Table 123

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Extraction Optimization Experiments of CPT-121 Extraction Procedure for Immune-Stimulating Activity of E. purpurea Whole Herb Powder

Sample Temperature Level Residence Time TNF-a (pg/ml)

1 Low Low 7424

2 Low High 7091

3 High Low 5542

4 High High 5649 E. purpurea Herb Starting Material N/A N/A 979 Control N/A N/A 192

Note: Data represent the mean TNF-a produced (pg/ml) of the cell culture supernatant after 24 hours of respective Echinacea extract stimulation. Each sample was used to treat three replicate wells of macrophage cells and the pooled supernatant run in three replicate ELISA wells.

N/A = not applicable.

(Figure 12.4). The CPT-121 extract contained over 80- and 10-fold less cichoric acid than the phenolic standardized extract and raw plant material, respectively. The production process for CPT-121 also virtually eliminated caftartic acid and reduced chlorogenic acid to undetectable levels (Figure 12.4). In addition, precautions were taken during processing to eliminate the possibility of bacterial growth to ensure that the subsequent immunostimulatory activity was not due to bacterial contamination. This was confirmed from subsequent testing of aerobic and anaerobic bacterial growth assays (Reninger et al., unpublished observations, 2000).

Testing of subsequent production batches of CPT-121 extract consistently demonstrated a similar level of the immunostimulatory potency. Dose-response experiments showed that the stimulatory activity was enhanced approximately 10-fold in comparison to the initial starting herb material (Figure 12.5).

Furthermore, this material was still approximately 20,000-fold less potent than bacterial LPS (Figure 12.5). In human PBMC viability assays, the activity of CPT-121 was found to be optimal at 100 ng/ml, 10-fold lower than the starting E. purpurea herb material. These data verified that the CPT-121 extract also stimulates human immune cells (Table 12.4).

Finally, to provide evidence that the active immunostimulatory constituents of the CPT-121 extracts would be absorbed from oral dosing, differentiated Caco-2 monolayers were employed as a predictive in vitro model of human absorption to determine the apparent permeability (Papp) of the active constituents (Artursson and Karlsson, 1991; Delie and Rubas, 1997; Yee, 1997). The use of the Caco-2 monolayer methodology has become prevalent in the pharmaceutical industry where it is used in combination with analytical detection to select potential lead compounds with good absorption and bioavailability (Taylor et al., 1997). This cell system has also recently been employed to determine the absorption of components of other medicinal herbal extracts (Kamuhabwa et al., 1999; Walgren et al., 1998). Nitric oxide production was quantified as a measure of macrophage activation produced from the extract constituents that permeated the monolayer. A dose-response treatment of RAW264.7 macrophage cells with CPT-121 was performed to estimate the absorption of the CPT-121 extract through differentiated Caco-2 monolayers via nitric oxide production. Utilizing the dose-response relationship, it was determined that approximately 0.9 mg of the CPT-121 extract activity had diffused through the Caco-2 monolayer. A Papp value was then calculated for the CPT-121 extract to be 12.8 x 10-6 (Table 12.5). Based on extensive analyses comparing Papp values derived from Caco-2 cells with drugs with known human absorption, this result would predict that the immunostimulatory constituents of the CPT-121 extract would be well absorbed (> 70%), based on the criteria set forth by Yee (1997).

summary

In this chapter, we provided a review of the scientific evidence from in vitro model systems to support the pharmacological activities of Echinacea. The majority of evidence to date indicates that Echinacea contains subsets of constituents that have antiinflammatory and antioxidant activities and those that are directly immunostimulatory to nonspecific immune cells. In addition, it has highlighted how in vitro bioassays have been applied to develop an Echinacea extract (CPT-121) optimized to immunostimulatory activity.

The design and implementation of in vitro experimental approaches are of central importance to define pharmacological profiles of herbal medicines and provide credible evidence for their efficacy to be assessed in clinical studies. Extracts from other popular herbal medicines including Hypericum perforatum (St. John's wort) and Serenoa repens (saw palmetto) that possess a defined in vitro pharmacology have subsequently been proven to have efficacy in human clinical trials as an antidepressant and for a treatment of benign prostate hyperplasia, respectively (Chatterjee et al., 1998a; Chatterjee et al., 1998b; Iehle et al., 1995; Müller et al., 1998; Plosker and Brogden, 1996;

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