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Paracelsian, Incorporated, Ithaca, New York
Correspondence: Joseph A. Rininger, Curagen Corp., 322 East Main Street, Branford, CT 06405. E-mail: JRininger{at}curagen.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, interleukin (IL)-1, IL-1ß, IL-6, IL-10, and nitric oxide.
Echinacea herb and root powders were found to stimulate
murine macrophage cytokine secretion as well as to significantly
enhance the viability and/or proliferation of human PBMCs in
vitro. In contrast, Echinacea extracts chemically
standardized to phenolic acid or echinocaside content and fresh pressed
juice preparations were found to be inactive as immunostimulatory
agents but did display, to varying degrees, anti-inflammatory and
antioxidant properties.
Key Words: tumor necrosis factor • interleukins • nitric oxide • lipopolysaccharide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The clinical effectiveness of Echinacea is greatly questioned because some studies have shown clinical benefit [1 2 3 4 ], whereas others have not [2 , 5 6 7 ]. The inability to demonstrate convincing evidence of efficacy with Echinacea administration can be attributed to several factors. These include the use of different Echinacea preparations (including combinations with other herbs) that were not pharmacologically characterized, poor study design, differing routes of administration (injection or orally), small study size, monitoring responses of healthy subjects, and the methodology employed for determining effectiveness [2 , 6 ]. For example, the majority of clinical studies finding no beneficial effect from Echinacea administration were evaluating Echinacea as a preventive medication. In addition, because of combination formulations the specific clinical effectiveness of Echinacea cannot be determined in certain trials. In contrast to the question of efficacy, Echinacea administration (pressed juice) was shown to be nontoxic in animals and has produced few documented adverse events in humans [8 , 9 ].
Laboratory studies have shown that Echinacea purpurea
herb and purified polysaccharides from Echinacea purpurea
cell cultures possessed immunostimulatory activity to murine and human
macrophages and mononuclear cells [10
11
12
13
].
Echinacea-activated macrophages and natural killer (NK)
cells displayed cytokine [tumor necrosis factor (TNF-),
interleukin (IL)-1, IL-6] production, enhanced phagocytic activity,
cellular proliferation, and the capacity to kill tumor cells as well as
effectively eliminate bacterial and fungal pathogens in
vitro [1
, 12
13
14
]. The purified
polysaccharides were subsequently shown to protect immunocompromised
mice from both fungal and bacterial infection [15
,
16
]. Testing of the Echinacea-derived
polysaccharides in human subjects also demonstrated activation of
phagocytic cells similar to that seen in mice [10
,
11
, 14
]. However, the polysaccharides
purified from Echinacea cell cultures may be different than
ones endogenously found in the plant [12
,
14
]. Furthermore, the test preparation used for the
in vivo studies was administered via injection, thereby
bypassing the digestive tract. Today, oral dosing of
Echinacea is the most predominant route of administration.
Therefore, it was of importance to determine which of the many
different Echinacea raw materials possess detectable
immunostimulatory activity after preparation through a simulated
digestion protocol.
In this report we provide results from screening various
Echinacea products and raw materials for macrophage
stimulatory activity after subjecting the material to a simulated
digestion protocol to emulate oral dosing of Echinacea
products. Macrophage activation was determined via quantification of
TNF- produced from cultures of treated murine macrophages.
