Echinacea as an antiinflammatory agent: the influence of physiologically relevant parameters.

Abstract

Numerous Echinacea preparations are available on the market for the prevention and treatment of cold and 'flu symptoms and inflammatory conditions associated with infections. Most of these preparations are consumed orally in the form of aqueous or ethanol extracts and tinctures. Since the recommended consumption normally involves a brief local exposure to the diluted preparation at an unspecified time in relation to the actual infection, then it is important that experimental models for the evaluation of Echinacea reflect these limitations. A line of human bronchial epithelial cells, in which rhinoviruses stimulate the production of pro-inflammatory cytokines, was used to evaluate several relevant parameters. The chemically characterized Echinacea preparation (Echinaforce) was capable of inhibiting completely the rhinovirus induced secretion of IL-6 (interleukin-6) and IL-8 (chemokine CXCL-8) in these cells, regardless of whether the Echinacea was added before or after virus infection, and in response to a range of virus doses. This inhibitory effect was also manifest under conditions resembling normal consumption with respect to the duration of exposure to Echinacea and the Echinacea dilution. It is concluded that under real life conditions of Echinacea consumption, the virus-induced stimulation of pro-inflammatory cytokines can be effectively reversed or alleviated.

Full text

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ECHINACEA AS AN INFLAMMATORY AGENT1
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. (2008)
Received 20 June 2008
DOI: 10.1002/ptr
Revised 27 August 2008
Copyright © 2008 John Wiley & Sons, Ltd.
PHYTOTHERAPY RESEARCH
Phytother. Res. (2008)
Published online in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/ptr.2714
Echinacea as an Antiinflammatory Agent: The
Influence of Physiologically Relevant Parameters
M. Sharma1, R. Schoop2 and J. B. Hudson1*
1Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada
2A. Vogel Bioforce AG, Roggwil, Switzerland
Numerous Echinacea preparations are available on the market for the prevention and treatment of cold and
’flu symptoms and inflammatory conditions associated with infections. Most of these preparations are con-
sumed orally in the form of aqueous or ethanol extracts and tinctures. Since the recommended consumption
normally involves a brief local exposure to the diluted preparation at an unspecified time in relation to the
actual infection, then it is important that experimental models for the evaluation of Echinacea reflect these
limitations. A line of human bronchial epithelial cells, in which rhinoviruses stimulate the production of pro-
inflammatory cytokines, was used to evaluate several relevant parameters. The chemically characterized
Echinacea preparation (Echinaforce®) was capable of inhibiting completely the rhinovirus induced secretion
of IL-6 (interleukin-6) and IL-8 (chemokine CXCL-8) in these cells, regardless of whether the Echinacea
was added before or after virus infection, and in response to a range of virus doses. This inhibitory effect was
also manifest under conditions resembling normal consumption with respect to the duration of exposure to
Echinacea and the Echinacea dilution. It is concluded that under real life conditions of Echinacea consump-
tion, the virus-induced stimulation of pro-inflammatory cytokines can be effectively reversed or alleviated.
Copyright © 2008 John Wiley & Sons, Ltd.
Keywords: Echinacea; antiinflammatory; cytokines; chemokines; IL-6 (interleukin 6); IL-8 (CXCL8).
Accepted 9 September 2008
* Correspondence to: Dr J. B. Hudson, Department of Pathology and
Laboratory Medicine, University of British Columbia, C-360 Heather
Pavilion, 2733 Heather Street, Vancouver V5Z 1M5, Canada.
E-mail: jbhudson@interchange.ubc.ca
INTRODUCTION
Different species and parts of Echinacea (Asteraceae)
plants have been used traditionally in North America
for the treatment of various symptoms of ‘colds’ and
‘flu’, as well as other applications (Barnes et al., 2005).
A number of well known marker compounds have been
characterized, including polysaccharides, specific caffeic
acid derivatives and alkylamides (Bauer, 1998; Binns
et al., 2002), and these have all demonstrated biological
activities in various tests in vitro and in vivo (Bauer,
1998; Vimalanathan et al., 2005; Sharma et al., 2006;
Woelkart and Bauer, 2007; Altamirano-Dimas et al.,
2007). The variety of these bio-activities suggests that
the interactions between Echinacea and cells are com-
plex, and may involve different levels of action and
different compounds.
