**5. Salograviolide A: Overview of biological activities**

To date, *C. ainetensis* water extract was shown to have antifungal, antibacterial, anti-viral, anticancer and anti-inflammatory activities. Sal A on the other hand, was only shown to have antifungal, anticancer and anti-inflammatory properties. The group of Vajs et al. discovered in 1999 the antifungal activity of Sal A while Ibsar faculty revealed the two others. It is unfortunate that no study has so far assessed whether Sal A is responsible for the antimicrobial and antiviral potentials exhibited by *C. ainetensis* water extract.

Following its isolation from *C. nicolai,* Sal A was tested against seven fungal species: *Aspergillus niger, A. ochraceus, Penicillium ochrochloron, Cladosporium cladosporoides, Fusarium tricinctum, Phomopsis helianthi* and *Trichoderma viride* (Vajs et al., 1999). Each strain was inoculated in the center of a plate with or without Sal A addition to the nutritional agar media. After incubation at 20°C for three weeks, the percentage of fungi inhibition was determined by comparing the diameter of each fungal strain colony inoculated in the presence of Sal A to that of the control inoculated without Sal A. The results showed that Sal A inhibited all the fungi strains except *Trichoderma viride*. Subsequently the use of different concentrations of Sal A enabled the determination of the Minimum Inhibitory Concentration (MICs) to inhibit the mycelial growth of the respective fungal species.

To test for the inflammation potential of *C. ainetensis* water extract, the pro-inflammatory cytokine interleukin-6 (IL-6) was chemically induced in mammary epithelial cells (CID-9 and Scp2) by treatment with endotoxin (ET)). The ability of the extract to reverse or prevent IL-6 production was then assessed and the results demonstrated that *C. ainetensis* water extract inhibited IL-6 in a dose-dependent manner (Talhouk et al., 2008). It was also shown that the extract reversed chemically induced paw edema signs in ET-pretreated Sprague-Dawley rats as well as thermal hyperalgesia in rats subjected to the hot plate test. Sal A was isolated from the water extracts and shown to be responsible for its observed bioactivity. In

Salograviolide A: A Plant-Derived Sesquiterpene

extract's cancer preventive activity.

Lactone with Promising Anti-Inflammatory and Anticancer Effects 379

parallel, the anti-inflammatory property of Sal A was demonstrated in a murine intestinal epithelial cell model (Mode K cells) treated with IL-1 and in *vivo* in a rat model of colonic inflammation induced by rectal injection of iodoacetoamide (Al-Saghir et al., 2009). The results showed that, similarly to *C. ainetensis* water extract, Sal A significantly reduced

*C. ainetensis* water extract and Sal A were also tested against different types of cancers (El-Najjar et al., 2007; Ghantous et al., 2007). *In vitro*, they were both non cytotoxic to normal primary murine keratinocytes while they preferentially inhibited neoplastic benign tumors and squamous cell carcinoma growth in a dose-dependent manner. The selective antiproliferative effects were confirmed in leukemic cells at the metastatically invasive stages as well as in human colon carcinomas. Sal A was also tested in combination with another sesquiterpene lactone, the Iso-seco-tanapartholide (TNP) extracted from *Achillea damascene,* against human colon cancer cell lines (Gali-Muhtasib, unpublished findings). The study demonstrated a synergistic apoptotic effect of both sesquiterpene lactones that failed to induce apoptosis when tested alone at the same low concentrations. Finally *in vivo*, the intraperitoneal water extract injection in Balb/c mice before chemically inducing colon cancer reduced drastically the mean size of aberrant crypt foci which is indicative of the

Natural products derivatives have contributed over the last 25 years to approximately one fourth of the anti-inflammatory drugs used in the clinic (Newman & Cragg, 2007). In 2008, 18 additional natural product derived drugs were being tested at different clinical stages (Harvey, 2008). Terpenoids including sesquiterpene lactones have also reached clinical trials as potential anti-inflammatory agents. For instance, andrographolide; a labdane diterpenoid derived from the plant *Andrographis paniculata* of the Acanthacaea family, reached phase II clinical trials for rheumatoid arthritis. The sesquiterpene lactone parthenolide that made it to phase I cancer clinical trials, has also reached phase II and III clinical trials for the

Drugs with anti-inflammatory activities have common targets and modes of action that lead to the downregulation of the signs of inflammation as well as the chemical mediators controlling the mechanisms of inflammation. Elements of the complement system, angiogenic factors, prostaglandins, cytokines such as interleukins and matrix

