Notes: Echinacea samples tested included a tincture mixture containing E. angustifolia and E. purpurea root (EC-1); glycerine extract containing E. angustifolia root (EC-2); tincture containing E. angustifolia root, E. purpurea root, flower head, and seed, Hydrastis canadensis root, Berberis aquilolium root, Berberis spp. bark, Hypericum perforatum buds, and propolis extract (EC-3); and tincture containing E. angustifolia root and E. purpurea root, flower head, and seed (EC-4).

a Zone of inhibition in mm.

bition zone for sample EC-3 (tincture mixture) was 6 mm, one half that of sulfizoxazole. QC disc results were consistent with the expected values (Table 13.1). A minimal amount of direct antimicrobial action was observed for EC-3 against S. pyogenes. EC-1 and EC-4 produced activity against S. pyogenes comparable to that of the prescription drug sulfizoxazole. No direct antimicrobial activity against E. coli or S. pyogenes was observed for any organic solvent fraction or the crude aqueous extract (tea) from bulk material tested.

antineoplastic activity: potato tumor induction assay

The potato tumor induction assay measures the ability of an extract or chemical to inhibit tumor formation. This assay uses Agrobacterium tumefaciens as a tumor initiator; the bacterium is a gramnegative rod and is the causative agent of crown gall disease in plants (Agrios, 1997; Anand and Heberlein, 1977; Lippincott and Lippincott, 1975). Crown gall disease causes a mass of tissue (callus) bulging from stems and roots of woody and herbaceous plants. These masses (tumors) may be spongy or hard, and may or may not cause a deleterious effect on the plant.

During infection of plant material with A. tumefaciens, a tumor-producing plasmid (Ti-plasmid) found in the bacterial DNA is incorporated into the plant's chromosomal DNA. When plant tissue is wounded, it releases phenols and other chemicals that stimulate the Ti-plasmid. The Ti-plasmid causes the plant's cells to multiply rapidly without going through apoptosis, resulting in the formation of tumors that are similar in nucleic acid content and histology to human and animal cancers (Agrios, 1997). Tumorigenesis in plants and animals involves similar mechanisms and common nucleic acid components (Agrios, 1997; Ferrigini et al., 1982). The tumor tissue in plants is known as callus tissue and may eventually differentiate into vascular tissue, just as animal tumors will mutate to produce blood vessels.

The potato tumor induction assay may identify agents that damage or stop the synthesis of DNA, preventing cellular division. It may identify compounds that stop mitosis, also preventing cellular division, thereby halting tumor growth. For example, etoposide, a semisynthetic derived from podophyllin, directly damages the DNA in the cell nucleus; vincristine and vinblastine are active in blocking the synthesis of the spindle in mitosis, where paclitaxel is active in blocking disassembly of the mitotic spindle (Riley, 1999).

Ferrigni et al. (1982) used the potato tumor induction assay to determine possible antitumor activity of several plant extracts (e.g., members of the Euphorbiaceae) with A. tumefaciens as the tumor initiator. In 1993, McLaughlin et al. used the potato tumor disc assay to evaluate several other plant extracts. In both of these studies, the potato tumor induction assay was compared to the 3PS in vivo tumor assay, the standard test for new antitumor agents. In the 3PS in vivo tumor assay, leukemic mice are treated with possible antitumor agents (McLaughlin et al., 1991, 1993). Life span differences of the leukemic mice compared to healthy mice are used as a measure of antitumor activity. A major problem in using the 3PS in vivo tumor assay is that high concentrations of antitumor agent often prove fatal to the subjects. The potato tumor induction assay eliminates this problem (Ferrigni et al., 1982; McLaughlin et al., 1991, 1993).

This assay is sensitive to the promotion and progression stages of carcinogenesis. Stage 1 (initiation stage) involves a mutation in a single cell that leads to increased proliferation. Stage 2 (promotion) involves reversible growth stimulation and requires promoting factors that are not carcinogenic themselves, but cause abnormal cell proliferation. This stage may be reversed if promoting factors are removed. Stage 3 (progression) involves irreversible growth; cells become immortal and proliferate at an exaggerated rate. Stage 4 involves invasion and metastasis, where cells invade underlying tissue, break off, and move to other areas.

Experimental Protocol

Plant samples consisted of an ethanolic tincture and a glycerol extract that were derived from whole E. purpurea plants, and a capsule derived from roots of E. purpurea and E. angustifolia. These products were purchased from a local health food store.

Discs were cut from disinfested Russet potato cylinders and placed in 24-well culture plates containing water agar. Standardized suspensions of A. tumefaciens were added to the wells; controls included the bacterium alone, camptothecin (a known tumor inhibitor), and solvents with and without the bacterium. After 12 days of incubation at room temperature, the discs were stained with Lugol's reagent (I2KI), which reacts with starch in the disc. The tumors do not react with Lugol's reagent and appear as white- to cream-colored masses against a dark purple or black background, and can be counted using a dissecting microscope.

Results and Discussion

The potato tumor induction assay was used to determine whether Echinacea products inhibit or promote tumor formation. A. tumefaciens alone served as a negative inhibitory control and with camptothecin at 0.1 ppm served as a positive inhibitory control (Figure 13.3). The ethanolic tincture (E1) at 0.1 ppm, 1.0 ppm, and 10 ppm showed no activity compared with Agrobacterium alone (negative inhibitory control), but was significantly different from the camptothecin sample (positive inhibitory control) (Table 13.2). Glycerol extracts (E2) at 0.1 ppm, 1.0 ppm, and 10 ppm were not significantly different from each other, but E2 at 0.1 ppm (13 tumors observed) was significantly different from all concentrations of E1 (18.0 to 18.6 tumors observed) and the negative inhibitory control (11.9 tumors observed). E2 at 1.0 ppm significantly inhibited tumor induction over the negative control, but inhibitory activity was not significant at 10 ppm. The dried root complex (E3) dilutions (0.1 ppm, 1.0 ppm, and 10 ppm) were not significantly different from each other or from the positive inhibitory control, but were significantly different from the negative inhibitory control (11.9 tumors observed on average). As a group, E3 exhibited an average of 14.0 to 15.6 tumors observed, whereas Agrobacterium alone (negative inhibitory control) induced an average 20.4 tumors. All dried root

FIGURE 13.3 Tumors induced by Agrobacterium tumefaciens (transformation control) (left). Tumors appear as white to cream-colored nodules on the surface of the potato disc (dark purple or black in the photo above). No tumors appear when camptothecin was added (positive inhibitory control) (right).
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