Immunopharmacological activity of Echinacea preparations following simulated digestion on murine macrophages and human peripheral blood mononuclear cells

Joseph A. Rininger, Shirley Kickner, Padmasree Chigurupati, Anne McLean, and Zsoa Franck Paracelsian, Incorporated, Ithaca, New York

Abstract: We have investigated the immunostimulatory, anti-inammatory, and antioxidant activities of various Echinacea raw materials and commercially available products on murine macrophages and human peripheral blood mononuclear cells (PBMCs). To emulate oral dosing, a simulated digestion protocol was employed as a means of sample preparation. Echinacea-induced macrophage activation was used as a measure of immunostimulatory activity determined via quantitative assays for macrophage-derived factors including tumor necrosis factor , interleukin (IL)-1, IL-1, IL-6, IL-10, and nitric oxide. Echinacea herb and root powders were found to stimulate murine macrophage cytokine secretion as well as to signicantly 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-inammatory and antioxidant properties. J. Leukoc. Biol. 68: 503510; 2000.
Key Words: tumor necrosis factor lipopolysaccharide

interleukins

nitric oxide

INTRODUCTION
Echinacea represents one of the most common traditional American herbal medicines that is commonly believed to act as an immunostimulant and marketed commercially as a cold and u remedy. There are three predominant species of Echinacea used for such purposes: angustifolia, purpurea, and pallida. In addition, a denitive lack of pharmacological knowledge as to which preparations are believed to be most effective has given rise to a myriad of different raw material forms that consist of dried herb or root powders, pressed juice, and chemically standardized extracts. This array of raw material types results in an even larger number of varying formulations of the species used and portions of the plant that are processed. The clinical effectiveness of Echinacea is greatly questioned because some studies have shown clinical benet [1 4], whereas others have not [2, 57]. The inability to demonstrate convincing evidence of efcacy with Echinacea administration can be attrib-

uted 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 nding no benecial effect from Echinacea administration were evaluating Echinacea as a preventive medication. In addition, because of combination formulations the specic clinical effectiveness of Echinacea cannot be determined in certain trials. In contrast to the question of efcacy, 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 puried polysaccharides from Echinacea purpurea cell cultures possessed immunostimulatory activity to murine and human macrophages and mononuclear cells [10 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, 1214]. The puried 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 puried 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

Correspondence: Joseph A. Rininger, Curagen Corp., 322 East Main Street, Branford, CT 06405. E-mail: JRininger@curagen.com Received October 30, 1999; revised March 13, 2000; revised May 13, 2000; accepted May 14, 2000.

Journal of Leukocyte Biology Volume 68, October 2000 503

digestion protocol to emulate oral dosing of Echinacea products. Macrophage activation was determined via quantication of TNF- produced from cultures of treated murine macrophages. Immunostimulatory properties of active materials were further dened and compared with bacterial lipopolysaccharide (LPS) for secretion of other macrophage-derived mediators [IL-1, IL-1, 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-inammatory 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-inammatory and antioxidant properties. This division amongst Echinacea pharmacological activities may explain varying results pertaining to Echinaceas clinical effectiveness. Our ndings 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.

mycin. Activation assays were set up in either 24- or 96-well tissue culture plates with the cells plated at a cell density of 1 106 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 proling, the same protocol was followed with the exception that the cell plating density was changed to 1 105 cells/mL to compensate for cell growth and potential stress from exhausted medium. Sample supernatants were taken at 24, 30, and 48 h posttreatment, ash 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 conrmed 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 5 106 cells/mL, and plated (100 L/well) into 96-well microtiter plates. The cells were then treated with test agents and incubated at 37C 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 510 min of development time the samples were read at an optical density wavelength of 540 nm.

MATERIALS AND METHODS Materials

RAW264.7 macrophage cells were obtained from ATCC (Rockville, MD) and HIV- and hepatitis-screened human PBMCs and lymphocyte growth medium were from Clonetics (San Diego, CA). Dulbeccos modied Eagles medium (DMEM) growth medium and murine recombinant interferon- (IFN-, 1 106 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-96 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 certied 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 ll 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).

