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

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

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 in nutrition and use for general health purposes.

*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 cited in Talhouk et al., 2008).

Salograviolide A: A Plant-Derived Sesquiterpene

(El-Najjar et al, 2007).

Lactone with Promising Anti-Inflammatory and Anticancer Effects 375

The plant was collected during its flowering season and water extract was obtained from it by decoction. Briefly, the plant was air dried and soaked either entirely or only its ground flower head in hot boiling water for 20-30 min with a ratio of plant material weight to water volume of 1/8 (1 g of plant for every 8 ml of water). The solution was filtered either through 3 mm Whatman filter or through Sterile Gauze sponges 30x30 cm which yielded a residue and a filtrate named the "crude water extract". The resulting aqueous layer was sterilized

*C. ainetensis* water extract is claimed in Lebanese folk literature to have anti-inflammatory effects. However, no research paper prior to 2004 investigating this plant's biological activity had ever been published. A screening of 29 plants reported by traditional medicine practitioners to have anti-inflammatory effects led to the validation of this activity for *C. ainetensis* water extract (Talhouk et al., 2008). Moreover, a screening of 109 wild Lebanese plant extracts, including 41 crude water, 34 methanol and 34 chloroform extracts, has resulted in the identification of selective and anti-proliferative bioactivity against several cancer cell lines in four wild Lebanese plant species namely, *Achillea damascene* also known as *Achillea falcata*, *Centaurea ainetensis*, *Onopordum cynarocephalum* and *Ranunculus myosuroudes* (Table 2). Although, *in vitro*, the three other plant extracts showed higher activities than *C. ainetensis*, the latter extract demonstrated the highest tumor growth inhibition ranging from 73 to 79% when tested *in vivo* in a mouse model of colorectal cancer

It was also shown later, that *C. ainestensis* water extract was mildly toxic but largely

Finally the extract was tested against the Infectious Bursal Disease (Gumboro) Virus (IBDV) in broilers and showed a mild reduction of IBDV viral antigens in the Bursa of Fabricius as

In an attempt to unravel the underlying causes for *C. ainetensis* water extract activity, we isolated the bioactive compound Sal A. The plant was subjected to bio-assay guided fractionation which consisted of testing the anti-tumor, anti-inflammatory and cytotoxicity effects of each fraction of the plant extract. Fractions of the crude water extract were inactive; however, Sal A was obtained from a fractionation of the methanol crude extract. Sal A manifested the same biological activities as the crude water extract with greater efficacy at lower concentrations. A parallel study was conducted to assess the effect of both the water crude extract and methanol crude extract of *C. ainetensis* along with 26 other indigenous Lebanese plants against nine microbial species. The results showed that the crude water extract was inactive against all microbial species whereas the methanol extract

Fig. 3 summarizes the acid-base extraction procedure used to fractionate the methanol crude extract and isolate Sal A. First the methanol crude extract was obtained by soaking the dried

The mixture was then incubated on a shaker for 2 h at 20°C. The extract was filtered and yielded a residue and a filtrate named the "methanol crude extract". For further fractionation, the residue issued from the methanol extraction was soaked in EtOAc mixture in a ratio of 10/1 w/v. It was then separated by filtration into a residue and a filtrate consisting of fat and waxes and numbered I.1. To the crude methanol extract, concentrated H2SO4 solution was then added drop-wise till the pH reached 2. Following, a mixture of CHCl3-H2O (2:1 ratio) was added. The CHCl3 phase enriched with terpenoids and phenols was collected and labeled as I.2. The aqueous layer, on the other hand, was basified to pH 10

inhibited metastasis of leukemic cells (El-Sabban, unpublished findings).

well as a mild reduction in bursal lesions (Barbour, unpublished findings).

was effective against 88.8% of the tested microorganisms (Barbour et al., 2004).

plant flowers in methanol with w/v of 1/10 for 16 h.

using 0.2 µm non-pyrogenic sterile-R filter before storing it at -20°C until use.

(a) Left: Photo of the plant *Centaurea salonitana* of Portuguese origin. Right: The aerial part of the plant

(b) Procedure for extraction of Sal A, C17H20O6

(c) Chemical structure of Sal A.

Fig. 2. Illustration of *Centaurea salonitana*, the procedure of Sal A extraction and its chemical structure.

(a) Left: Photo of the plant *Centaurea salonitana* of Portuguese origin. Right: The aerial part of the plant

Aerial parts of the plant

Methanol extraction

Purification of powder by chromatography techniques

‐ IR ‐ X‐ray ‐ MS ‐ NMR 1H/ 13C

**H**

**15**

‐ TLC ‐ CC Analyticalmethods

‐ MeOH (x3, 24 h, 1 L) ‐ Dissolvation in H2O‐CHCl3 (1:1) ‐ Aqueous layer extraction x3 with CHCl3

**AcO**

Drying Powder

(b) Procedure for extraction of Sal A, C17H20O6

**<sup>10</sup> <sup>9</sup>**

**H OH**

**<sup>7</sup> <sup>6</sup>**

**8**

**Salograviolide A**, C17H20O6

**14**

(c) Chemical structure of Sal A. Fig. 2. Illustration of *Centaurea salonitana*, the procedure of Sal A extraction and its chemical

**O**

**12 13 11**

**OH**

**O**

structure.