Many clinical trials have been conducted in indivi-
duals suffering from natural or experimentally induced
rhinovirus infections, but with variable results (Barnes
et al., 2005; Schoop et al., 2006; Woelkart and Bauer,
2007; Shah et al., 2007). This variability may be due to
differences in the therapeutic products and experi-
mental protocols. Thus the question of clinical efficacy
will remain unresolved until some of the variables have
been considered systematically. There has also been
considerable discussion about the timing of Echinacea
consumption, in relation to cold or ’flu symptoms, what
kind of formulation should be consumed, and in what
dosage. It should be possible to resolve most of these
issues by judicious use of appropriate model systems.
Rhinoviruses have been implicated as major players
in common colds and various types of allergic rhinitis
and bronchial syndromes (Message and Johnston, 2004;
Schaller et al., 2006). However, numerous studies have
shown that rhinovirus infection in cultured epithelial
cells, and in nasal epithelial tissues in vivo, results in
relatively low levels of virus replication and cytopathology,
apparently due to the small number of cells supporting
virus replication (Gwaltney, 2002; Mosser et al., 2005),
yet in spite of this there is substantial induction of
secretion of certain pro-inflammatory cytokines and
chemokines, particularly IL-6 and IL-8 (Message and
Johnston, 2004, Sharma et al., 2006; Schaller et al., 2006;
Edwards et al., 2007). Thus the typical symptoms of a
common cold, such as sneezing, coughing, runny nose,
stuffed nasal passages, etc. (Gwaltney, 2002), are not
the direct result of viral pathology, but rather the
indirect stimulation of pro-inflammatory cytokines and
chemokines, which are secreted and attract the various
inflammatory leukocytes to the site of infection. Conse-
quently, the successful treatment of colds and ’flu might
be obtained by appropriate use of an antiinflammatory
material. This can be evaluated in an experimental
cell culture system, provided it bears resemblance to
the in vivo situation.
To carry out such an analysis, the rhinovirus infected
BEAS-2B epithelial cell system was used, together
with a standardized and chemically characterized pre-
paration of Echinacea purpurea, to evaluate the role of
various experimental parameters relevant to a natural
infection involving symptoms of the ‘common cold’.

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Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. (2008)
DOI: 10.1002/ptr
2 M. SHARMA ET AL.
MATERIALS AND METHODS
Echinacea source. The test material was Echinaforce®
(A. Vogel Bioforce AG, Switzerland), a 65% ethanol
extract of freshly harvested aerial parts of Echinacea
purpurea supplemented with 5% E. purpurea roots. This
preparation was essentially free of polysaccharides, and
contained the following caffeic acids and alkylamides
(caffeic acid 0, caftaric acid 264.4 μg/mL, chlorogenic
acid 40.2 μg/mL, cichoric acid 313.8 μg/mL, cynarin 0,
echinacoside 6.9 μg/mL, PID 8/9 36.3 μg/mL). The
composition was determined (courtesy J. T. Arnason,
University of Ottawa) by HPLC as described in Binns
et al. (2002).
Cells and viruses. BEAS-2B human epithelial cells, origi-
nally obtained from ATCC, were grown in Dulbecco
MEM (DMEM) in 10% fetal bovine serum. For the
experiments, the cells were sub-cultured in 6-well trays,
and when confluent the medium was changed to
DMEM, HBSS (Hanks balanced salt solution), or PBS
(phosphate buffered saline), without serum. Under these
conditions the cultures remained viable for at least 5 days.
The H-1 sub clone of HeLa cells (ATCC) and the human
lung epithelial cell line A-549 (ATCC), were grown in
DMEM + 5% fetal bovine serum. Human skin fibroblasts
(courtesy Dr Aziz Ghahary) in their sixth passage were
also cultivated in DMEM with 10% serum. No antibiotics
or antimycotic agents were used.