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 avonoids [17]. The assay was performed by serially diluting the test material in triplicate wells of a 96-well microtiter

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 uid (37 mM NaCl, 0.03 N HCl, 3.2 mg/mL pepsin) and incubated at 37C in a shaker incubator for 2 h. The acidity of the simulated gastric uid was then neutralized by adding an equinormal amount of NaOH (2.2 N, 0.5 mL). Then 15.5 mL of a 2 simulated intestinal uid (30 mM K2HPO4, 160 mM NaH2PO4, pH 7.4 plus 20 mg/mL pancreatin) was added and then the material returned to a 37C shaker incubator for an additional 2 h. Samples were then aliquotted and ash 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/strepto-

Fig. 1. Macrophage activation induced by Echinacea purpurea herb dissolved in dimethyl sulfoxide (DMSO) or prepared through the simulated digestion protocol at a concentration of 20 g/mL. Data represent the mean TNF- produced (pg/mL) SD of the cell culture supernatant after 24 h of Echinacea stimulation. Data are representative of experiments (n 3) in which each test sample was used to treat three replicate wells of macrophage cells and the pooled supernatant run in triplicate ELISA wells. *Statistically different than placebo capsule digestion control P 0.001.

504

Journal of Leukocyte Biology Volume 68, October 2000

http://www.jleukbio.org

plate. The DPPH was then added to the wells to a nal concentration of 150 M 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 avonoid antioxidants rutin and quercetin were used as positive controls.

Anti-inammatory assay
Anti-inammatory 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 1 106 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 signicance between the individual treatment groups and control was then assessed with Students t test.

RESULTS
Initial investigations were targeted at the immunomodulatory effects reported for Echinacea on stimulation of macrophage function [1113, 18]. RAW264.7 macrophage cells have been well characterized to have prototypical macrophage responses such as cytokine release and NO production from stimulation with LPS or IFN- [19 21]. Treatment of RAW264.7 cells with Echinacea purpurea herb test material dissolved in traditional

Fig. 3. Dose-response of macrophage activation of Echinacea purpurea herb after simulated digestion preparation. Data represent the mean standard deviation for TNF- (pg/mL, n 6) and nitrites (M, n 12) quantied from the cell culture supernatant after 24 h of Echinacea stimulation. *Statistically different than control cells, which were treated with placebo capsules (20 g/mL equivalent dilution) processed through the simulated digestion protocol P 0.01.

Fig. 2. Reproducibility of Echinacea purpurea herb prepared via a simulated digestion protocol to stimulate RAW264.7 macrophage cell TNF- production in comparison to identically processed placebo capsules. Data represent the mean SD of TNF- produced the pooled tissue culture supernatant from three replicate wells of macrophage cells treated with the same Echinacea purpurea herb or placebo capsule control sample preparation. There was only a 12% variation in the Echinacea-induced TNF- production.

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 specic, a dose-response study was initiated and found that the Echinacea-induced stimulation of TNF- and NO was dose-dependent and was statistically signicant versus placebo capsule control at doses as low as 5 g/mL (P0.001) 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).
505

Rininger et al. Characterization of Echinacea immunopharmacology

TABLE 1. Cell Viability of RAW264.7 Macrophages After 24 h of Stimulation With Simulated Digested Echinacea purpurea Herb, Simulated Digested Placebo Capsules, or LPS ( N 5 Replicate Wells)
Mean SD optical density readings (570 nM) Calculated percent viability

Sample treatment (dose)

Nontreated cells Digested Echinacea purpurea herb (1280 g/mL) Digested placebo capsules (same dilution as Echinacea purpurea herb) LPS (0.1 g/mL)

1.069 0.145 1.246 0.102 1.058 0.041 0.998 0.055

100% 116%* 99% 93%*

* Statistically different ( P 0.05) from nontreated cells, which represent the 100% viability control.