The plant was collected during its flowering season and water extract was obtained from it by decoction. Briefly, the plant was air dried and soaked either entirely or only its ground flower head in hot boiling water for 20-30 min with a ratio of plant material weight to water volume of 1/8 (1 g of plant for every 8 ml of water). The solution was filtered either through 3 mm Whatman filter or through Sterile Gauze sponges 30x30 cm which yielded a residue and a filtrate named the "crude water extract". The resulting aqueous layer was sterilized using 0.2 µm non-pyrogenic sterile-R filter before storing it at -20°C until use.

*C. ainetensis* water extract is claimed in Lebanese folk literature to have anti-inflammatory effects. However, no research paper prior to 2004 investigating this plant's biological activity had ever been published. A screening of 29 plants reported by traditional medicine practitioners to have anti-inflammatory effects led to the validation of this activity for *C. ainetensis* water extract (Talhouk et al., 2008). Moreover, a screening of 109 wild Lebanese plant extracts, including 41 crude water, 34 methanol and 34 chloroform extracts, has resulted in the identification of selective and anti-proliferative bioactivity against several cancer cell lines in four wild Lebanese plant species namely, *Achillea damascene* also known as *Achillea falcata*, *Centaurea ainetensis*, *Onopordum cynarocephalum* and *Ranunculus myosuroudes* (Table 2). Although, *in vitro*, the three other plant extracts showed higher activities than *C. ainetensis*, the latter extract demonstrated the highest tumor growth inhibition ranging from 73 to 79% when tested *in vivo* in a mouse model of colorectal cancer (El-Najjar et al, 2007).

It was also shown later, that *C. ainestensis* water extract was mildly toxic but largely inhibited metastasis of leukemic cells (El-Sabban, unpublished findings).

Finally the extract was tested against the Infectious Bursal Disease (Gumboro) Virus (IBDV) in broilers and showed a mild reduction of IBDV viral antigens in the Bursa of Fabricius as well as a mild reduction in bursal lesions (Barbour, unpublished findings).

In an attempt to unravel the underlying causes for *C. ainetensis* water extract activity, we isolated the bioactive compound Sal A. The plant was subjected to bio-assay guided fractionation which consisted of testing the anti-tumor, anti-inflammatory and cytotoxicity effects of each fraction of the plant extract. Fractions of the crude water extract were inactive; however, Sal A was obtained from a fractionation of the methanol crude extract. Sal A manifested the same biological activities as the crude water extract with greater efficacy at lower concentrations. A parallel study was conducted to assess the effect of both the water crude extract and methanol crude extract of *C. ainetensis* along with 26 other indigenous Lebanese plants against nine microbial species. The results showed that the crude water extract was inactive against all microbial species whereas the methanol extract was effective against 88.8% of the tested microorganisms (Barbour et al., 2004).

Fig. 3 summarizes the acid-base extraction procedure used to fractionate the methanol crude extract and isolate Sal A. First the methanol crude extract was obtained by soaking the dried plant flowers in methanol with w/v of 1/10 for 16 h.

The mixture was then incubated on a shaker for 2 h at 20°C. The extract was filtered and yielded a residue and a filtrate named the "methanol crude extract". For further fractionation, the residue issued from the methanol extraction was soaked in EtOAc mixture in a ratio of 10/1 w/v. It was then separated by filtration into a residue and a filtrate consisting of fat and waxes and numbered I.1. To the crude methanol extract, concentrated H2SO4 solution was then added drop-wise till the pH reached 2. Following, a mixture of CHCl3-H2O (2:1 ratio) was added. The CHCl3 phase enriched with terpenoids and phenols was collected and labeled as I.2. The aqueous layer, on the other hand, was basified to pH 10

Salograviolide A: A Plant-Derived Sesquiterpene

bioactivity after it was purified using Solid Phase Extraction.

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

analysis.

its mechanisms of action.