Rhinovirus types 1A and 14 (RV 1A and RV 14, from
ATCC) were propagated and assayed, by plaque assay,
in H-1 cells. The stock viruses had titers of between
2 × 107 and 1 × 108 pfu/mL.
Test system. Details of the test system were described
previously (Sharma et al., 2006). BEAS-2B cells, and
other cells, were grown in complete medium, in 6-well
trays, to produce confluent monolayers. The medium
was then replaced with PBS (phosphate buffered
saline) or other serum-free media for the experiments.
Virus was added to the cells at a multiplicity of infec-
tion 1.0 infectious virus per cell (1 pfu/cell), unless noted
otherwise, for 1 h at 35 °C, followed by a 1:100 dilution
of Echinaforce in PBS. Culture supernatants were
harvested at the indicated times for measurement of
cytokines, by ELISA tests.
ELISA assays were carried out according to the in-
structions supplied by the companies (either Immunotools,
Germany, or e-Bioscience, USA).
RESULTS
Kinetics of pro-inflammatory cytokine secretion in
different media
In order to establish a relevant, consistent and reliable
culture system in which to evaluate antiinflammatory
activity, the study compared the kinetics of secretion of
several cytokines in uninfected and RV- infected BEAS-
2B cells in different media (MEM, the normal culture
medium; HBSS, Hank’s balanced salt solution; PBS,
phosphate buffered saline) over a period of several days.
During this time the level of IL-6 and IL-8 secretion
Figure 1. Effect of Echinacea on RV-induced cytokine secretion.
BEAS-2B cells were grown to confluent monolayers, and half
the cultures were infected with RV 1A (1 pfu/cell) for 1 h. The
media on both infected and uninfected cultures were then re-
placed with phosphate buffered saline with or without Echinacea
at 1:100 dilution. After 48 h, culture supernatants were removed
for assay of IL-6 and IL-8. C, control (uninfected) cultures with-
out Echinacea; CE, control cultures plus Echinacea treatment;
RV, infected cultures without Echinacea; RV + E, infected cultures
plus Echinacea treatment.
changed relatively little in uninfected cells, particularly
in PBS or HBSS. Virus infection, however, resulted
in substantial cytokine induction, first observed at 4 h
after infection, but in greater amounts at 24–96 h, for
both IL-6 and IL-8 (Figs 1 and 2). After this time the
levels of cytokines declined.
Figure 1 shows the comparison between the relative
amounts of IL-6 and IL-8 at 48 h after infection. Although
the maximum amount of induced cytokine was usually
somewhat higher in the presence of complete medium,
the differential between control and virus infected cells
was consistently larger in PBS and HBSS (data not
shown). Therefore it was decided to use PBS for most
of the subsequent experiments. In addition as simple a
medium as possible was used to avoid possible interac-
tions between medium and Echinacea components.
Figure 1 also shows the dramatic inhibitory effect
of Echinacea on induced IL-6 and -8, which were often
reduced to control levels. Similar results were obtained
for any of the time points chosen, 24–96 h (not shown).
However, there was no significant effect of Echinacea
on the levels of these cytokines in control uninfected
cells (Fig. 1).
These results were unaffected by the passage number
of the BEAS-2B cells; passage 8 and passage 54 cells
showed similar responses to RV infection and to Echinacea
inhibition (not shown). In addition rhinovirus type 1A
(RV 1A), which uses a different cellular receptor from
RV 14 (LDL instead of ICAM-1), showed results simi-
lar to RV 14.
Furthermore similar results were obtained when the
BEAS-2B cells were replaced by A549 human lung
epithelial cells, or by human skin fibroblast cells (data
not shown).
Time of addition of the Echinacea
Since the exact time of a natural rhinovirus infection can
never be certain, or the interval between infection and

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ECHINACEA AS AN INFLAMMATORY AGENT3
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. (2008)
DOI: 10.1002/ptr
Figure 2. Time course of RV-induced cytokines ± Echinacea. BEAS-2B cells were grown to confluent monolayers, and half the cultures
were infected with RV 14 (1 pfu/cell) for 1 h. The media on both infected and uninfected cultures were then replaced with phosphate
buffered saline with or without Echinacea at 1:100 dilution. Periodically, culture supernatants were removed for assay of IL-6 and IL-
8. For simplicity only the values for RV-infected cultures are shown. RV14, infected cultures without Echinacea; RV14 + E, infected
cultures plus Echinacea at 1:100 dilution.