Immunostimulatory properties of active materials were further defined
and compared with bacterial lipopolysaccharide (LPS) for secretion of
other macrophage-derived mediators [IL-1, IL-1ß, IL-6, IL-10, and
nitric oxide (NO)]. Echinacea preparations were also
assayed for enhancement of cell viability of human peripheral blood
mononuclear cells (PBMCs). In addition, anti-inflammatory and
antioxidant properties of these materials were also compared as
potential modes of action. The data indicate that the functional
activity of Echinacea preparations can be broken into two
distinct categories: immunostimulatory or anti-inflammatory and
antioxidant properties. This division amongst Echinacea
pharmacological activities may explain varying results pertaining to
Echinacea’s clinical effectiveness. Our findings also
demonstrate that the immunostimulatory attributes of
Echinacea are far less potent and only transient compared to
LPS and may explain the low incidence of reported side effects from
Echinacea administration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-, 1 x 106 units/mL) were
purchased from GIBCO (Long Island, NY), and fetal bovine serum was
supplied through Gemini Bioproducts (Carlsbad, CA). All quantitative
enzyme-linked immunosorbent assay (ELISA) kits for the detection of
murine cytokines were supplied through Endogen (Woburn, MA) and Cell
Titer-96TM cell proliferation assay reagents were from
Promega (Madison, WI). Limulus amebocyte lysate assay kits
for endotoxin contamination were supplied through BioWhitaker
(Walkersville, MD). Echinacea raw herb and root powders were obtained from organic certified farms. The aerial parts or roots of the plant were dried and then milled to no. 60 mesh screen particle size. Standardized Echinacea extract and pressed juice preparations were obtained from various suppliers, whereas other Echinacea products were purchased from local pharmacy and health food stores. Placebo capsules utilized for negative controls were kindly provided by R. P. Scherer (Tampa, FL) and consisted of common fill material for softgel capsules, soybean oil, lecithin, and beeswax. LPS from Escherichia coli (serotype 0127:B8), concanavalin A, and all other reagents were purchased through Sigma (St. Louis, MO).
Sample preparation
Test samples were either dissolved in DMSO or subjected to a
simulated digestion protocol. The simulated digestion methodology was
to weigh 750 mg of Echinacea raw material and add to 15 mL
of simulated gastric fluid (37 mM NaCl, 0.03 N HCl, 3.2 mg/mL pepsin)
and incubated at 37°C in a shaker incubator for 2 h. The acidity
of the simulated gastric fluid was then neutralized by adding an
equinormal amount of NaOH (2.2 N, 0.5 mL). Then 15.5 mL of a 2x
simulated intestinal fluid (30 mM K2HPO4, 160
mM NaH2PO4, pH 7.4 plus 20 mg/mL pancreatin)
was added and then the material returned to a 37°C shaker incubator
for an additional 2 h. Samples were then aliquotted and flash
frozen with liquid N2 until further analysis. LPS was
dissolved in a 50% ethanol/water solution at a concentration of 1
mg/mL.
RAW264.7 cell culture and assay conditions for macrophage
activation
RAW 264.7 cells were routinely cultured in DMEM supplemented
with 10% fetal bovine serum, 2 mM L-glutamine, and 100
units/mL penicillin/streptomycin. Activation assays were set up in
either 24- or 96-well tissue culture plates with the cells plated at a
cell density of 1 x 106 cells/mL. All test samples
were cleared of particulate matter by centrifugation (10,000
g for 5 min) before dilution. Twenty-four hours after the
cells were plated, test agents were added and the supernatant from
replicated treatment wells pooled and assayed for TNF- 24 h
posttreatment. For dose-response and time course experiments and
cytokine secretion profiling, the same protocol was followed with the
exception that the cell plating density was changed to 1 x
105 cells/mL to compensate for cell growth and potential
stress from exhausted medium. Sample supernatants were taken at 24, 30,
and 48 h posttreatment, flash frozen with liquid N2 so
that all samples could be assayed for cytokines at the same time. Cell
viability was routinely monitored by visual inspection and confirmed
with a Cell Titer 96 Nonradioactive Cell Proliferation Assay Kit.
Human PBMC viability
Human PBMCs isolated from normal human donors (HIV-1-, HIV-2-,
hepatitis-, bacteria-free) were purchased from Clonetics (San Diego,
CA) and stored in liquid nitrogen until use. Cells were then thawed,
resuspended in lymphocyte growth medium (Clonetics) at a cell density
of 5 x 106 cells/mL, and plated (100 µL/well) into
96-well microtiter plates. The cells were then treated with test agents
and incubated at 37°C for 72 h. Viability of the cells was
assessed with a Cell Titer 96 Nonradioactive Cell Proliferation Assay
Kit.
Determination of NO production
NO production was detected by assaying for the presence of
nitrites (NO2) in culture medium by mixing an equal volume
of medium and Griess reagent in 96-well microtiter plates. A standard
curve was prepared by dissolution of potassium nitrite into culture
medium. After 5–10 min of development time the samples were read at an
optical density wavelength of 540 nm.