Lactone with Promising Anti-Inflammatory and Anticancer Effects 377

by adding concentrated NH4OH drop-wise and then resuspended in a CHCl3-MeOH mixture (3:1 ratio) to be later separated into two organic and aqueous layers labeled I.3 containing alkaloids and I.4, respectively. I.1, I.2, I.3 and I.4 were evaporated to dryness under reduced pressure and weighed. A known amount of each subfraction was dissolved in a known volume of suitable solvent for further chromatographic and or/bioassays

Fraction "I.2" was the only of three other fractions to exhibit activity against cancer cells and in models of inflammation. Therefore TLC, thick layer chromatography and CC were used to further subdivide the I.2 bioactive fraction and yielded six subfractions I.2.1-I.2.6. Additional testing showed that only subfraction I.2.2 exhibited the anti proliferative, antiinflammatory activity observed with the fraction I.2. This subfraction also maintained its

Finally, UV, IR, NMR and MS enabled the identification of the bioactive compound Sal A. In conclusion, three research papers on *C. ainetensis* water extracts and three on Sal A antiinflammatory and anti-tumor activities have been so far published by Ibsar while a fourth one is in progress (Al-Saghir et al., 2009; Barbour et al., 2004; El-Najjar et al., 2007; Ghantous et al., 2007; Saliba et al., 2009; Talhouk et al., 2008). Prior to our work, there was only one published article on Sal A's biological activity since its isolation (Vajs et al., 1999). Therefore, Ibsar has had a major contribution in identifying Sal A's biological activity and determining

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

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

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

the antimicrobial and antiviral potentials exhibited by *C. ainetensis* water extract.

(MICs) to inhibit the mycelial growth of the respective fungal species.


Table 2. Representation of the four Lebanese plant extracts with anticancer potentials. The following results were obtained for the water extracts of *A. damascene*, *C. ainetensis* and *O. cynarocephalum* and the methanol extract of *R. myosuroudes*. Cell proliferation and cytotoxicity were determined using the CellTiter 96 Non-Radioactive Cell Proliferation Assay and the CytoTox 96 Non-Radioactive Cytotoxicity Assay (both kits from Promega, Madison, WI) according to the manufacturer's suggestions.

\* % = Volume extract/ Volume media

**Growth Inhibition (%)** 

65

85 80 70

60

60 80 50

50

70 50 50

**Cell line**

Breast: Scp2

Breast: Scp2

Breast: Scp2

Skin: PMK SP1 308 PAM212 17

Colon/intestine: HCT-116 HT-29 Mode K

Colon/intestine: HCT-116 HT-29 Mode K

Colon/intestine: HCT-116 HT-29 Mode K Skin: PMK SP1 308 PAM212 17

**Concentration** 

**(%)\*** 

3 3 3

3

3 3 5

0.1

0.5 0.5 1.5

Table 2. Representation of the four Lebanese plant extracts with anticancer potentials. The following results were obtained for the water extracts of *A. damascene*, *C. ainetensis* and *O.* 

*cynarocephalum* and the methanol extract of *R. myosuroudes*. Cell proliferation and cytotoxicity were determined using the CellTiter 96 Non-Radioactive Cell Proliferation Assay and the CytoTox 96 Non-Radioactive Cytotoxicity Assay (both kits from Promega,

**Plant Name in** 

*Centaurea ainetensis* 

*Onopordum cynarocephalum* 

*Ranunculus myosuroudes* 

*Achillea damascene* Akhilia zat al-

**Arabic** 

alf waraqah 0.5

Quanturyun Aynata/ Shawk al-dardar

Aqsun harshafi al ra's

Hawdhan

Madison, WI) according to the manufacturer's suggestions.

\* % = Volume extract/ Volume media

by adding concentrated NH4OH drop-wise and then resuspended in a CHCl3-MeOH mixture (3:1 ratio) to be later separated into two organic and aqueous layers labeled I.3 containing alkaloids and I.4, respectively. I.1, I.2, I.3 and I.4 were evaporated to dryness under reduced pressure and weighed. A known amount of each subfraction was dissolved in a known volume of suitable solvent for further chromatographic and or/bioassays analysis.

Fraction "I.2" was the only of three other fractions to exhibit activity against cancer cells and in models of inflammation. Therefore TLC, thick layer chromatography and CC were used to further subdivide the I.2 bioactive fraction and yielded six subfractions I.2.1-I.2.6. Additional testing showed that only subfraction I.2.2 exhibited the anti proliferative, antiinflammatory activity observed with the fraction I.2. This subfraction also maintained its bioactivity after it was purified using Solid Phase Extraction.

Finally, UV, IR, NMR and MS enabled the identification of the bioactive compound Sal A. In conclusion, three research papers on *C. ainetensis* water extracts and three on Sal A antiinflammatory and anti-tumor activities have been so far published by Ibsar while a fourth one is in progress (Al-Saghir et al., 2009; Barbour et al., 2004; El-Najjar et al., 2007; Ghantous et al., 2007; Saliba et al., 2009; Talhouk et al., 2008). Prior to our work, there was only one published article on Sal A's biological activity since its isolation (Vajs et al., 1999). Therefore, Ibsar has had a major contribution in identifying Sal A's biological activity and determining its mechanisms of action.
