**2. Sesquiterpene lactones in traditional and conventional medicine**

Sesquiterpene lactones (SLs) are generally colourless bitter phytochemicals of lipophylic nature. Thousands of molecules are classified in the SLs subfamily of the terpenoids group of plant secondary metabolites. They are predominantly isolated from leaves or flowering heads of plants of the sunflower family Asteraceae and to a limited extent from Umbelliferae and Magnoliaciae (Heywood et al., 1977). The percentage of SLs per plant dry weight often exceeds 1% (Heywood et al., 1977).

The benefits of many plants enriched with SLs have been described in depth in Mediterranean folk literature with emphasis on their laxative values as well as their potential for the treatment of sores, wounds, sprains, fever, pain, headaches, malaria, anaemia, microbial infections, arthritis, cough, bronchitis, diabetes, hypertension and inflammation (Awadallah, 1984; Moukarzel, 1997).

Similarly, scientific literature supports most of the SLs activities attributed to their plants of origin in traditional medicine such as the hypoglycemic (Genta et al., 2010), antibacterial (Bach et al., 2011), antifungal (Vajs et al., 1999), antiplasmodial (Medjroubi et al., 2005), antinociceptive, antipyretic (Akkol et al., 2009) and anti-inflammatory (Al-Saghir et al., 2009) effects.

There are to date around 1500 publications that have reported the anticancer and antiinflammatory properties of SLs. Three major SLs and/or many of their synthetic derivatives have reached phase I-II cancer clinical trials, namely thapsigargin from *Thapsia garganica* (Apiaceae), artemisinin and artesunate from *Artemisia annua*, and parthenolide from *Tanacetum parthenium* (Fig. 1) (reviewed in Ghantous et al., 2010). These SLs have properties that enable them to target tumor cells and cancer stem cells while sparing normal cells. They also affect different cancers or inflammation conditions. For example, thapsigargin demonstrated promising results against advanced solid tumors (breast, kidney and intestine) while parthenolide had an effect on blood and lymph nodes tumors (reviewed in Ghantous et al., 2010). Artemisinin showed efficacy against lupus nephritis, metastatic breast and colorectal cancer while clinical evidence indicated that artesunate is effective against nonsmall cell lung cancer, metastatic uveal melanoma and laryngeal squamous cell carcinoma (Berger et al., 2005; Christensen et al., 2009; Efferth, 2006; Guzman et al., 2007, as cited in Ghantous et al., 2010). The chemical basis for the observed biological activities of SLs has been reviewed by our group recently (Ghantous et al., 2010).

*Centaurea* is one of the largest genera of the Asteracae family with almost 250 species (Font et al., 2008). Plants belonging to the *Centaurea* genus are native to Eurasia. They were

Lebanon falls within the Levantine Uplands center of diversity. Compared to other Mediterranean countries, it stands second after Turkey in its floristic diversity with 2600 plant species distributed around its humble 10452 km2 (Nehmeh, 1977). About 311 plants corresponding to 12% of the total plant species are endemic to Lebanon and have been used in part by Lebanese folk medicine practitioners and Lebanese people for preventive and therapeutic purposes (Nehmeh, 1977). As members of the Nature Conservation Center for Sustainable Futures (IBSAR), we have been leading research over the past 10 years for the understanding and evaluation of Lebanese indigenous plant properties against various conditions, especially cancer. The following chapter aims at retracing our adventure with "Salograviolide A" (Sal A), and bringing to light this peculiar molecule that exhibits both

**2. Sesquiterpene lactones in traditional and conventional medicine** 

Sesquiterpene lactones (SLs) are generally colourless bitter phytochemicals of lipophylic nature. Thousands of molecules are classified in the SLs subfamily of the terpenoids group of plant secondary metabolites. They are predominantly isolated from leaves or flowering heads of plants of the sunflower family Asteraceae and to a limited extent from Umbelliferae and Magnoliaciae (Heywood et al., 1977). The percentage of SLs per plant dry

The benefits of many plants enriched with SLs have been described in depth in Mediterranean folk literature with emphasis on their laxative values as well as their potential for the treatment of sores, wounds, sprains, fever, pain, headaches, malaria, anaemia, microbial infections, arthritis, cough, bronchitis, diabetes, hypertension and

