Journal of Undergraduate Research
Volume 1, Issue 2 - November 1999

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 % NaCl), 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 1mg/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% CO2. After incubation, the wells were radiolabled with 1mCi 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.

Trial	0	1	10	100	1000
Table 1 The Raw Data, in Counts per Minute (CPM) ( for three trials using Centrum echinacea with 5 different concentrations in µg/ml.)
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
average	4818	10810	8324.667	4826	150
standard dev.	787.2947	2104.083	1023.476	2294.088	63.3798
Trial 3	3071	5113	4508	1174	144
	2232	5618	5581	2406	153
	2421	6282	3712	2392	234
average	2574.667	5671	4600.333	1990.667	177
standard dev.	440.1026	586.2994	937.9149	707.2887	49.5681

Trial	0	1	10	100	1000
Table 2 The Raw Data, in CPM, for Three Trials using Nature's Herbs Echinacea with 5 Different Concentrations in µg/ml.
Trial 1	31390	47071	47018	25443	3275
	34596	41394	49097	26199	1982
	40956	45509	40929	30219	2742
average	35647.33	44658	45681.33	27287	2666.333
standard dev.	4868.887	2932.615	4244.887	2567.168	649.8125
Trial 2	4634	6448	8111	3926	182
	4139	8664	10191	4726	182
	5681	9170	5957	4451	257
average	4818	8094	8086.333	4371	207
standard dev.	787.2947	1447.756	2117.108	401.0299	43.30127
Trial 3	3071	4961	4579	992	154
	2232	6372	4184	1277	126
	2421	5824	5456	3055	168
average	2574.667	5719	4739.667	1774.667	149.3333
standard dev.	440.1026	711.3361	651.0425	1117.921	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 2follow. 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.

Trial	.355%	3.55%
Table 3 The Raw Data, in CPM, for Three Trials Demonstrating the Effect of Methanol
Trial 1	21189.33	25409.67
Trial 2	5402.667	2324
Trial 3	1530.333	517
average	9374.111	9416.889
standard dev.	10413.85	13879.59

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 1mg/ml, while Nature's Herbs echinacea exhibited highest stimulation at 10 mg/ml.

Figure 1. Percent stimulation with 5 different concentrations of Centrum Echinacea and PMA.

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

German Commission E Monographs
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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
Pepping J. Alternative Therapies Echinacea. American Journal of Health-Systems Pharmacists. 1999:121-122
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
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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
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
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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