Echinacea purpurea-induced macrophage activation as assessed by TNF- production was then expanded to examine secretion of other macrophage-derived cytokines: IL-1, IL1, 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 g/mL) stimulation. Echinacea purpurea herb processed through simulated digestion and LPS produced time- and dose-dependent induction of TNF-, NO, IL-1, IL-1, 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-1, 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-1 and IL-10, although the magnitude of this response was small compared with the other cytokines that were examined (Table 2). Echinaceainduced 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 g/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 g/mL) than that of LPS at a concentration of 0.1 g/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. Based upon these results, we then examined different lots of Echinacea purpurea raw material (herb powder and root powder) from a single supplier to compare immunostimulatory activity using TNF- 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 ve had activity similar to our original test material (Fig. 5B). Comparison of endotoxin, bacterial load, fungus, and mold in these samples demon-

Fig. 4. (A) TNF- (pg/mL) and (B) nitric oxide (M nitrites) dose and time course proles of Echinacea purpurea herb after simulated digestion and comparison to LPS. Data represent the mean SD of the respective biomarker produced by RAW264.7 macrophages in cell culture supernatant and are representative of experiments (n 2) in which each test sample was used to treat three replicate wells of macrophage cells and the pooled supernatant run in three replicate ELISA wells. Data represent the mean SD of each sample time point run in triplicate wells. All data points shown were statistically different from digested placebo capsule control at the respective time point P 0.05.

506

Journal of Leukocyte Biology Volume 68, October 2000

http://www.jleukbio.org

TABLE 2. Dose-Response and Time Course of Cytokine Induction by Echinacea purpurea Herb and LPS Compared to Placebo Control Capsules
Time point Treatment 24 h 30 h 48 h

IL-1 LPS (0.1 g/mL) Echinacea (320 g/mL) Echinacea (80 g/mL) Echinacea (20 g/mL) Echinacea (5 g/mL) Control IL-1 LPS (0.1 g/mL) Echinacea (320 g/mL) Echinacea (80 g/mL) Echinacea (20 g/mL) Echinacea (5 g/mL) Control IL-6 LPS (0.1 g/mL) Echinacea (320 g/mL) Echinacea (80 g/mL) Echinacea (20 g/mL) Echinacea (5 g/mL) Control IL-10 LPS (0.1 g/mL) Echinacea (320 g/mL) Echinacea (80 g/mL) Echinacea (20 g/mL) Echinacea (5 g/mL) Control

82 4** 42 12** 30 11** 12 4 10 3 71 7 0.4** 6 0.6** 3 0.4** 0 0 0 582 50** 123 5** 59 8 48 19 29 12 27 9 97 9* 77 19 63 29 57 17 45 26 49 8
SD

116 2** 31 8** 20 3* 19 14 73 82 12 1.6** 10 1.5** 7 0.4** 1 1.1 0 0 762 189** 186 19** 81 2 49 7 41 2 45 8 107 9** 59 4** 44 2 32 5 24 2 42 4

204 7** 63 7** 36 7** 17 1 12 4 10 1 24 1.3** 14 0.9** 5 0.7** 1 0.6* 0 0 1563 20** 234 5** 75 5** 28 5* 40 8** 23 3 137 22** 29 6 44 7* 29 9 18 5 37 5

cell types such as T and B lymphocytes, NK cells, and neutrophils [2224]. 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 signicantly enhanced the viability of PBMCs (Fig. 7A). The Echinacea-induced enhancement of PBMC viability was found to be dose-dependent with optimal stimulation by Echinacea at concentrations of 1 g/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).

Data represent the mean pg/mL 0.05, ** P 0.01.

for each cytokine ( n 3), * P

strated 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 Echinaceas immunostimulatory activity. Sampling and testing of various Echinacea products and additional raw materials clearly demonstrated that the macrophage activating capacity of Echinacea was found in Echinacea purpurea herb and root powders (Fig. 6). Echinacea angustifolia herb and root powders also showed varying levels of immunostimulatory activity (data not shown). Finished products and raw materials comprised of Echinacea extracts that had been standardized to contain 4% phenolic compounds such as chlorogenic acid and chicoric acid or to echinocasides/ alkylamides were inactive for induction of macrophage cytokine production (Fig. 6) even at doses 250 g/mL (data not shown). Chlorogenic acid was also tested as an individual compound and was found to be negative for macrophage activation (data not shown). Echinacea purpurea fresh pressed juice powders and liquids, and full spectrum extracts containing polysaccharides (fructofuranosides) and phenolic compounds were also found to be inactive in the macrophage activation assay. Cytokines such as TNF-, IL-1, and IL-6 were originally characterized as growth and activation factors for other immune

Fig. 5. Macrophage stimulation after simulated digestion of different lots of Echinacea purpurea herb (A) and root (B) raw material powders (20 g/mL Echinacea concentration). Data represent the mean TNF- produced (pg/ mL) SD of the cell culture supernatant after 24 h of Echinacea stimulation. Each sample was used to treat three replicate wells of macrophage cells and the pooled supernatant run in three replicate ELISA wells.

