Helena Sestakova and Bohumil Turek

contents

Introduction Protocol

Results and Interpretation References introduction

The plants of the genus Echinacea possess a number of known bioactive properties, including antioxidant and anticarcinogenic effects. The study of parts of plants of genus Echinacea or of their components, in terms of their capacity to influence the immune mechanisms of an organism, is therefore very important for the assessment of protection against various pathogens.

Immunologic studies are generally concerned with the response of an organism to foreign (extraneous) substances entering the body. The basic function of the immune system is to differentiate between "foreign" and "one's own" molecules, and to protect against extraneous proteins. An immune reaction takes the form either of a specific response mediated by T and B cells, or of a nonspecific (natural) response mediated by macrophages, natural killer cells, and polymorpho-nuclear leukocytes (PMNLs). A positive or negative effect on immunity of substances obtained from plants or of nutritional factors is therefore very important for maintaining the integrity of an organism. One of the most important mechanisms underlying the natural defense of an organism is phagocytosis. Quantitative as well as qualitative insufficiency of the system of phagocytosis results, among other things, in an increased sensitivity of an individual to infectious agents. PMNLs are responsible for natural defense, and actively emigrate from the circulation to the site of inflammation in response to a signal in the form of a chemotactic factor (Dahlgren, 1989; Schiffmann and Gallin, 1979; Wilkinson, 1983). In addition to affecting the mobility of phagocytes, chemotactic factors can trigger the oxidative metabolism of these cells, with subsequent formation of oxygen free radicals and the release of lysosomal enzymes (Badwey and Karnovsky, 1980; Dahlgren, 1989; Klebanoff, 1980). PMNLs are activated by various phagocytotic stimuli, including bacteria and allergens, and by carcinogenic substances (Klein et al., 1991). The activation of the PMNL membrane is followed by the so-called burst of oxidative metabolism (respiratory flare-up) usually associated with phagocytosis. Ligands binding to receptors in the cytoplasmic membranes of the phagocytes disturb their structure, activating NADPH oxidases. These oxidases catalyze electron transport from NADPH to oxygen, reducing it to a superoxide radical. This, in turn, is reduced to hydrogen peroxide either spontaneously or through catalysis by superoxide dismutase. The superoxide anion gives rise not only to hydrogen peroxide but also to other cytotoxic forms of oxygen. These forms of oxygen are not dependent on the fusion of a phagosome with a lysosome in the phagocyte. However, once this fusion occurs, the enzyme myeloperoxidase can enter the phagolysosome, forming, together with hydrogen peroxide and the halide cofactor (Cl-, I-), one of the most potent microbicidal systems of PMNLs (De Chatelet et al., 1982; Thomas et al., 1988).

While monitoring chemiluminescent activity, we were mainly interested in the possibility of influencing the endogenic induction of free oxygen radicals, as well as the possibility of its application in radical chain reactions and oxidative processes in cell membranes and organelles. The "interface" between pro-oxidant and antioxidant processes is controversial and speculative. A significant role is played by the actual state of antioxidant activity as well as by interactions with other substances, when even antioxidants can, under certain circumstances, act in a pro-oxidant manner, which results in a significant change of their biological activity.

Extracts from various parts of the plants of genus Echinacea (E. purpurea, E. angustifolia, E. pallida) have become known primarily for their capability to strengthen the activity of an unspecified part of the immune system. North American Indians used these plants to treat febrile conditions and open wounds as well as insect or snake bites (Bauer, 1994). It has also been shown that an extract from the fresh plant, its top and root, acts as an immunostimulant when used in conditions such as the common cold, inflammatory processes, and malignant growths. The genus Echinacea contains substances similar in composition and character of effect. Pronounced immunostimulant, antibacterial, and virostatic effects have been associated primarily with polysaccharides, glycopro-teins, alkamides, echinacoside (a glycoside with a pronounced analgesic effect), and caffeic acid derivatives (cichoric acid) (Bauer, 1996; Facino et al., 1995). The phagocytic activity PMNL in healthy volunteers was significantly enhanced by the alcoholic extract of E. purpurea radix (Mel-chart et al., 1995). The antiinflammatory effect is due to alkamides that inhibit the metabolism of the arachidonic acid (Muller-Jakic et al., 1994). The polysaccharide fraction increases the production of the "tumor necrosis factor" (TNF-a) and the induction of interleukins IL-1 and IL-6 (Roesler et al., 1991). On the German market, about 300 preparations containing Echinacea exist at present, indicated for use, for example, in atopic eczema, injuries, burns, and infections, as well as in polyarthritis and psoriasis. Most importantly, these preparations are recommended to strengthen the defensive capabilities, that is, immunity, of the organism (Bauer, 1994).

