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Echinacea Stimulates Human Lymphocytes
Herbal products of echinacea (Picture 1, Picture 2) have been found to have immunomodulatory properties. By
using proliferation assays with peripheral blood mononuclear cells (PBMC's) the immunomodulation was
measured. Immunostimulation was observed at the lower concentrations and immunosuppression was observed
at the higher concentrations of echinacea.
United States expenditures on herbal medicinal products totaled over $441.5 million in 1997.1 The highest
selling herbal product was echinacea(1). Echinacea, also known as purple coneflower, is endogenous to
midwest North America. Echinacea was first used in the US by Native Americans to treat ailments such as
snake bites, bowel pain, toothaches, rabies, burns, seizures, wound infections, septic conditions, cancer,
tonisilitis, cough, and sore throat (2,3), US physicians began using Echinacea in the 1880's to treat common
colds. However, the use of this herbal remedy decreased after the creation of sulfa drugs and common antibiotics (4).
There are three echinacea species used for health care purposes: Echinacea purpurea, Echinacea pallida,
and Echinacea angustifolia (3). Commercial preparations are chemically different because they are prepared from
one or more of the three species and contain a variety of plant parts including stem, flower, leaf, and/or
root. Therefore, echinacea is a generic term for a multitude of echinacea derivatives found in over-the-counter
(OTC) drug products. Echinacea is also a generic term used in research. Researchers generally manufacture their
own extracts from varying species and plant parts. This makes it very difficult to compare echinacea study
results from different labs. It is also impossible to correlate data from research manufactured echinacea with
Research by Brinkeborn et al. found echinacea to be significantly effective for treating patients with the common
cold (7). However, several clinical studies on echinacea have reported different results. Some researchers found
their preparations of echinacea to have no significant change on their patient's incidence of upper
respiratory infections or common colds (2,3). Burger et al. found an increase in interleukin-1 (IL-1), tumor
necrosis factor-a (TNF-a), interleukin-6 (IL-6), and interleukin-10 (IL-10) production by PBMCs stimulated
with various concentrations of Echinacea (8). Roesler et al. studied the effect of their purified echinacea in
healthy volunteers. They found an increase in the motility of polymorphonuclear cells (PMN), the ability of PMN to
kill bacteria, and proliferation of PBMCs after intravenous (i.v.) administration of echinacea(9). These data
suggest that echinacea increases acute inflammatory responses.
Research utilizing OTC preparations of echinacea are most easily translated to clinical outcomes. Burger et al
used unfractionated, marketed echinacea in their study and found immunostimulation, as stated previously (8).
In this study we used two commonly used OTC echiancea products. Each of these products contained
echinacea purpurea root and herb (above ground parts).
Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
Peripheral blood was collected from volunteers by venipuncture. PBMCs were isolated using a density gradient
called Histopaque-1077" (Sigma, St. Louis, MO). The viability of the cells was determined by trypan blue
(Sigma) exclusion stain. The sample was diluted 1x106 cells/ml with RPMI 1640 (Sigma) tissue culture
media containing 25 mM HEPES, L-glutamine, sodium bicarbonate, 3 ml/ 100 ml media of antibiotic
antimycotic (contains 10,000 units penicillin, 10 mg streptomycin and 25 mg amphotericin B/mL in 0.9 % NaCI),
and 10% fetal calf serum (FCS).
Echinacea Extraction and Preparation of Standards
Two OTC echinacea products were used: Centrum Echinacea and Natures's Herbs Echinacea containing
Echinacea Purpurea root and herb. The echinacea was extracted with a 1:1 water:methanol solvent.