Rininger et al. Characterization of Echinacea immunopharmacology

507

these immunomodulatory functions. In contrast, chemically standardized Echinacea extracts were found to possess antioxidant and anti-inammatory activities. A high degree of variability was found amongst similarly standardized extracts and formulated products for these activities. Based upon this in vitro data, Echinacea-induced transient stimulation of nonspecic 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

Fig. 6. Macrophage stimulation of commercial Echinacea products after simulated digestion. Data represent the mean TNF- released (pg/mL) SD into the cell culture supernatant after 24 h of Echinacea stimulation. Each sample was used to treat three replicate wells of macrophage cells with a nal Echinacea concentration of 20 g/mL and the pooled supernatant run in triplicate TNF- ELISA wells.

The marker compounds commonly standardized for in Echinacea extracts are phenolic (caffeic) acid derivatives. These compounds have antioxidant and anti-inammatory properties [2527]. Therefore, we compared different Echinacea raw materials and nished products for free radical scavenging potential, and for inhibition of arachidonic acid conversion to prostaglandins. As can be seen from Table 3 and Table 4, the standardized extracts of Echinacea were superior to E. purpurea herb powder for these functional activities. However, the antioxidant activity from different products containing standardized Echinacea (3 4% phenolics) was highly variable, with EC50 values ranging from 23 to 137 g/mL (Table 3). These differences could arise from extract preparation, specicity and sensitivity of analytical testing methodology, age and storage conditions of the extract at time of manufacture, as well as product form, tablet, two-piece hardshell gelatin capsule, or soft-gelatin capsule.

DISCUSSION
In this report we have demonstrated that Echinacea purpurea whole herb and root powders can act as innate immune stimulators based upon macrophage activation and enhancement of human PBMC viability. The capacity of Echinacea to produce these immunostimulatory effects was observed after subjecting the test material to a simulated digestion protocol. Dissolution in more typical solvents used for in vitro investigations were not able to extract immunostimulatory activity. Echinacea immunostimulatory activity varied signicantly from lot-to-lot of raw material from the same supplier and may reect growth conditions, time of harvest, milling, and storage conditions. Furthermore, standardized Echinacea extracts as well as puried chemical standards for the production of Echinacea extracts were found to be inactive for
508 Journal of Leukocyte Biology Volume 68, October 2000

Fig. 7. Enhancement of human PBMC viable cell numbers by Echinacea purpurea herb and concanavalin A. (A) Mean responses from three different PBMC donors. (B) Comparison of PBMC viability responses to different Echinacea products after preparation through the simulated digestion protocol and compared at a concentration of 1 g/mL. Echinacea samples 13 represent Echinacea purpurea products consisting of whole herb powders and stimulated macrophage TNF- secretion. Samples 4 6 represent Echinacea products containing standardized extracts (4% phenolic compounds) prepared through simulated digestion and were inactive for macrophage activation. *Statistically different from digested placebo capsule control Echinacea products (P 0.001).

http://www.jleukbio.org

TABLE 3. Free Radical Scavenging Activity of Echinacea Raw Materials and Finished Products With Identied Standardization
Echinacea material tested EC50 (g/mL)
a

Standardized extract Standardized extractb Standardized extractc Echinacea tablet brand Aa Echinacea tablet brand Bb Echinacea capsules brand Cb Echinacea capsules brand Db Echinacea purpurea herb capsules Echinacea purpurea herb capsules Caffeic acid Chlorogenic acid
a b

56.1 18.8 35.3 157.0 79.0 139.0 23.0 144.0 175.0 8.3 5.9

Standardized to contain phenolic acids, alkylamides, and polysaccharides. Standardized to contain a minimum of 3 4% phenolic acids. c Echinacea purpurea pressed juice powder standardized to contain a minimum of 3% phenolic acids. Data represent the calculated EC50 (concentration of Echinacea that quenched 50% of the free radical DPPH).

