undecaenediynoic acid isobutylamide treatment, the antagonisticeffect of T0070907 on IL-2 secretion is lost, as would be expected inincreasing concentrations of a competitive agonist and a fixed con-centration of antagonist.

3.3. Cell survival of Jurkats after treatment with undecaenediynoic acidisobutylamide and undecaenediynoic acid isobutylamide/T0070907does not account for IL-2 modulation

Wenext determined if the IL-2 changes seen in Jurkats treated in theexperimental conditions were due to the effects of undecaenediynoicacid isobutylamide and undecaenediynoic acid isobutylamide/T0070907 and not due to significant changes in cell number underthese treatment conditions. Cells from the same treatment conditions inthe same experiment in which supernatants were harvested for IL-2ELISA were assayed for cell viability by the XTT assay. A statisticallysignificant (p<0.05) proliferative effect of undecaenediynoic acidisobutylamide on the Jurkat E6.1 cells was observed (Fig. 3). Thus, theIL-2 inhibition induced by undecaenediynoic acid isobutylamide is notdue to cell death, as IL-2 inhibition occurs despite the increase in cellnumber. In addition, these data demonstrate that the increase in IL-2concentration observed in wells of Jurkat cells treated with the PPARγselective antagonist T0070907 are not due to an increase in cell number. 3.4. Treatment of 3T3-L1 preadipocytes with undecaenediynoic acid

isobutylamide indicates PPARγ involvement

The 3T3-L1 cell line, which is PPARγ dependent for differentiationinto adipocytes, has been widely established as a model to probe forPPARγ activity [17,18]. To verify PPARγ involvement in the inhibitionof IL-2 by undecadiynoic acid in T cells, 3T3-L1 differentiation in thepresence of controls or undecaenediynoic acid isobutylamide wasanalyzed to confirm that this alkylamide is able to induce PPARγ-dependent preadipocyte differentiation.

3T3-L1 cells were grown to confluence and then exposed toexperimental conditions including the TZD positive control (10 μM),insulin negative control (10 μg/mL), vehicle control, or treatmentwithundecaenediynoic acid isobutylamide (+insulin 10 μg/mL) at in-creasing concentrations (5.0 μg/mL, 7.5 μg/mL and 10 μg/mL).

Differentiation was assessed morphologically by the relativelyround shape of the adipocytes and by the presence of the obviousprominent lipid vacuoles seen bymicroscopy. The vehicle control (VC)and the insulin control exhibit no differentiation, while undecaene-diynoic acid isobutylamide induced a dose dependent adipogenesis in3T3-L1 cells that was morphologically indistinguishable from thatinduced by the selective PPARγ agonist positive control (TZD) (Fig. 4).

4. Discussion

PPARγ plays a role in a variety of diseases including diabetes,atherosclerosis, inflammation, cancer and autoimmune disorders [19–23]. Thus, novel PPARγ ligands may offer further therapeutic optionsfor a wide array of diseases. PPARγ ligands include fatty acidderivatives, as well as the selective PPARγ agonists known as thethiazolidinediones (TZDs). Both classes of compounds have beenshown to reduce IL-2 levels in T cells [6,16,24–26]. This effect has beenshown via reporter assays to be due to the PPARγ nuclear receptor [8].Moreover, endocannabinoids, which share common structure with

Fig. 2. PPAR-γ antagonist T0070907 attenuated the undecaenediynoic acid isobutyla-mide (UDA) induced inhibition of IL-2 secretion by suboptimally stimulated Jurkat E6.1cells. Cells were subjected to suboptimal mitogenic stimulation (PMA 1.25 ng/mL, PHA0.25 μg/mL) after serum starvation, for induction of IL-2 in all cases except in the notreatment group (No Tx). The selective agonist troglitazone (TZD) represents a positivecontrol (black bar). Stripped bars represents the combination of 2.5 μM of T0070907with the matched treatments (vehicle-VC, undecaenediynoic acid isobutylamide, TZD)shown in the left bar of each pair. Results show a dose dependent response of IL-2 byundecaenediynoic acid isobutylamide and the positive control TZD. The T0070907treatments dose-dependently attenuate the IL-2 inhibition (up to 1 μg/mL undecaene-diynoic acid isobutylamide) that occurs upon treatment of Jurkat cells with increasingdoses of undecaenediynoic acid isobutylamide (grey bars). The positive control (TZDblack bar), also shows significant inhibition of IL-2 which is blocked by T0070907.Values are mean+S.E. of experiments performed in triplicate. One-way ANOVA wasused to determine statistical significance between the vehicle (VC) versus the varioustreatments (undecaenediynoic acid isobutylamide or TZD) treated groups (*p<0.05)and the undecaenediynoic acid isobutylamide versus the undecaenediynoic acidisobutylamide/T0070907 treated groups (†p<0.05).