Similarly, scientific literature supports most of the SLs activities attributed to their plants of origin in traditional medicine such as the hypoglycemic (Genta et al., 2010), antibacterial (Bach et al., 2011), antifungal (Vajs et al., 1999), antiplasmodial (Medjroubi et al., 2005), antinociceptive, antipyretic (Akkol et al., 2009) and anti-inflammatory (Al-Saghir et al., 2009)

There are to date around 1500 publications that have reported the anticancer and antiinflammatory properties of SLs. Three major SLs and/or many of their synthetic derivatives have reached phase I-II cancer clinical trials, namely thapsigargin from *Thapsia garganica* (Apiaceae), artemisinin and artesunate from *Artemisia annua*, and parthenolide from *Tanacetum parthenium* (Fig. 1) (reviewed in Ghantous et al., 2010). These SLs have properties that enable them to target tumor cells and cancer stem cells while sparing normal cells. They also affect different cancers or inflammation conditions. For example, thapsigargin demonstrated promising results against advanced solid tumors (breast, kidney and intestine) while parthenolide had an effect on blood and lymph nodes tumors (reviewed in Ghantous et al., 2010). Artemisinin showed efficacy against lupus nephritis, metastatic breast and colorectal cancer while clinical evidence indicated that artesunate is effective against nonsmall cell lung cancer, metastatic uveal melanoma and laryngeal squamous cell carcinoma (Berger et al., 2005; Christensen et al., 2009; Efferth, 2006; Guzman et al., 2007, as cited in Ghantous et al., 2010). The chemical basis for the observed biological activities of

*Centaurea* is one of the largest genera of the Asteracae family with almost 250 species (Font et al., 2008). Plants belonging to the *Centaurea* genus are native to Eurasia. They were

SLs has been reviewed by our group recently (Ghantous et al., 2010).

anti-inflammatory and anticancer effects.

weight often exceeds 1% (Heywood et al., 1977).

inflammation (Awadallah, 1984; Moukarzel, 1997).

effects.

introduced to North America around the late 1800's and can now be found all around the world. *Centaurea* extracts have also been used in traditional medicine for their effects as stimulants, diuretics, analgesics, anti-rheumatics, anti-microbial, anti-diabetics and antiinflammatory (refer to www.ibsar.org). Anecdotally; the genus' name is a dedication to the centaur Chiron who, according to Greek mythology, had discovered the curative properties of these medicinal plants (Nehmeh, 1977). Scientists eventually investigated the medicinal properties traditionally attributed to the *Centaurea* genus and isolated a multitude of SLs in addition to other various types of compounds such as alkaloids, lignans, acetylenes and flavonoids. When entering on PubMed the search terms "Centaurea and sesquiterpene lactones", and adding to them the hits from the search "Centaurea and cancer", 46 results get displayed. We investigated the number, nature and biological activity of SLs in the different species and summarized some of them in Table 1.

Fig. 1. Illustration of the sesquiterpene lactones that have reached clinical trials, namely thapsigargin extracted from *Thapsia garganica*, artemisinin from *Artemisia annua* L., and parthenolide from *Tanacetum parthenium*. The plants pictures are courtesy of Mr. Luigi Rignanese, Mr. Peter Griffee and Mr. Paul Drobot, respectively.

Table 1 indicates that there are at least 89 SLs in 10 *Centaurea* species. As expected, the amount of the SLs as well as their nature is species-specific. Also, the activities of the represented SLs cover most of those attributed to the plants in traditional medicine. Aside

Salograviolide A: A Plant-Derived Sesquiterpene

Lactone with Promising Anti-Inflammatory and Anticancer Effects 373

species *salonitana* from which it was isolated, "gravi" in allusion for the region Gravia and

The first procedure used for the isolation and characterization of Sal A (Fig. 2b) included drying the plant and grinding it to a powder. This was followed by dividing the dry material (600 g) into three parts that were each soaked in 1 l of methanol (MeOH) for 24 h. The extracts were afterwards combined, evaporated (7.5 g) and dissolved in a mixture of equal amounts of water and chloroform (H2O-CHCl3 (1:1)). The aqueous layer was extracted three times with chloroform alone and the final step consisted of evaporating the extract and subjecting it to two separate chromatography techniques for optimal purification, thin layer chromatography (TLC) and column chromatography (CC). Infra Red (IR), high resolution Mass Spectrum (MS) and 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy techniques enabled the identification of the structure of Sal A that was later confirmed by