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 ndings 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 inammatory cytokines such as TNF-, IL-1, and IL-6. It would seem unlikely that Echinacea preparations to be this potent at stimulating inammatory cytokines without the occurrence of signicant 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 Echinaceainduced 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 g/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 identied weak activity against hyaluronidase, an enzyme used by certain bacteria to penetrate tissue [30], antioxidant activity [31, 32], anti-inammatory 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-inammatory activity, consistent with prior ndings with these agents. The design and implementation of in vitro experimental approaches are of central importance to contemporary research and scientic discovery in molecular and cellular biology, pharmacology, and toxicology. However, the true scientic impact of in vitro results depends upon their relevance and efcacy in vivo. The clinical effectiveness of Echinacea preparations for treatment (provide faster resolution), prevention, or alleviation of symptoms associated with colds and u are inconclusive. For example, Hoheisel et al. [4] found that Echinacea pressed juice signicantly reduced the severity of colds, whereas Grimm et al. [7], using the same formulation, found no statistically benecial effect. In addition, two recently reported studies using different Echinacea root extracts showed no signicant benet [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 reviewers 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 benet [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 prole 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 benet relating to the pharmacological activity of the test materials. Results demonstrating activation of macrophages and NK cells using Echinacea purpurea herb powder and polysaccharides puried 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

TABLE 4. Comparison of Echinacea purpurea Herb to a Standardized Echinacea Extract (4% Phenolic Acids) Anti-inammatory Activity Measured by Inhibition of Prostaglandin Production
Percent inhibition of PGE2 synthesis

Test material

Standardized Echinacea extract Echinacea purpurea herb Indomethacin (0.1 g/mL)

40.6 0.5 89.7

Samples were subjected to simulated digestion and tested at a concentration of 20 g/mL. Standard deviation ( n 3) was 3.0%.

Rininger et al. Characterization of Echinacea immunopharmacology

509

vivo for TNF-, IL-1, IL-6, and the immunomodulator imiquimod [2224, 37 40]. This is further supported by animal studies in which in vitro-characterized immunostimulatory Echinacea materials were proven efcacious 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 Echinaceas 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 nonspecic immune cells and responses to pathogen challenge. This work will resolve into human clinical trials employing Echinacea with known pharmacological activities.

17.

18.

19.

20.

21.

22.