Prostaglandins play a role in the modulation of blood flow. They are derived from the arachidonic acid due to the action of two prostaglandins H synthases isoforms known as the cyclooxygenases 1 and 2 (COX-1 and COX-2). COX-1 is constitutively expressed in different tissues while COX-2 is induced by inflammatory stimuli and is therefore thought to be the only isoform involved in propagating the inflammatory response (Larsen & Henson, 1983). IL-6 secreted by the macrophages releases proteinases and elastases which bind to IL-6 receptor and generate signals implicated in humoral inflammation (Heinrich et al., 2003). IL-1, on the other hand, induces the mobilization of the arachidonic acid and its metabolism into prostaglandins thus contributing to cellular inflammation. It was also shown that IL-1 induces the synthesis of COX-2 through the activation of the nuclear factor Kappa B (NF-κB) transcription factor (Larsen & Henson, 1983). NF-κB is a key modulator of inflammation and is implicated in inflammation-induced tumor formation as well (Karin & Greten, 2005). It is

inflammatory cytokines and acted as a preventive agent to reduce inflammation.

**6. Salograviolide A and inflammation: Mechanisms and targets** 

treatment of allergic contact dermatitis (refer to http://www.clinicaltrials.gov/).

metalloproteinases (MMPs) are some of the most common inflammatory mediators.

(a) *Centaurea ainetensis* and its taxonomy

(b) The bioguided fractionation procedure

Fig. 3. Illustration of the indigenous Lebanese plant *Centaurea ainetensis* and the bioguided fractionation procedure that enabled the isolation of Salograviolide A. The plant picture is courtesy of Mr. Khaled Sleem.

(a) *Centaurea ainetensis* and its taxonomy

Solvent extraction

**I.3**

**No activity**

**CHCl3-MeOH extract (Alkaloids) I.3**

> Cancer, Inflammation

**I.2.3 I.2.4 I.2.5 I.2.6**

Cancer, Inflammation

**MeOH extract (Polar extract) I.4**

> Cancer, Inflammation

**Methanol Crude Extract**

Further Fractionation (TLC & C.C)

**No activity No activity**

**CHCl3-H2O extract (Terpenoids and Phenols) I.2**

> Cancer, Inflammation

**Bioactive**

Purification (SPE)

Cancer, Inflammation

**No activity Bioactive No activity**

**Purification and structure elucidation of Bioactive Compound Sal A**

(b) The bioguided fractionation procedure Fig. 3. Illustration of the indigenous Lebanese plant *Centaurea ainetensis* and the bioguided fractionation procedure that enabled the isolation of Salograviolide A. The plant picture is

courtesy of Mr. Khaled Sleem.

Cancer, Inflammation

**I.2.1 I.2.2**

Cancer, Inflammation

**EtOAc extract (Fat, waxes) I.1**

parallel, the anti-inflammatory property of Sal A was demonstrated in a murine intestinal epithelial cell model (Mode K cells) treated with IL-1 and in *vivo* in a rat model of colonic inflammation induced by rectal injection of iodoacetoamide (Al-Saghir et al., 2009). The results showed that, similarly to *C. ainetensis* water extract, Sal A significantly reduced inflammatory cytokines and acted as a preventive agent to reduce inflammation.

*C. ainetensis* water extract and Sal A were also tested against different types of cancers (El-Najjar et al., 2007; Ghantous et al., 2007). *In vitro*, they were both non cytotoxic to normal primary murine keratinocytes while they preferentially inhibited neoplastic benign tumors and squamous cell carcinoma growth in a dose-dependent manner. The selective antiproliferative effects were confirmed in leukemic cells at the metastatically invasive stages as well as in human colon carcinomas. Sal A was also tested in combination with another sesquiterpene lactone, the Iso-seco-tanapartholide (TNP) extracted from *Achillea damascene,* against human colon cancer cell lines (Gali-Muhtasib, unpublished findings). The study demonstrated a synergistic apoptotic effect of both sesquiterpene lactones that failed to induce apoptosis when tested alone at the same low concentrations. Finally *in vivo*, the intraperitoneal water extract injection in Balb/c mice before chemically inducing colon cancer reduced drastically the mean size of aberrant crypt foci which is indicative of the extract's cancer preventive activity.