Free radical scavenging assay
Antioxidant activity was determined based upon the ability of
test materials to scavenge the free radical
1,1-diphenyl-2-picrylhydrazyl (DPPH), a method documented to strongly
correlate with the antilipoperoxidant and antinecrotic activity
associated with plant flavonoids [17
]. The assay was
performed by serially diluting the test material in triplicate wells of
a 96-well microtiter plate. The DPPH was then added to the wells to a
final concentration of 150 µM and a blank (no test compound added)
was prepared under the same conditions. Scavenging of the DPPH free
radical yielded a decoloration of the compound that was read
spectrophotometrically at an optical density wavelength of 540 nm.
Samples were run at six different concentrations to calculate a 50%
effective scavenging concentration (EC50) based upon the
percentage of decoloration compared to the blank. The flavonoid
antioxidants rutin and quercetin were used as positive controls.
Anti-inflammatory assay
Anti-inflammatory activity was determined based upon disruption
of arachidonic acid metabolism resulting in decreased production of
prostaglandin E2 (PGE2). RAW264.7 macrophage
cells were plated at 1 x 106 cells/mL in 24-well
tissue culture plates and treated with 10 units/mL IFN- to initiate
PGE2 production concurrently with the test
Echinacea sample. The cell medium was then assayed for
PGE2 production utilizing quantitative ELISA kits from
Oxford Biomedical Research (Oxford, MI) 24 h poststimulation.
Statistical analysis
Response measurements to various treatments were subjected to
analysis of variance for means comparisons utilizing JMP v2 statistical
software (SAS Institute, Cary, NC). Statistical significance between
the individual treatment groups and control was then assessed with
Student’s t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[19
20
21
]. Treatment of RAW264.7
cells with Echinacea purpurea herb test material dissolved
in traditional solvents for in vitro studies, such as
dimethyl sulfoxide, were inactive for production of TNF- and NO as
activation biomarkers. However, when materials were prepared through a
simulated digestive protocol, the immunostimulatory activity was
detected as shown in Figure 1
. The assay methodology demonstrated good reproducibility with an
inter-assay TNF- response variation of 10–15% (Fig. 2
). Similar variability was obtained from separate simulated
digestion preparations of the same Echinacea material (data
not shown). To demonstrate that the response was specific, a
dose-response study was initiated and found that the
Echinacea-induced stimulation of TNF- and NO was
dose-dependent and was statistically significant versus placebo capsule
control at doses as low as 5 µg/mL (P<0.001) for both
biomarkers (Fig. 3
, A and B). Furthermore, MTT measurements
indicated no loss of viability from the digested sample treatment and
that the Echinacea test material enhanced RAW264.7 cell
proliferation, whereas LPS inhibited cell growth (Table 1
).
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production was then expanded to examine secretion of other
macrophage-derived cytokines: IL-1, IL-1ß, IL-6, and IL-10, as
well as NO production in a time- and dose-dependent fashion.
Echinacea-induced responses were compared to LPS (0.1
µg/mL) stimulation. Echinacea purpurea herb processed
through simulated digestion and LPS produced time- and dose-dependent
induction of TNF-, NO, IL-1, IL-1ß, and IL-6 (Fig. 4
and Table 2
) with TNF- and NO being the most sensitive biomarkers. TNF-
levels produced by Echinacea stimulation peaked at
approximately 30 h poststimulation and then declined sharply by
48 h, whereas TNF- levels from LPS stimulation did not drop
dramatically (Fig. 4A) . In contrast, IL-1ß, IL-6, and NO continued to
rise over the 48-h time-course (Fig. 4B
and Table 2
).
Echinacea-induced IL-1 production rose at 24 h,
dipped at 30 h, and then was slightly elevated at the 48-h time
period, whereas LPS-induced IL-1 levels rose continually over the
time course (Table 2)
. Echinacea and LPS also induced
IL-1ß and IL-10, although the magnitude of this response was small
compared with the other cytokines that were examined (Table 2)
.