REFERENCES
1. Coeugniet, E. G., Elek, E. (1987) Immunomodulation with Viscum album and Echinacea purpurea extracts. Onkologie 10, 2733. 2. Melchart, D., Linde, K., Worku, F., et al. (1995) Results of ve randomized studies on the immunomodulatory activity of preparations of Echinacea. J. Altern. Complement. Med. 1, 145160. 3. Scaglione, F., Lund, B. (1995) Efcacy in the treatment of the common cold of a preparation containing an Echinacea extract. Int. J. Immunother. 11, 163166. 4. Hoheisel, O., Sandberg, M., Bertram, S., Bulitta, M., Schaffer, M. (1997) Echinagard treatment shortens the course of the common cold: a doubleblind, placebo-controlled clinical trial. Eur. J. Clin. Res. 9, 261268. 5. Elsasser-Belle, U., Willenbacher, W., Bartsch, H. H., Gallati, H., SchulteMonting, J., von Kleist, S. (1996) Cytokine production in leukocyte cultures during therapy with Echinacea extract. J. Clin. Lab. Anal. 10, 441 445. 6. Melchart, D., Walther, E., Linde, K., Brandmaier, R., Lersch, C. (1998) Echinacea root extracts for the prevention of upper respiratory tract infections. Arch. Fam. Med. 7, 541545. 7. Grimm, W., Muller, H-H. (1999) A randomized controlled trial of the effect of uid extract of Echinacea purpurea on the incidence and severity of colds and respiratory infections. Am. J. Med. 106, 138 143. 8. Lersch, C., Zeuner, M., Bauer, A., et al. (1992) Nonspecic immunostimulation with low doses of cyclophosphamide (LDCY), thymostimulin, and Echinacea purpurea extracts (Echinacin) in patients with far advanced colorectal cancers: preliminary results. Cancer Invest. 10, 343348. 9. Mengs, U., Clare, C. B., Poiley, J. A. (1991) Toxicity of Echinacea purpurea. Acute, subacute and genotoxicity studies. Drug Res. 41, 1076 1081. 10. See, D. M., Broumand, N., Sahl, L., Tilles, J. G. (1997) In vitro effects of Echinacea and ginseng on natural killer and antibody-dependent cell cytotoxicity in healthy subjects and chronic fatigue syndrome or acquired immunodeciency syndrome patients. Immunopharmacology 35, 229 235. 11. Roesler, J., Emmendorffer, A., Steinmuller, C., Luettig, B., Wagner, H., Lohmann-Matthes, M. L. (1991) Application of puried polysaccharides from cell cultures of the plant Echinacea purpurea to test subjects mediates activation of the phagocyte system. Int. J. Immunopharmacol. 13, 931941. 12. Wagner, H., Stuppner, H., Schafer, W., Zenk, M. (1988) Immunologically active polysaccharides of Echinacea purpurea cell cultures. Phytochemistry 27, 119 126. 13. Stimpel, M., Proksch, A., Wagner, H., Lohmann-Matthes, M. L. (1984) Macrophage activation and induction of macrophage cytotoxicity by puried polysaccharide fractions from the plant Echinacea purpurea. Infect. Immun. 46, 845 849. 14. Luettig, B., Steinmuller, C., Gifford, G. E., Wagner, H., Lohmann-Matthes, M. L. (1989) Macrophage activation by the polysaccharide arabinogalactan isolated from plant cell cultures of Echinacea purpurea. J. Natl. Cancer Inst. 81, 669 675. 15. Roesler, J., Steinmuller, C., Kiderlein, A., Emmendorffer, A., Wagner, H., Lohmann-Matthes, M. L. (1991) Application of puried polysaccharides from cell cultures of the plant Echinacea purpurea to mice mediates protection against systemic infections with Listeria monocytogenes and Candida albicans. Int. J. Immunopharmacol. 13, 2737. 16. Steinmuller, C., Roesler, J., Grottrup, E., Franke, G., Wagner, H., Lohmann-Matthes, M. L. (1993) Polysaccharides isolated from plant cell

23. 24.

25.

26. 27.

28.

29.

30.

31.

32.

33.

34.

35.

36. 37. 38.

39.

40.