One would reasonably expect greater amounts of virus
to produce more intense symptoms.
To test this increasing concentrations of virus (RV14)
were used, from 0.01 to 1.0 pfu/cell, which resulted
in successively greater amounts of cytokine induction,
for both IL-6 and IL-8 (Fig. 4), although even at a
multiplicity of infection of 1.0 infectious virus per 100
cells there was still a substantial induction in cytokine
secretion after 48 h. Nevertheless, in all cases Echinacea
was able to reverse these responses and bring the
cytokine levels down to the control levels previously
indicated in Fig. 2. These data are shown for A-549 cells:
but similar results were obtained for BEAS-2B cells.
appearance of symptoms, then it is necessary to know
if the Echinacea can work at any time during this period.
In the standard treatment (Fig. 1), Echinacea was added
immediately after virus infection, and incubated for
an additional 24–48 h. However, when the addition
of Echinacea was delayed for 4, 24 or 48 h after virus
infection, and the exposure to Echinacea continued
for a further 24 h, the virus-induced cytokine induction
was still inhibited (Fig. 2) for both cytokines. In addi-
tion Echinacea can also be added 24 h before infection,
and still inhibit subsequent RV-induced stimulation of
cytokines (see below).
Echinacea dose effect
A common question about Echinacea consumption is:
how much is appropriate for its success in counteract-
ing cold symptoms?
To determine if the inhibitory capacity of Echinacea
was dose dependent, as might be expected, the anticytokine
effect of different doses was examined, using the same
experimental conditions described above. The previous
tests (Fig. 1) utilized a 1:100 dilution of Echinacea,
equivalent to a final concentration of 160 μg/mL. How-
ever inhibition was still observed with dilutions up to
1:400, and occasionally at 1:800, but there was clearly a
dose response effect, as shown in Fig. 3. This suggests
that the preparation could be diluted considerably and
should still be active. None of the Echinacea doses used
in these studies appeared to have adverse effects on
the cells.
Effect of virus concentration (multiplicity
of infection, MOI)
Another variable that could affect the outcome of a
cold is the amount of virus acquired in the infection.
Figure 3. Echinacea dose effect. BEAS-2B monolayers were
produced as usual, and half the cultures were infected with RV
14, 1 pfu/cell, followed by exposure of infected and uninfected
cultures to various dilutions of Echinacea for 48 h, in PBS.
Culture supernatants were then removed for the assay of IL-6
and IL-8. Data are shown only for the infected cultures; all the
control values were very low (<100 pg/mL). RV, virus only with
no Echinacea; RE 20-RE 400, infected cultures plus indicated
dilution of Echinacea.

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Copyright © 2008 John Wiley & Sons, Ltd.Phytother. Res. (2008)
DOI: 10.1002/ptr
4 M. SHARMA ET AL.
Figure 4. Effect of virus concentration. A549 cells were grown
to confluent monolayers, and half the cultures were infected
with RV 14, at various multiplicities of infection, 10−2, 10−1 or
1.0 pfu/cell. After 48 h, supernatants were removed from all
cultures for assay of IL-6 and IL-8. Data are shown only for
infected cultures. Control values were all low (<200 pg/mL).
Figure 5. Duration of Echinacea exposure. BEAS-2B monolayers
were produced as usual, and half the cultures were infected
with RV 1A, 1 pfu/cell, followed by exposure of infected and
uninfected cultures to Echinacea at 1:100 dilution for various
times, followed by washing to remove excess Echinacea. All
cultures were then incubated for the balance of the 48 h period,
at which point supernatants were removed and assayed for
IL-6 and IL-8. Data are shown only for the infected cultures; all
the control values were very low (<200 pg/mL). RV 1A, virus
only, no Echinacea; E5, E30, E1H, E48H, indicate times of expo-
sure of infected cultures to Echinacea at 1:100 dilution.