In our work, we tested Echinacea preparations using the chemiluminescence method to measure the activity of stimulated granulocytes. It is a dynamic test that demonstrates the formation of microbicidal substances in the phagocytes and evaluates their function. Specific surfaces on phagocytes form the first defense barrier against various pathological conditions of the macroorganism.

protocol

Test animals were female mice (6 weeks old), weighing 20 to 22 g, of the Balb/c strain (Biotest, Konarovice, Czech Republic). A commercial Echinacea product (distributed by Profitness, Ontario, Canada), consisting of the dried root and leaf of plants of several species, were dissolved into fine gelatinous matter in redistilled water, and applied by lavage. Echinacea was administered to the mice in daily doses of 83 mg/kg. Polymorphonuclear leukocytes were obtained from the peritoneum of six mice per group, 4 hours after giving 5 ml of glycogen by intraperitoneal injection. A veronal buffer at pH 7.3 containing two units of heparin per milliliter was used for washing out the peritoneum. The obtained cells were washed in the veronal buffer twice without heparin by centrifugation for 10 minutes at 300 g. The final concentration of cells was adjusted to 5.75 x 106 per ml in the veronal buffer without heparin. Each of four cm2 polystyrene tubes contained a blended mixture of 0.4 ml of veronal buffer, 0.1 ml of dilute luminol, 0.4 ml of cell suspension, and 0.1 ml of 1% zymosan as stimulant (0.1 ml of veronal buffer replace zymosan in controls). Chemi-luminescence activity was measured at room temperature at 5-minute intervals over a period of 90 minutes in an analytical luminometer.

results and interpretation

In the first experiment (Figure 11.1), Echinacea was administered for 5 days and we began with the chemiluminescence investigation on the third day after termination. In the course of the following 4 days of testing we observed chemiluminescence values to be on the average 1.2 times greater in the group of mice treated with Echinacea compared with the control group. These values remained relatively consistent even on day 6 after termination of the Echinacea treatment. At each sampling interval, there was a statistically significant difference between the treatment and control groups of mice (third, fifth, sixth day p < 0.01; fourth day, p < 0.05).

In the second experiment (Figure 11.2), Echinacea was administered continuously for 16 days. The mice were tested daily for 5 days between Days 12 and 16. Chemiluminescence activity was an average 1.7 times higher (Day 16) than in the control group. After termination of Echinacea (Day 16) Days 19 and 22 after beginning treatment, the chemiluminescence values dropped to the levels similar to those found in Figure 11.1. All chemiluminescence values in the treatment group (Figure 11.2) were significantly higher (p < 0.01) than in the control group.

Chemiluminescence is an ideal test for monitoring the formation of free oxygen radicals in PMNL. On the one hand, the capability of PMNL to luminesce differs during inflammatory reactions and phagocytosis in response to bacteria, but on the other hand, it also reflects an increased risk, occurring with an overabundance of free radicals observed particularly in cases of insufficient antioxidant defense. Induction of oxygen radicals is relevant in relation to both the atherogenic and oncogenic processes. In the final stages of oncogenesis, the molecular switch is made that determines whether a cancer cell will continue its progression toward a tumor or, instead, destroy itself (apoptosis). The latter event involves cells of the immune system.

The administration of an extract from E. purpurea was followed by increased phagocytosis of Candida albicans by granulocytes and monocytes in healthy subjects as well as by an increase in

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