cultures of Echinacea purpurea enhance resistance of immunosuppressed mice against systemic infections with Candida albicans and Listeria monocytogenes. Int. J. Immunopharmacol. 15, 605 614. Joyeux, M., Lobstein, A., Anton, R., Mortier, F. (1995) Comparitive antilipoperoxidant, antinecrotic and scavenging potencies of terpenes and biavones from Ginkgo and some avonoids. Planta Med. 61, 126 129. Burger, R. A., Torres, A. R., Warren, R. P., Caldwell, V. D., Hughes, B. G. (1997) Echinacea-induced cytokine production by human macrophages. Int. J. Immunopharmacol. 19, 371379. Riese, J., Hoff, T., Nordhoff, A., Dewitt, D. L., Resch, K., Kaever, V. (1994) Transient expression of prostaglandin endoperoxide synthase-2 during mouse macrophage activation. J. Leukoc. Biol. 55, 476 482. Xia, Y., Zweier, J. L. (1997) Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc. Natl. Acad. Sci. USA 94, 6954 6958. Sakai, S., Ochiai, H., Kawamata, H., et al. (1997) Contribution of tumor necrosis factor and interleukin-1 on the production of macrophage inammatory protein-2 in response to respiratory syncytial virus infection in a murine macrophage cell line, RAW264.7. J. Med. Virol. 53, 145149. Ranges, G. E., Zlotnick, A., Esperik, T., Dinarello, C. A., Cerami, A., Pallandino, M. A. Jr. (1988) Tumor necrosis factor /cachectin is a growth factor for thymocytes: synergistic interactions with other cytokines. J. Exp. Med. 167, 14721478. Billiau, A. (1986) BSF-2 is not just a differentiation factor. Nature 324, 415. Ghiara, P., Boraschi, D., Nencioni, L., Ghezzi, P., Tagliabue, A. (1987) Enhancement of in vivo immune response by tumor necrosis factor. J. Immunol. 139, 3676 3679. Exon, J. H., Magnuson, B. A., South, E. H., Hendrix, K. (1998) Effect of dietary chlorogenic acid on multiple immune functions and formation of abberrant crypt foci in rats. J. Toxicol. Environ. Health 53, 375384. Hirazawa, H., Okada, T. (1995) Antioxidant substances in coffee extracts. 16th International Scientic Colloquium on Coffee 1, 240 248. Fernandez, M. A., Saenz, M. T., Garcia, M. D. (1998) Anti-inammatory activity in rats and mice of phenolic acids isolated from Scrophularia frutescens. J. Pharm. Pharmacol. 50, 11831186. Yokota, S., Geppert, T. D., Lipsky, P. (1988) Enhancement of antigen- and mitogen-induced human T lymphocyte proliferation by tumor necrosis factor-. J. Immunol. 140, 531536. Scheurich, P., Thoma, B., Ucer, U., Pzenmaier, K. (1987) Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)-: induction of TNF receptors on human T cells and TNF--mediated enhancement of T cell responses. J. Immunol. 138, 1786 1790. Facino, R. M., Carini, M., Aldini, G., et al. (1993) Direct characterization of caffeoyl esters with antihyaluronidase activity in crude extracts from Echinacea angustifolia roots by fast atom bombardment tandem mass spectrometry. Farmaco 48, 14471461. Facino, R. M., Carini, M., Aldini, G., Saibene, L., Pietta, P., Mauri, P. (1995) Echinacoside and caffeoyl conjugates protect collagen from freeradical-induced degradation: a potential use of Echinacea extracts in prevention of skin photodamage. Planta Med. 61, 510 514. Kim, S., Lee, C., Kang, S., Jung, H., Choi, J. (1997) Chlorogenic acid, an antioxidant principle from the aerial parts of Artemisia iwayomogi that acts on 1,1-diphenyl-2-picrylhydrazyl radical. Arch. Pharmacol. Res. 20, 148 154. Robinson Jr., W. E., Reinecke, M. G., Abdel-Malek, S., Jia, Q., Chow, S. A. (1996) Inhibitors of HIV-1 replication that inhibit HIV integrase. Proc. Natl. Acad. Sci. USA 93, 6326 6331. Robinson Jr., W. E., Cordiero, M., Abdel-Malek, S., et al. (1996) Dicaffeoylquinic acid inhibitors of human immunodeciency virus integrase: inhibition of the core catalytic domain of human immunodeciency virus integrase. Mol. Pharmacol. 50, 846 855. Melchart, D., Linde, K., Worku, F., Bauer, R., Wagner, H. (1994) Immunomodulation with EchinaceaA systemic review of controlled clinical trials. Phytomed. 1, 245254. Barrett, B., Mohnmann, M., Calabrese, C. (1999) Echinacea for upper respiratory infection. J. Fam. Pract. 48, 628 635. Wong, G. H. W., Goeddel, D. V. (1986) Tumor necrosis factors and inhibit virus replication and synergize with interferons. Nature 323, 819 822. DeChiara, T. M., Young, D., Semionow, R., et al. (1986) Structure-function analysis of murine interleukin 1: biologically active polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of a 270-amino acid precursor. Proc. Natl. Acad. Sci. USA 83, 8303 8307. Reiter, M. J., Testerman, T. L., Miller, R. L., Weeks, C. E., Tomai, M. A. (1994) Cytokine induction in mice by the immunomodulator imiquimod. J. Leukoc. Biol. 55, 234 240. Delno, D., DAdamio, F., Migliorati, G., Riccardi, C. (1991) Growth of murine natural killer cells from bone marrow in vitro: role of TNF- and IFN-. Int. J. Immunopharmacol. 13, 943954.

510

Journal of Leukocyte Biology Volume 68, October 2000

http://www.jleukbio.org