Seventy milligrams of each echinacea product was extracted with 10 mL of solvent. Each sample was sonicated for
30 minutes and then centrifuged at 500g for 15 minutes. The supernatant was then removed and filtered through
a 0.2 mm Nalgene filter. This stock solution concentration was 7mg/ml, assuming 100% recovery. Four
standard concentrations were made by serial dillution in RPMI (1000 mg/ml, 100mg/ml, 10mg/ml, and img/
ml). Methanol controls were made in RPMI to mimic the methanol concentrations in the 1000 mg/ml and 100mg/
PBMC Proliferation Measurement
PBMCs were stimulated by adding 80 ng/ml of phorbol-12-myristate 13-acetate (PMA) (Sigma). The stimulated
cells were plated on a 96 microtiter well plate in triplicate with an equal volume of echinacea standards. The final
well volume was 200 ml. The controls included stimulated cells with RPMI, stimulated cells with
methanol, unstimulated cells with RPMI, and RPMI alone. The well plate was covered and transferred to a
humidified incubator for 48 hours at 37C with 5% C02. After incubation, the wells were radiolabled with imCi
of methyl-tritiated-thymidine ([3H]-thymidine) (NENTM Life Science Products, Inc, Boston, MD). The plate
was transferred back to the incubator for an additional 24 hours. The cells were harvested using a Skatron
Semi-automatic cell harvester. The uptake of [3H]-thymidine was determined by adding National Diagnostic
(Atlanta, GA) Ecoscint-O (R) scintillation fluid and reading radioactivity in counts per minute on a Beckman
(Fullerton, CA) LS 6500 Scintillation Counter.
The triplicates were then averaged to give one number for each concentration or control. The percent stimulation
(%S) was then calculated according to the following equation.
%S = stimulated cells (exposed to echinacea) - stimulated cells * 100
where stimulated cells = no echinacea was added [0 concentration]
Both Centrum and Nature's Herbs Echinacea effected the immunoassays in a dose dependent manner. Table 1
shows the raw data for Centrum Echinacea. The lower concentrations (1 and 10 m g/ml) show immunostimulation
in all three trials using Centrum Echinacea. The higher concentrations (100 and 1000 mg/ml)
show immunosuppression in all three trials. As shown in Table 2, the Nature's Herbs echinacea data show the
The Raw Data, in Counts per Minute (CPM)
(for three trials using Centrum echinacea with 5 different concentrations in pg/ml.)
Trial 0 1 10 100 1000
Trial 1 31390 51447 46550 27582 2394
34596 46960 51233 30953 1278
40956 48373 45953 33477 1898
average 35647.33 48926.67 47912 30670.67 1856.67
standard dev. 4868.887 2294.167 2891.519 2957.624 559.147
Trial 2 4634 10550 7705 2426 142
4139 8848 9506 5055 91
5681 13032 7763 6997 217
4818 10810 8324.667 4826 150
The Raw Data, in CPM, for Three Trials using Nature's Herbs Echinacea with 5 Different
Concentrations in pg/ml.
Trial 0 1 10 100 10
Trial 1 31390 47071 47018 25443 32
One of the controls plated was 100 ml of unstimulated cells and 100 ml of RPMI 1640. The unstimulated cells
was 495 ï¿½ 227 cpm. The RPMI control was <228 ï¿½ 53 cpm. The methanol control concentrations were
3.55% and .355% in the 1000 and 100 mg/ml respectively. As Table 3 demonstrates the raw data for the
methanol controls follows the same general trend that the echinacea data in Tables 1 and
follow. Immunosuppression in the methanol controls increases as methanol percentage increases. However,
the immunosuppression is greater with the Echinacea and stimulated cells than with the methanol and
The Raw Data, in CPM, for Three Trials Demonstrating the Effect of
Trial .355% 3.55%
Trial 1 21189.33 25409.67
Trial 2 5402.667 2324
Trial 3 1530.333 517
The average data from Table 1 and 2 were used to calculate the percent stimulation. The %S represents a
single value therefore no error bars could be assigned.(fig.1 and 2) Centrum echinacea exhibited highest
stimulation at img/ml, while Nature's Herbs echinacea exhibited highest stimulation at 10 mg/ml.
Cell Response to Stimulaiion with Centrum Echinacea and PMA
S.' O 1000 1 00
E idinaeas coinenataions in ugrml
Figure 1. Percent stimulation with 5 different concentrations of Centrum Echinacea and PMA.
C, i RttrsEW ID o SiiuilIi i wt1 Niatii's H Etn F cdw mi mdl PMA
tin c- i cTretn u2lf
Figure 2. Percent stimulation with 5 different concentrations of Nature's Herbs Echinacea and PMA.