Echinacea-induced IL-10 peaked at 24 h and then
declined, whereas LPS-induced IL-10 continued to rise over the 48-h
time period. In this series of experiments, Echinacea at
concentrations 
20 µg/mL did not induce secretion of detectable
levels of these cytokines. This was due to the use of 10-fold fewer
cells (see Methods). As can be seen from Table 2
, the Echinacea
purpurea-induced activation of cytokine production from RAW264.7
macrophage cells was weaker at the highest concentration tested (320
µg/mL) than that of LPS at a concentration of 0.1 µg/mL and were
transient for TNF- and IL-1 (Fig. 4A
, Table 2
). The transient
nature of the Echinacea response could represent an
explanation for lack of reported toxicity from Echinacea
administration.
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induction as a biomarker for activity. There was a wide
degree of variation amongst both Echinacea purpurea herb and
Echinacea purpurea root samples. Two out of seven different
Echinacea purpurea herb lots had activity similar to the
original Echinacea purpurea herb material (Fig. 5A
). Of the root powders, one out of five had activity similar to
our original test material (Fig. 5B)
. Comparison of endotoxin,
bacterial load, fungus, and mold in these samples demonstrated no
correlation with the immunostimulatory response of RAW264.7
macrophages. With the demonstrated reproducibility of the test system
(Fig. 2)
, these data highlight the variability of natural products and,
for Echinacea, could represent non-optimal harvest time,
environmental conditions, or storage factors that govern production and
preservation of Echinacea’s immunostimulatory activity.
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, IL-1, and IL-6 were originally characterized
as growth and activation factors for other immune cell types such as T
and B lymphocytes, NK cells, and neutrophils [22
23
24
].
To demonstrate that Echinacea preparations could stimulate
proliferation of various immune cell types, human PBMCs were treated
with Echinacea or placebo capsules prepared through the
simulated digestion protocol without other stimulation and cellular
viability assessed after 72 h. In the absence of proliferative
stimulation, PBMC viability dropped steadily over 72 h (data not
shown). Echinacea materials that stimulated TNF-
production in RAW264.7 macrophage cells significantly enhanced the
viability of PBMCs (Fig. 7
A). The Echinacea-induced enhancement of PBMC viability was
found to be dose-dependent with optimal stimulation by
Echinacea at concentrations of 1 µg/mL and was effective
with different donors (data not shown). Echinacea
preparations that did not stimulate macrophage TNF- production did
not enhance PBMC viability (Fig. 7B)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based upon this in vitro data, Echinacea-induced
transient stimulation of nonspecific immune cells such as macrophages
to produce TNF-, IL-1, IL-6, and NO could serve to augment the
immune response and more rapidly destroy invading pathogens
[15
, 16
]. The macrophage-derived cytokines
immunologically function to initiate cascades of immune cell type
activation such as T and B lymphocytes to up-regulate major
histocompatibility complex (MHC) expression and produce additional
cytokines such as IFN- [29
, 30
].
In vitro, this was modeled by assessing the viability of
human PBMCs in the absence of proliferative stimulation, and supports
findings that Echinacea treatment produced expansion of
other immune parameter effects in vivo [11
].
The immunostimulating activities we have described for
Echinacea are similar to those of Burger et al.
[18
] with a notable exception. Although describing
similar macrophage activating effects to those described above, these
investigators found Echinacea (pressed juice containing
fructofuranosides) to be active at nanogram concentrations and more
potent at stimulating macrophage cytokine synthesis than bacterial
endotoxin (LPS). Endotoxin shock represents a potentially fatal
clinical condition characterized by excessive production of
inflammatory cytokines such as TNF-, IL-1, and IL-6. It would seem
unlikely that Echinacea preparations to be this potent at
stimulating inflammatory cytokines without the occurrence of
significant toxicity in vivo. In fact, Echinacea
preparations (pressed juice) have been shown to possess very low
toxicity [9
]. The data generated from our investigations
are in agreement with prior investigators and show that
Echinacea-induced cytokine secretion was far weaker and
transient at 3200-fold higher concentrations in comparison to those of
LPS (Table 2)
. Additional experiments in our laboratory have found that
LPS at concentrations as low as 1 ng/mL produce greater responses for
TNF- secretion than Echinacea at 20 µg/mL, a
20,000-fold difference.