Fig. 3. Cell survival assay (XTT assay) of Jurkat E6.1 cells treated with undecaenediynoicacid isobutylamide (UDA) and T0070907. Cells were subjected to identical conditionsas the IL-2 assays. The selective agonist troglitazone (TZD) represents a positivecontrol (black bar). Stripped bars represent the combination of 2.5 μM of T0070907with the matched treatments (vehicle-VC; undecaenediynoic acid isobutylamide; orTZD) shown in the left bar of each pair. A proliferative effect by undecaenediynoic acidisobutylamide (grey bars) on Jurkat cells is observed. A trend showing inhibition ofproliferative effects is observed with the addition of the selective PPARγ antagonistT0070907. The positive control, selective PPARγ agonist TZD combined withT0070907, shows a statistically significant inhibition of proliferation as compared toTZD alone (black bar). One-way ANOVA was used to determine statistical significancebetween the vehicle (VC) versus the undecaenediynoic acid isobutylamide — or TZD-treated groups (*p<0.05), and the undecaenediynoic acid isobutylamide versus theundecaenediynoic acid isobutylamide/T0070907 treated groups (†p<0.05).

1262 K. Spelman et al. / International Immunopharmacology 9 (2009) 1260–1264


some of the unsaturated alkylamides, have been shown to reduce IL-2levels via PPARγ [7,8].

While cannabinoid 2 receptor (CB2) activity has been shown forthe 2,4-diene olefinic alkylamides, the 2-ene alkylamide undecaene-diynoic acid isobutylamide (Fig. 1) has shown relatively no affinity forCB2 [11]. With this in mind, we probed for PPARγ activity withundecaenediynoic acid isobutylamide. The effect of undecaenediynoicacid isobutylamide treatment on PPARγ activation was assessed usingtwo well established PPARγ mediated biological responses, IL-2production and PPARγ-dependent adipogenesis in 3T3-L1 cells.

As shown in Fig. 2, the selective PPARγ antagonist T0070907 at2.5 μM is effective in attenuating the IL-2 inhibitory effects ofundecaenediynoic acid isobutylamide at alkylamide concentrationsbetween 330 ng/mL and 1 μg/mL. This is in range of relevantphysiological alkylamide concentrations based on in vivo humanpharmacokinetic data [27]. T0070907 appears to lose its ability toattenuate the IL-2 inhibition at higher concentrations of undecaenediy-noic acid isobutylamide. This apparent loss of antagonist activity is anexpected observation of competitive agonism. The fixed dose ofT0070907 (2.5 μM) appears to be overwhelmed by increasing concen-trations of undecaenediynoic acid isobutylamide ranging two orders ofmagnitude. It is also possible that at high undecaenediynoic acidisobutylamide concentrations, IL-2 inhibition occurs independently ofPPARγ activity. Alternatively, recent data suggests that the PPARγreceptor,whichpresents a particularly large binding cavity as comparedto other nuclear receptors,may bindmore than one ligand at a time[26].For example, rosiglitazone, a selective PPARγ agonist structurally similarto the agonist used in these studies, occupies only about 40% of theligand-binding site in the ternary complex of PPARγ, leaving adequate

room for other ligands [28]. Crystal structures of PPARγ demonstratethat PPARγ can bind two 9-(S)-hydroxyoctadecadienoic acidmoleculesconcurrently [29]. Thus a more involved explanation of the loss of theantagonism by T0070907 at higher undecaenediynoic acid isobutyla-mide concentrations may be that PPARγ is binding both T0070907 andundecaenediynoic acid isobutylamide at the same time and this mayresult in an overall IL-2 inhibitory effect. It is also possible these resultscould be explained by undecaenediynoic acid isobutylamide acting as apartial agonist, asmost recently suggested for the alkylamide hexadeca-2E,9Z,12Z,14E-tetraenoic acid isobutylamide in a fibroblast in vitromodel [13].