Many researchers have found in vitro stimulation with laboratory preparations of echinacea (6,8,9). However, there
is only one other in vitro study done using an OTC product which was manufactured, unfractionated
echinacea product (8). The lack of data comparing the efficacy of commonly used OTC echinacea products in
their original form prompted our investigation. Many of the OTC echinacea products are utilizing different
echinacea forms. We chose Centrum and Nature's Herbs echinacea because they are comparable in ingredients.
Each of the products contain echinacea Purpurea root and herb.
The percent stimulation graphs show immunostimulation at the lower concentrations (1 mg/ml and 10 mg/ml)
for both Natures herbs and Centrum Echinacea. (Fig. 1 and 2) Also, as shown in Tables 1 and 2 Centrum
echinacea has a slightly higher stimulation at lower concentrations then the Nature's Herbs
product. Immunosuppression was exhibited at the higher concentrations for both products. This could be due
to either the echinacea compounds or the increasing methanol concentration causing cell death. The
methanol concentration in the 1000 mg/ml well was 3.5% and .35% in the 100 mg/ml well. Methanol
concentrations greater than 1% may cause interruption of T lymphocyte proliferation and result
Our data show that low dose Echinacea can cause as much as 120% stimulation. This supports the idea that
OTC echinacea stimulates the immune system.
Future in vitro and clinical trials should address some additional issues pertaining to echinacea products.
More research is needed to deduce the potency of these products. Data on extra-cellular serum levels after
oral intake would also be helpful for future in vitro and clinical trials.
Photos by John Elderkin
I would like to thank the following for their assistance with this project:
The University of Florida Undergraduate Scholars Program
Dr. Janet L. Karlix and the University of Florida College of Pharmacy
The Department of Pharmacy Practice
Dr. Nicholas Bodor's Lab and Dr. Fubao Ji for use of a sonicator
Dr. Joe Walker
Ms. Clara Johary
I would like to thank Heather Myers, lab manager, for her assistance in teaching me the lab expertise needed
to answer the scientific questions.
1. German Commission E Monographs
2. Grimm W, Muller H. A Randomized Controlled Trial of the Effect of Fluid Extract of Echinacea Purpurea on
the Incidence and Severity of Colds and Respiratory Infections. The American Journal of Medicine. 1999;106:138-143
3. Melchart D, Walther E, Linde K, Brandmaier R, Lersch C. Echinacea Root Extracts for the Prevention of
Upper Respiratory Tract Infections. Archives of Family Medicine. 1998;7:541-545
4. Pepping J. Alternative Therapies - Echinacea. American Journal of Health-Systems Pharmacists. 1999:121-122
5. Lersch C, Zeuner M, Bauer A, Siemens M, Hart R, Drescher M, Fink U, Dancygier H, Classen M.
Nonspecific Immunostimulation with Low Doses of Cyclophosphamide, Thymostimulin, and Echinacea
Purpurea Extracts (Echinacin) in Patients with Far Advanced Colorectal Cancers: Preliminary Results. Title.
6. See DM, Broumand N, Sahl L, Tilles JG. In Vitro Effects of Echinacea and Ginseng on Natural Killer and
Antibody-dependent Cell Cytotoxicity in Healthy Subjects and Chronic Fatigue Syndrome or
Acquired Immunodeficiency Syndrome Patients. Immunopharmacology. 1997;35:229-235
7. Brinkeborn RM, Shah DV, Degenring RH. Echinaforce" and Other Echinacea Fresh Plant Preparations in the
Treatment of the Common Cold. Phytomedicine. 1999;6(1):1-5
8. Burger RA, Torres, AR, Warren RP, Caldwell, VD, Hughes BG. Echinacea-induced Cytokine Production by
Human Macrophages. International Journal of Immunopharmacology. 1997;19(7):371-379
9. Roesler J, Emmendorffer A, Steinmuller C, Luettig B, Wagner H, Lohmann-Matthes M. Application of
Purified Polysaccharides from Cell Cultures of the Plant Echinacea Purpurea to Test Subjects Mediates Activation
of Phagocyte System. International Journal of Immunopharmacology. 1991;13(7):931-941
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