In an effort to ensure reproducibility of product preparations, standardized extracts of Echinacea have become more prevalent. These extracts are typically standardized to phenolic compounds, such as chlorogenic acid, which are actually common constituents of many plant species [26 ]. Literature review of these compounds has identified weak activity against hyaluronidase, an enzyme used by certain bacteria to penetrate tissue [30 ], antioxidant activity [31 , 32 ], anti-inflammatory activity [26 , 27 ], as well as potent inhibitory activity toward the HIV integrase enzyme [33 , 34 ]. Furthermore, in vivo studies assessing immune parameters in laboratory animals have found no evidence of immune stimulatory activity of chlorogenic acid as a single agent [25 ]. The data generated from our in vitro studies, incorporating simulated digestion, found that standardized Echinacea extracts and chlorogenic acid alone did not stimulate macrophage cytokine secretion nor enhanced PBMC viability. We found that standardized Echinacea extracts displayed antioxidant and anti-inflammatory activity, consistent with prior findings with these agents.
The design and implementation of in vitro experimental approaches are of central importance to contemporary research and scientific discovery in molecular and cellular biology, pharmacology, and toxicology. However, the true scientific impact of in vitro results depends upon their relevance and efficacy in vivo. The clinical effectiveness of Echinacea preparations for treatment (provide faster resolution), prevention, or alleviation of symptoms associated with colds and flu are inconclusive. For example, Hoheisel et al. [4 ] found that Echinacea pressed juice significantly reduced the severity of colds, whereas Grimm et al. [7 ], using the same formulation, found no statistically beneficial effect. In addition, two recently reported studies using different Echinacea root extracts showed no significant benefit [6 ]. A critical review of the clinical literature on Echinacea found that only 8 out of 26 clinical trials earned a 50% or better rating on the reviewer’s quality score system for trial design [35 ]. A more recent review on Echinacea use for upper respiratory infections have concluded that there is some suggestive evidence of benefit [36 ]. Two key problems with the clinical literature associated with Echinacea, irrespective of the study design, are (1) the administration of different Echinacea extracts, each with a different phytochemical profile and (2) lack of in vitro studies characterizing the immunomodulating activity of the test material [2 , 6 ]. Our assessment of the variability in activity amongst material from the same supplier and equivalently standardized extracts clearly demonstrates the importance of such knowledge before clinical evaluation and could potentially explain negative clinical results. Such basic pharmacological information should be determined in order to design trials that assess benefit relating to the pharmacological activity of the test materials.
Results demonstrating activation of macrophages and NK cells using
Echinacea purpurea herb powder and polysaccharides purified
from cell culture cultivation of Echinacea are at the core
of literature for the immunostimulatory effects of Echinacea
[10
, 13
, 15
]. The secretion of
macrophage-derived cytokines suggest that Echinacea may
augment the immune response, as has been previously reported both
in vitro and in vivo for TNF-, IL-1, IL-6, and
the immunomodulator imiquimod [22
23
24
,
37
38
39
40
]. This is further supported by animal studies in
which in vitro-characterized immunostimulatory
Echinacea materials were proven efficacious by protecting
animals from lethal bacterial and fungal infections [15
,
16
]. However, the Echinacea test material was
administered as an intraperitoneal injection, surpassing digestion and
absorption. Our investigations have shown that Echinacea’s
immunostimulatory activity can be predicted to survive digestion
in vivo. Our laboratory is continuing our work with
elucidation of pharmacological activities of Echinacea raw
materials, evaluation of bioavailability using in
vitro-based models, and in vivo activation of
nonspecific immune cells and responses to pathogen challenge. This work
will resolve into human clinical trials employing Echinacea
with known pharmacological activities.
Received October 30, 1999; revised May 13, 2000; accepted May 14, 2000.
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and interleukin-1 on the production of macrophage inflammatory protein-2 in response to respiratory syncytial virus infection in a murine macrophage cell line, RAW264.7 J. Med. Virol. 53,145-149[Medline]
/cachectin is a growth factor for thymocytes: synergistic interactions with other cytokines J. Exp. Med. 167,1472-1478 J. Immunol. 140,531-536[Abstract]
: induction of TNF receptors on human T cells and TNF--mediated enhancement of T cell responses J. Immunol. 138,1786-1790[Abstract]
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and IFN- Int. J. Immunopharmacol. 13,943-954[Medline]
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