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Echinacea Stimulates Human Lymphocytes

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Title:
Echinacea Stimulates Human Lymphocytes
Creator:
Momary, Kathryn Marie
Karlix, Janet ( Mentor )
Place of Publication:
Gainesville, Fla.
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University of Florida
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English

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serial ( sobekcm )

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University of Florida
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University of Florida
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Echinacea Stimulates Human Lymphocytes

Kathryn Momary


ABSTRACT


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.



INTRODUCTION


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

OTC preparations.



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).



METHODS


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/

ml standards.



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.



Calculations


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

stimulated cells

where stimulated cells = no echinacea was added [0 concentration]




RESULTS



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

same trends.




Table 1

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


average






standard dev.


Trial 3


787.2947


3071

2232

2421


2574.667

440.1026


average

standard dev.


2104.083


5113

5618

6282


5671

586.2994


1023.476


4508

5581

3712


4600.333

937.9149


2294.088


1174

2406

2392


1990.667

707.2887


63.3798


144

153

234


177

49.5681


Table 2

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


average

standard dev.


Trial 2







average

standard dev.


Trial 3







average

standard dev.


34596

40956


35647.33

4868.887


4634

4139

5681


4818

787.2947


3071

2232

2421


2574.667

440.1026


41394

45509


44658

2932.615


6448

8664

9170


8094

1447.756


4961

6372

5824


5719

711.3361


49097

40929


45681.33

4244.887


8111

10191

5957


8086.333

2117.108


4579

4184

5456


4739.667

651.0425


26199

30219


27287

2567.168


3926

4726

4451


4371

401.0299


992

1277

3055


1774.667

1117.921


100

75


1982

2742


2666.333

649.8125


182

182

257


207

43.30127


154

126

168


149.3333

21.38535


One of the controls plated was 100 ml of unstimulated cells and 100 ml of RPMI 1640. The unstimulated cells

was 495 � 227 cpm. The RPMI control was <228 � 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






stimulated cells.


Table 3

The Raw Data, in CPM, for Three Trials Demonstrating the Effect of

Methanol

Trial .355% 3.55%

Trial 1 21189.33 25409.67


Trial 2 5402.667 2324


Trial 3 1530.333 517


average


9374.111 9416.889

10413.85 13879.59


standard dev.


Percent stimulation


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.







DISCUSSION


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

in immunosuppression.



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.



CONCLUSION


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






ACKNOWLEDGEMENTS


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.






REFERENCES


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.

1992;Vol:343-348

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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