**5.2 Feverfew PFE inhibits cellular inflammation**

UV irradiation has been shown to induce the release of various inflammatory cytokines such as IL-1α, IL-6, TNF-α, that are involved in the pathophysiology of UV-induced inflammation (Aubin, 2003). Inflammation has been linked to epithelial skin tumors, and antiinflammatory drugs are being studied for the prevention and treatment of non-melanoma skin cancers (Mueller, 2006).

Feverfew PFE can reduce the release of pro-inflammatory mediators through inhibition of enzymes involved in production and regulation of inflammation. Feverfew PFE directly inhibited the activity of 5-lipoxygenase (5-LOX), phosphodiesterase-3 (PDE3) and phosphodiesterase-4 (PDE4) with IC50 values 11.8 ± 4.8 μg/ ml, 35.2 ± 12.3 μg/ml and 20.8 ± 9.4 μg/ml respectively (Martin et al., 2008).

Feverfew PFE also reduced PGE2 secretion from human skin equivalents and inhibited p38 MAP kinase activation in vitro (Martin et al., 2005). Feverfew PFE had no direct effect on COX-2, this indicates the mechanism of inhibiting PGE2 formation may be upstream to COX-2. Human skin equivalents were pretreated with Feverfew PFE and then thoroughly washed prior to UV exposure. In the absence of treatment, UV irradiation induced inflammatory cytokine release. Pretreatment with Feverfew PFE significantly reduced UVinduced cytokine release by more than 60% over placebo treated control skin equivalents.

Topical application of Feverfew PFE was examined in an investigator blinded, placebocontrolled clinical study for their effect on UV-induced erythema. Subjects were exposed to UVB irradiation of 0.5 to 1.5 MED (Minimal Erythema Dose), followed by daily applications

The Botanical Extract Feverfew PFE Reduces

**6.1 Antioxidant Response Element (ARE)** 

initiates base excision DNA repair.

**6.2 Transcription factor Nrf2** 

**DNA damage** 

events taking place between UV irradiation and DNA damage.

DNA Damage and Induces DNA Repair Processes 537

PFE decreased UV induced TT dimer formation by nearly 50% compared to UV alone (Martin etal 2008). Thus the antioxidant properties of Feverfew PFE can block the cascade of

In addition to having direct effects on mitigating DNA damage, Feverfew PFE may also aid the DNA repair process via an indirect mechanism, such as induction of the Nrf2/ARE pathway and downstream activation of several genes involved in oxidative stress response.

**6. The NRF2/ARE pathway and its effect on reducing oxidative damage and** 

The antioxidant response element is a *cis*-acting enhancer sequence that mediates transcriptional activation of genes in cells exposed to oxidative stress. It was initially identified in the promoters of the cell detoxification enzymes, GSTA2 (glutathione Stransferase A2) and NQO1 (NADPH: quinone oxidoreductase 1) (Friling et al., 1990; Li and Jaiswal, 1992; Rushmore and Pickett, 1990). The ARE possesses structural and biological features that characterize its unique responsiveness to oxidative stress, and its consensus sequence was identified to be 5'-TGACnnnGC-3' (Rushmore et al., 1991). In addition to being involved in inducible gene expression, the antioxidant response element is also responsible for the low-level basal expression of several genes, and is therefore crucial for maintaining cellular redox homeostasis under a variety of cell conditions. Proteins that are encoded by the ARE include enzymes associated with glutathione biosynthesis (Moinova and Mulcahy, 1998; Wild et al., 1998), redox proteins with active sulfhydryl moieties (Ishii et al., 2000; Kim et al., 2001), and drug-metabolizing enzymes (Favreau and Pickett, 1991; Rushmore and Pickett, 1990). Several of these proteins have an endogenous role in protecting the cells from oxidative damage, for example, enzymes such as GST, NQO1, and HO-1 (heme oxygenase-1) function to detoxify harmful by-products of oxidative stress. Other phase II enzymes induced by ARE activation include aldehyde dehydrogenase, glutathione peroxidase, glutathione transferases, superoxide dismutase, quinone reductase, epoxide hydrolase, UDP-glucuronosyl transferases, and gamma-glutamylcysteine synthetase, etc. The human 8-oxoguanine DNA glycosylase (OGG1) enzyme has also been shown to contain the binding sites for transcription factor Nrf2 in its promoter region (Dhenaut et al., 2000). Human OGG1 functions to remove 8-oxoG, a mutagenic base byproduct which occurs as a result of exposure to reactive oxygen, from damaged DNA and

Transcription factor Nrf2 (Nuclear factor E2–related factor 2) binds to and induces activation of the ARE. Nrf2 was first isolated by an expression cloning procedure using an oligonucleotide containing the NF-E2 DNA binding motif as a probe to screen for closely related proteins (Moi et al., 1994). Nrf2 belongs to the cap-and-collar family of basic region– leucine zipper transcription factors, and is an essential component of the ARE-mediated transcriptional machinery. It has been shown that Nrf2 mediates both the basal and inducible activity of the ARE, and the loss of Nrf2 results in a profound reduction in the enzyme activities of NQO1 and certain GST isoenzymes (Itoh et al., 1997). These observations also correlate well with the ubiquitous expression of Nrf2 at steady-state levels

of Feverfew PFE. Chromameter, diffused reflectance spectroscopy measurements and independent dermatologist assessment concluded that Feverfew PFE significantly reduced the UV-induced erythema at 24 and 48 hrs after UV exposure (Tierney et al., 2005). This clinical study clearly demonstrates that Feverfew PFE can reduce the skin inflammation and damage resulting from UV exposure.

Fig. 2. Feverfew PFE mitigates UV-induced erythema.