Sal A is a 3β-acetoxy-8α, 9β-dihydroxy-lαH, 5αH, 6βH, 7αH-guaian-4(15), 10(14), 11(13) trien-6, 12-olide with the molecular formula C17H20O6. It is classified according to its carbocyclic skeleton in the guaianolides group, one of the major groups of SLs. Comprised of 15 carbons (15-C) as indicated by the prefix "sesqui", Sal A has 3 isoprene (5-C) units and a lactone group (cyclic ester) (Fig. 2c). The presence of this α-methylene-γ-lactone is thought to be responsible for the biological activity of Sal A because of its ability to react with

Sal A was subsequently isolated from other *Centaurea* species, namely from *C. nicolai* Bald (Vajs et al., 1999) and from *C. ainetensis* Bois by bioguided fractionation following an

Ibsar, AUB's nature conservation center for sustainable futures, is an interdisciplinary and interfaculty center founded in the year 2002 by AUB faculty. Ibsar's mission is "to promote the conservation and sustainable utilization of biodiversity in arid and Mediterranean regions by providing an open academic platform for innovative research and development", and its vision is "for societies to become guardians and primary beneficiaries of biodiversity

Very early in its establishment, Ibsar recognized that the Lebanese floristic richness also represents an untapped resource for the potential discovery of new therapeutic agents and/or useful dietary supplements. As a result, one of the key program areas in Ibsar has been to integrate traditional knowledge and biotechnology. The objective of this program is to discover useful therapeutic agents that may be hidden in wild Lebanese plants and to develop products attractive to biotechnology industries. Towards this end, plants from the region are collected, extracted and tested for their potential effects on major diseases such as cancer, inflammation, microbial infections, skin diseases and diabetes as well as their value

*C. ainetensis* (Arabic name; Qanturyun Aynata or Shawk al-dardar) whose specimen is deposited at the herbarium of the American University of Beirut (Lebanon), is an endemic plant to Lebanon. It flowers from May to June, has purplish tube of anthers and can only be found growing wild in stony, sterile or bushy places in particular areas in Lebanon, mainly Dayr-ul-Ahmar to Aynata region at elevations of 1200–1800 m above sea level respectively, and in Anti-Lebanon Mountain range above Ayn-Burday at 1250–1300 m (Dinsmore, 1932 as

finally the suffix "olide" indicating the presence of a lactone group.

Rychlewska et al. (1992) using crystal X-ray diffraction techniques.

nucleophiles by a Michael-type addition (Ghantous et al., 2010).

**4. IBSAR efforts for bringing Sal A to the forefront** 

in the region" (www.ibsar.org).

cited in Talhouk et al., 2008).

in nutrition and use for general health purposes.

extraction scheme adopted from Harborne (1998) (Saliba et al., 2009).

from Sal A, several articles have reported anticancer properties of SLs extracted from *Centaurea* species (Bruno et al., 2005; Chicca et al., 2011; Csupor-Löffler et al., 2009; El-Najjar et al., 2007; Ghantous et al., 2007; González et al., 1980; Koukoulista et al., 2002; Saroglou et al., 2005).


1: Bruno et al., 2005 – 2: Nowak et al., 1993– 3: Cho et al., 2004 – 4: Medjroubi et al., 2005– 5: Bach et al., 2011 – 6: Vajs et al., 1999 – 7: Djeddi et al., 2007, 2008 – 8: González et al., 1980 – 9: Ozçelik et al., 2009 – 10: Youssef et al., 1994, 1998 – 11: Akkol et al., 2011 – 12: Cheng et al., 1992 - 13: Gürbüz et al., 2007 – 14: Ozçelik et al., 2009 – 15: Saroglou et al., 2005 – 16: Lakhal et al., 2010 – 17: Fortuna et al., 2001 – 18: Lonergan et al., 1992.

Table 1. Sesquiterpene lactones of different *Centaurea* species and their biological activities.

