Introduction and immunostimulant activity have been confirmed (Bauer and Wagner, 1991; Hobbs, 1989). Formulations of Echinacea are found in salves, tinctures, capsules, or teas (Foster and Duke, 1990). Active ingredients include cichoric acid, echinaceine, echinolone, and echinacoside.

Several members of Echinacea are endangered species; thus, collection of plants for research and extraction of pharmaceuticals is not allowed, or at best restricted (Murdock, Southeast Region, U.S. Fish and Wildlife Service, Georgia, 1994, personal communication). Successful in vitro culture protocols have been established for E. purpurea (Coker, 1999; Coker and Camper, 2000).

Assessment of plant extracts or commercial products to verify folklore, anecdotal, or other types of information (ethnobotanical, observations, or serendipity) requires some type of initial screening followed by clinical studies. Detection of biologically active components in a medicinal plant extract by carefully designed screens or bioassays is an effective strategic plan to verify reported or claimed activity or traditional use. Screening bioassays must meet several criteria; they must be rapid, convenient, reliable, inexpensive, sensitive, require little material, and be able to identify a broad spectrum of activities. These criteria were recently verified for an antitumor bioassay, the potato tumor induction assay, and the assay would detect chemicals that disrupted the cell cycle at any point (Coker et al., 2003). Bioassays can also be used to direct extract fractionation that may lead to identification of active ingredients in a crude extract that exhibits specific biological activity. Additionally, bioassay results can identify extracts, or fractions thereof, that should be included in clinical studies.

Bioassay tests can provide valuable information about a plant extract or fraction and its biological activity. While bioassays do not deal specifically with the interactions between the organism and the extract or drug, a modification within the bioassay can assess the biotransformation and its subsequent effect on biological activity. This will provide some information about whether the organism will transform the extract or drug rendering it inactive biologically or converting it to a more active chemical form. In studies described herein, an additional treatment used a human microsomal fraction, which was rich in cytochrome P450 enzyme activity. This enzyme activity is found most abundantly in the liver, but is also found in small amounts in other body tissues. Cytochrome P450 enzymes are involved in biotransformation of drugs and other compounds in the body (detoxification and metabolism). Cytochrome P450 oxidative reactions result in a more water-soluble chemical, thus facilitating elimination from the body (Cupp and Tracey, 1998). Another potential result is transforming a chemical from a toxic form to a nontoxic form, or to convert a chemical from a tumor inducer to a tumor inhibitor. Thus, inclusion of a microsomal fraction treatment in the bioassays reported herein was intended to simulate passage of the plant extract through the body. Bioassays used in these studies were classified as "bench-top" bioassays that did not involve live animals or human subjects.

Selected bench-top bioassays used in studies with purple coneflower are discussed below. The discussion focuses on the type of information that can be gained, and how it might lead to further bioassay-directed fraction and clinical studies. Results obtained with various purple coneflower extracts and commercially available products are summarized.

antimicrobial activity

Extracts from purple coneflower are reported to have antimicrobial properties, as well as antiviral and immune-stimulating properties. Antimicrobial activity has been attributed to two chemical families, the polysaccharides and alkyl amides. Extracts may be used topically, orally, intravenously, or intramuscularly, and have been tested in Europe against upper respiratory tract diseases, wounds, urinary tract infections, Herpes simplex virus, and influenza. However, these tests were not all performed with sufficient quality control to merit acceptance in the U.S. (Hobbs, 1990).

The Kirby-Bauer sensitivity test (diagrammatically illustrated in Figure 13.1) was used to test various purple coneflower fractions and extracts with several different bacteria. Filter paper discs

Plant extract or drug

Plant extract or drug

Zone of inhibition

Black = bacterial growth

FIGURE 13.1 Schematic drawing of a Kirby-Bauer sensitivity test. Filter paper discs shown in grey; zones of inhibition are in white.

Zone of inhibition

Black = bacterial growth

FIGURE 13.1 Schematic drawing of a Kirby-Bauer sensitivity test. Filter paper discs shown in grey; zones of inhibition are in white.

are saturated with the test material and placed on an agar medium inoculated with the bacterial suspension. Clear zones around the discs indicate that as the test material diffuses from the disc, bacterial growth is inhibited (zone of inhibition). The presence of no clear zones around the discs indicates no inhibitory response. Measurement of the inhibition zone provides quantitative data enabling evaluation of test sample efficacy; for example, the larger the zone of inhibition, the more inhibitory or active the test sample.

Experimental Protocol

Samples tested included the following:

Three organic solvent fractions (n-butanol, methanol, and hexane) of bulk plant material (consisting of roots, stems, leaves, and flower tops purchased locally) A crude tea prepared from steeping bulk plant material in water for 15 minutes Four commercial products:

1. A tincture containing E. angustifolia and E. purpurea root (designated EC-1)

2. An alcohol-free sample in glycerine containing E. angustifolia root (designated EC-2)

3. A tincture containing E. angustifolia root, E. purpurea root, flower head, and seed, Hydrastis canadensis root, Berberis aquifolium root, Berberis spp. bark, Hypericum perforatum buds, and propolis extract (designated EC-3)

4. A tincture containing E. angustifolia root and E. purpurea root, flower head, and seed (designated EC-4)

Bacteria tested included Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pyogenes. Filter paper discs were saturated with each test sample and placed on plates inoculated with one of the test bacteria. For quality control, antimicrobial susceptibility test discs (QC discs) were obtained from Becton Dickinson. These disks were impregnated with drugs currently in use at specific concentrations, and were accompanied with expected susceptibility results for each organism tested (Figure 13.2).

FIGURE 13.2 Kirby-Bauer sensitivity. Petri dish inoculated with S. pyogenes shows zones of inhibition by commercially extracted E. purpurea products (left). Petri dish inoculated with S. pyogenes shows zones of inhibition around Difco Sensi-disk control drugs (right).

Results and Discussion

Bacterial suspensions were standardized (absorbance versus colony-forming units, an indication of bacterial growth) in order to establish an inoculum, which is critical for quality control of diffusion disc analysis. No effect of the commercial products on E. coli was observed; however, three of the commercial products were active against S. pyogenes (Table 13.1). For EC-1 (tincture of E. angus-tifolia and E. purpurea root tissue) and EC-4 (tincture of E. angustifolia and E. purpurea root, flower head, and seed), the inhibition zones (activity) averaged 14.4 mm and 13.4 mm, respectively, which is comparable to the activity of the prescription drug sulfizoxazole (Table 13.1). The inhi-

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