Undecaenediynoic acid isobutylamide, is considered a 2-enealkylamide, which commonly contain diacetylinic tails, and are morecharacteristic of the alkylamides found in E. angustifolia. This isopposed to the 2,4-diene alkylamides, more commonly found in E.purpurea, which are more commonly olefinic alkylamides. However,both Echinacea species contain olefinic and acetylinic classes ofalkylamides [30]. The olefinic 2,4-dienes have demonstratedmoderateto high CB2 affinity, while the diacetylinic 2-enes have thus far, withfew exceptions, demonstrated negligible CB2 affinity [11]. ConsideringChristensen's et al. [13] recent work demonstrating activation ofPPARγ in fibroblasts with olefinic alkylamides at high concentrations(40–100 μM) and our results showing PPARγ activation atmuch lowerconcentrations (1.4–14 μM) with a diacetylinic alkylamide, it ispossible that other diacetylinic alkylamides which have loweraffinities for CB2 than the olefinic alkylamides, may activate PPARγ.

Accordingly, it may be that E. angustifolia and E. purpurea differ in thedegree of PPARγ and CB2 mediated immunomodulation. Previousstudies report that the alkylamide investigated here, undecaenediynoic

Fig. 4. Dose-dependent response of 3T3-L1 cell differentiation by undeca-2E-ene-8,10-diynoic acid (UDA). Increasing concentrations of UDA enhance 3T3-L1 differentiation, asobserved by microscopy. 3T3-L1 cells were plated in 6 well plates with 1.5 mL of medium and grown to confluence over 5 days. After confluence was achieved, UDA, insulin positivecontrol (10 μg/mL), TZD (10 μM), or vehicle control (EtOH/DMSO) were added. Medium and treatment conditions were replenished every 2 days after PBS wash. Photomicrographswere taken using an Olympus inverted microscope at day 5. Results shown are representative of triplicate experiments.

1263K. Spelman et al. / International Immunopharmacology 9 (2009) 1260–1264


acid isobutylamide, comprises 5% of the alkylamide content of E.angustifolia root [31]. Furthermore, up to 55%ofE. angustifolia alkylamidespossess a diacetylenic tail like that of undecaenediynoic acid isobutyla-mide [32]. This is in contrast to E. purpurea root, which does not containundecaenediynoic acid isobutylamide in detectable concentrations [30],and contains less than 45% diacetylenic alkylamides overall [31]. Ofinterest, previous reports by the eclectic physicians of the early-mid 19thto mid 20th century, who brought Echinacea into clinical practice,suggested that the two species differed in effect [33]. Further research isneeded to evaluate howE. angustifolia andE. purpureavary in their effects,possibly due to differential actions on pathways such as PPARγ and CB2.

Our results should be interpreted carefully. There are otherproteins known to directly influence adipocyte differentiation otherthan PPARγ. This includes CCAAT/enhancer binding proteins (C/EBP),and the basic helix-loop-helix-leucine zipper transcription factorsterol regulator element-binding-protein-1c [29]. Furthermore, acti-vation of C/EBP is known to engage the PPARγ pathways in both 3T3-L1 cells [34] and T cells [35]. Of particular relevance, a recent geneexpression study using microarrays suggested C/EBPβ is an upstreamactivation node for many of the pathways activated by Echinacea [29].It remains to be determined whether C/EBPβ plays a role in the PPARγdependent alkylamide interactions reported here.

In summary, these experiments illustrate the involvement ofPPARγ in the inhibition of IL-2 secretion by T cells in response toundeca-2-ene-8,10-diynoic acid isobutylamide. We demonstrate adecrease in IL-2 levels in this model system starting at 330 ng/mL ofundeca-2E-ene-8,10-diynoic acid isobutylamide, which is reversed bythe addition of a PPARγ selective antagonist. However, the possibilitythat other targets are involved in this effect is not ruled out by thesedata. Thus, it is possible that there is a combination of effects due toPPARγ and other targets that inhibit IL-2 production and induceadipocyte differentiation. These results, coupled with previous resultsdemonstrating cannabinoid activity of the alkylamides, suggest thatthe immunomodulatory potential of the alkylamides is likely due topolyvalent actions. Further investigations are needed to elucidate therole of PPARγ and other potential alkylamide targets in the IL-2inhibitory response of T cells to undecaenediynoic acid isobutylamide.

Acknowledgements

These studies were supported by Research Corporation (CottrellCollege Science Award # CC6595), the National Science Foundation(MRI grant # 0420292), the UNC Research Competitiveness Fund anda faculty seed grant from Bastyr University. Special thanks to RonMorrison and Yashomati Patel at UNCG for technical advice on 3T3-L1differentiation and Julia Hartenstein, Chester Frazier and IrvingWainer at National Institute on Aging, National Institutes of Healthfor assistance on Western blots.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.intimp.2009.08.009.

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