#### **3. Salograviolide A: Isolation and chemical characterization**

In 1992, Daniewski et al. isolated an unusually hydroxylated SL from the aerial parts of the plant *Centaurea salonitana* Vis. of Bulgarian origin collected in the Greek region Gravia (Fig. 2a). The compound was baptised "Salograviolide A" with the prefix "salo" referring to the

from Sal A, several articles have reported anticancer properties of SLs extracted from *Centaurea* species (Bruno et al., 2005; Chicca et al., 2011; Csupor-Löffler et al., 2009; El-Najjar et al., 2007; Ghantous et al., 2007; González et al., 1980; Koukoulista et al., 2002; Saroglou et

*C. bella* 26 Repin Anti-tumor 1, 2

*C. nicolai* 5 Kandavanolide Antifungal 6

*C. musimomum* 11 Cynaropicrin Antiplasmodial

*C. napifolia* 4 Cnicin Antibacterial

*C. pullata* 10 Melitensin Antibacterial

*C. scoparia* 9 Chlorohyssopifolin Anti-tumor

*C. solstitialis* 7 Solstitialin A Hypoglycemic

*C. spinosa* 10 Malacitanolide Antibacterial

*C. tweediei* 4 Onopordopicrin Antibacterial

**3. Salograviolide A: Isolation and chemical characterization** 

*C. sulphurea* 3 Sulphurein - 16

1: Bruno et al., 2005 – 2: Nowak et al., 1993– 3: Cho et al., 2004 – 4: Medjroubi et al., 2005– 5: Bach et al., 2011 – 6: Vajs et al., 1999 – 7: Djeddi et al., 2007, 2008 – 8: González et al., 1980 – 9: Ozçelik et al., 2009 – 10: Youssef et al., 1994, 1998 – 11: Akkol et al., 2011 – 12: Cheng et al., 1992 - 13: Gürbüz et al., 2007 – 14: Ozçelik et al., 2009 – 15: Saroglou et al., 2005 – 16: Lakhal et al., 2010 – 17: Fortuna et al., 2001 – 18:

Table 1. Sesquiterpene lactones of different *Centaurea* species and their biological activities.

In 1992, Daniewski et al. isolated an unusually hydroxylated SL from the aerial parts of the plant *Centaurea salonitana* Vis. of Bulgarian origin collected in the Greek region Gravia (Fig. 2a). The compound was baptised "Salograviolide A" with the prefix "salo" referring to the

**Example of SLs Activity Reference** 

Cytotoxic

Cytotoxic

Antifungal

Antiviral Antimicrobial

Antiviral Antimicrobial

Cytotoxic

Antifungal Cytotoxic

3, 4

1, 5

7

15

5, 17, 18

8, 9, 10

11, 12, 13, 14

al., 2005).

Lonergan et al., 1992.

**Plant Number of** 

**SLs** 

species *salonitana* from which it was isolated, "gravi" in allusion for the region Gravia and finally the suffix "olide" indicating the presence of a lactone group.

The first procedure used for the isolation and characterization of Sal A (Fig. 2b) included drying the plant and grinding it to a powder. This was followed by dividing the dry material (600 g) into three parts that were each soaked in 1 l of methanol (MeOH) for 24 h. The extracts were afterwards combined, evaporated (7.5 g) and dissolved in a mixture of equal amounts of water and chloroform (H2O-CHCl3 (1:1)). The aqueous layer was extracted three times with chloroform alone and the final step consisted of evaporating the extract and subjecting it to two separate chromatography techniques for optimal purification, thin layer chromatography (TLC) and column chromatography (CC). Infra Red (IR), high resolution Mass Spectrum (MS) and 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy techniques enabled the identification of the structure of Sal A that was later confirmed by Rychlewska et al. (1992) using crystal X-ray diffraction techniques.

Sal A is a 3β-acetoxy-8α, 9β-dihydroxy-lαH, 5αH, 6βH, 7αH-guaian-4(15), 10(14), 11(13) trien-6, 12-olide with the molecular formula C17H20O6. It is classified according to its carbocyclic skeleton in the guaianolides group, one of the major groups of SLs. Comprised of 15 carbons (15-C) as indicated by the prefix "sesqui", Sal A has 3 isoprene (5-C) units and a lactone group (cyclic ester) (Fig. 2c). The presence of this α-methylene-γ-lactone is thought to be responsible for the biological activity of Sal A because of its ability to react with nucleophiles by a Michael-type addition (Ghantous et al., 2010).

Sal A was subsequently isolated from other *Centaurea* species, namely from *C. nicolai* Bald (Vajs et al., 1999) and from *C. ainetensis* Bois by bioguided fractionation following an extraction scheme adopted from Harborne (1998) (Saliba et al., 2009).