Duration of exposure to Echinacea
In order to mimic the natural consumption of Echinacea,
experiments were carried out in which Echinacea (at
the usual 1:100 dilution) was added to the RV-infected
cells for various times, and then the Echinacea washed
off the cells. Cytokine secretion was then measured 24 h
later. A short exposure of only 5 min did not signifi-
cantly affect the RV-induction of cytokines; however,
with increasing exposure to Echinacea, the more effec-
tive was the cytokine inhibition (Fig. 5). This suggests
that under normal conditions of oral Echinacea con-
sumption, inhibition of RV-induced cytokine secretion
could be substantial, even with a high virus input.
Although the results presented in Fig. 5 indicate that
a brief 5 min exposure to 1:100 Echinacea was not very
effective, nevertheless the results in Table 1 show that
a 5 min exposure to 1:10 Echinacea (a more realistic
dose in practice) gave a significant reduction in IL-6.
DISCUSSION
The present results show that Echinaforce, at a concen-
tration resembling its natural consumption, is capable
of completely reversing the rhinovirus induced secre-
tion of the pro-inflammatory cytokines IL-6 and IL-8.
These cytokines are prominent in numerous types
of infection and inflammatory conditions (Message
and Johnston, 2004; Schaller et al., 2006). In previous
studies (Sharma et al., 2006) it was shown that other
pro-inflammatory cytokines, which are induced to a
lesser degree in the epithelial cells, e.g. SCF, were also
inhibited by Echinacea preparations.
The degree of virus induced cytokine secretion was
affected, as might be anticipated, by the amount of
virus added to the cells, and this situation will reflect
the natural variation in virus ‘doses’ that individuals
receive during infection. It is unlikely, however, that a
person would be exposed to more than 1.0 pfu/cell, the
maximum dose used here, during normal exposure,
especially since the yields of rhinoviruses in vivo are
relatively low compared with other viruses (Gwaltney,
2002; Mosser et al., 2005). Therefore it is believed that
the efficacy of Echinaforce described in this study is
more than adequate to counteract the inflammatory
response in normal rhinovirus infections.
Considerable dilution of the Echinaforce was still
compatible with its efficacy; consequently the prescribed
dosage of commercial preparations (at least for E.
purpurea) is supported by these experiments.
A number of experiments were performed designed
to mimic the natural uses of Echinacea, in terms of
timing and duration of application. Echinaforce was
still very effective when applied before, as well as up to
48 h after, the virus infection, and the actual duration
of exposure was not a limiting factor. Thus prophylac-
tic and therapeutic usage is both supported. Therefore
the recommendations for taking Echinacea preparations
of this kind at the first sign of a cold are justified.
Table 1. Exposure to Echinacea: different concentrations and
times
IL-6 (pg/mL)
(mean ± SEM)Treatment
RV infection for 48 h, no Echinacea
RV infection followed by
Echinacea 1:100 for 48 h
Pre-exposure to Echinacea 1:200
for 24 h, followed by RV infection for 24 h
Pre-exposure to Echinacea 1:100 for 24 h,
followed by RV infection for 24 h
RV infection followed by Echinacea 1:10
for 5 min, incubated for 24 h
830 ± 3.4
64.7 ± 16.3
46.2 ± 2.2
21.3 ± 1.3
588 ± 12

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ECHINACEA AS AN INFLAMMATORY AGENT5
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. (2008)
DOI: 10.1002/ptr
The mechanisms of action of Echinacea are not under-
stood, since previous studies (Barnes et al., 2005) have
implicated possible roles for various constituents, such
as polysaccharides, caffeic acid derivatives and alkylamides.
Echinaforce is essentially free of polysaccharides, so
they can be ruled out as major players in the cytokine
effects, although it should be pointed out that Echinacea
preparations enriched in polysaccharides do have pro-
found effects on gene expression (Altamirano-Dimas
et al., 2007). Some of the other constituents possess
antiviral, antibacterial and other activities (Vimalanathan
et al., 2005; Sharma et al., 2008), and consequently differ-
ent compounds, including additional known constituents,
may be involved in the overall mechanism of action.
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