Bioassay-Guided Isolation of Sesquiterpene Coumarins from Ferula narthex Bioss: A New Anticancer Agent

The main objective of cancer management with chemotherapy (anticancer drugs) is to kill the neoplastic (cancerous) cell instead of a normal healthy cell. The bioassay-guided isolation of two new sesquiterpene coumarins (compounds 1 and 2) have been carried out from Ferula narthex collected from Chitral, locally known as “Raw.” Anticancer activity of crude and all fractions have been carried out to prevent carcinogenesis by using MTT assay. The n-hexane fraction showed good activity with an IC50 value of 5.434 ± 0.249 μg/mL, followed by crude MeFn extract 7.317 ± 0.535 μg/mL, and CHCl3 fraction 9.613 ± 0.548 μg/mL. Compounds 1 and 2 were isolated from chloroform fraction. Among tested pure compounds, compound 1 showed good anticancer activity with IC50 value of 14.074 ± 0.414 μg/mL. PASS (Prediction of Activity Spectra) analysis of the compound 1 was carried out, in order to predicts their binding probability with anti-cancer target. As a results the compound 1 showed binding probability with human histone acetyltransferase with Pa (probability to be active) value of 0.303. The compound 1 was docked against human histone acetyltransferase (anti-cancer drug target) by using molecular docking simulations. Molecular docking results showed that compound 1 accommodate well in the anti-cancer drug target. Moreover the activity support cancer chemo preventive activity of different compounds isolated from the genus Ferula, in accordance with the previously reported anticancer activities of the genus.


INTRODUCTION
Ferula narthex Bioss; family Apiaceae, is indigenous to Kandahar, Eastern Persia, Western Afghanistan and Pakistan (Kashmir and Baltistan; Indrayan et al., 2009). In Pakistan it is found in various localities like Gilgit, Chitral (Kamari, Damusar, Chilim, Gudai, Astore, and the hill of Majini Harai), locally it is known as "Raw" in Chitral (Shinwari and Gilani, 2003). Family Apiaceae (Umbelliferae) comprises of 275 genera and 2850 species (Indrayan et al., 2009). F. narthex Bioss. is a well-known carminative, anti-flatulence digestive spice, much essential for Ayurvedic formulation and even for the preparation of many Indian dishes i.e., Asafoetida botanically known as Ferula narthex Bioss (Sengupta et al., 2004). It is an oleogum-resin collected from rhizome and root of the plant. It is considered as very important agent in Indian system of medicine, especially for intestinal flatulence. It is acrid, bitter, stimulant and mostly useful in constipation-gaseous distention (colic pain; Sengupta et al., 2004). Local people used this plant for cough, asthama, toothache, gastric problems and in constipation, angina pectoris. Gum resin of Ferula narthex Bioss. is used in hysteria, treatment of habitual abortion, whooping cough and scorpion sting (Srinivasan, 2005;Anuar et al., 2008;Khan et al., 2011). Extracts and pure compounds from this plant showed anticancer (Saleem et al., 2001), antidiabetic (Iranshahy and Iranshahi, 2011), and anti-fertility effect (Kalita et al., 2011). A large number of active compounds have been isolated from genus Ferula. Mainly sesquiterpene, coumarins, and sulfur containing compounds have been previously reported (Buddrus et al., 1985;Appendino et al., 1993;El-Razek et al., 2003;Bandyopadhyay et al., 2006). Different substances like foods, pharmaceuticals or some cosmetic agents exhibited cytotoxic property. The main objective of cancer management with chemotherapy (anticancer drugs) is to kill the neoplastic (cancerous) cell instead of a normal healthy cell. Different types of cells present in human as part of immune system including natural killers, cytotoxic cells, and lymphokine activated cells are responsible to destroy abnormal and damaged cells (Cano et al., 2011). Any agent having cytotoxic activity can be used in various pathological conditions (inflammation, AIDS, infection, and cancer; Su et al., 2009). The current studies revealed the bioassay-guided isolation of two sesquiterpene coumarins and their anticancer potential against PC3 cells (prostate cancer).

Plant Material
Whole plant of F. narthex was collected from Chitral located in Khyber Pakhtunkhwa, Pakistan, in July 2010, and was identified by the Taxonomy section Department of Botany University of Peshawar. A voucher specimen (BOT 20002) was submitted to the herbarium section of the same department.

Anticancer Assay
Anticancer activity of compounds was evaluated in 96-well flatbottomed micro plates by using the standard reduction of MTT colorimetric assay (Dimas et al., 1998). For this purpose, PC3 cells (prostate cancer) were cultured in DMEM, containing 5% of FBS, 100 µg mL −1 of streptomycin and 100 IU mL −1 of penicillin in 25 cm 3 flasks, in an incubator at 37 • C under a 5% carbon dioxide atmosphere. The exponentially growing cells were counted with a haemocytometer and diluted to a concentration of 1 × 10 5 cells mL −1 . The diluted culture was then introduced into 96well plates (100 µL well −1 ) with various concentrations of the compounds in the range 1-100 µM and incubated overnight. After incubation, medium was separated and 50 µL MTT (2 mg mL −1 ) was added to each well and incubated further for 4 h. Subsequently, 100 µL of DMSO was added to each well. The MTT was reduced to formazan within viable cells and its absorbance was measured at 570 nm using a microplate ELISA reader (Spectra Max plus, Molecular Devices,CA, USA).

Molecular Docking
The molecular docking procedure was widely used to predict the binding interaction of the compounds in the binding pocket of the enzyme. The 3D crystal structure of human histone acetyltransferase was downloaded from Protein Data Bank (PDB ID: 4PZS) (Oikonomakos et al., 2000). All the ions and water molecules were removed and the hydrogen atoms were added to the enzyme by the 3D protonation using the MOE (Molecular Operating Environment) (www.chemcomp.com) software. The target enzyme were then energy minimized by the default parameters of the MOE for the stability and further assessment of the enzyme. The structures of the compounds were built in MOE and energy minimized using the MMFF94x forcefield and gradient: 0.05. The synthesized compounds were docked into the active site of the target enzyme in MOE by the default parameters i.e., Placement: Triangle Matcher, Rescoring: London dG. For each ligand ten conformations were generated. The top-ranked conformation of each compound was used for further analysis.

Statistical Analysis
IC 50 evaluation of only active compounds was done, serial dilutions of test compounds were prepared with different concentrations. A triplicate sample of each concentration was incubated using the standard procedure described. Percentage inhibition for each concentration was calculated. IC 50 valve is evaluated using EZ-FIT, Enzyme kinetics software (Perrella Scientific, Inc., Hillsborough, NH, USA).
IC 50 -value was presented as mean ± S.E.M (standard error of the mean) calculated by using the formula x i − x n = sample data set; x = mean valve of sample data; N = size of data.

Identification of Compounds
Compound 1 was isolated as a white amorphous solid from the chloroform-soluble part of the crude extract of F. Narthex Boiss. Its molecular formula C24H30O4 was deduced with the help of 13C NMR as well as HRESI-MS. Compound 1 (Figure 1) was found to be a sesquiterpene coumarin. The detail spectroscopic data of compound 1 were already reported in our previous article (Bashir et al., 2014). All of the spectral data of compound 2 have molecular formula C24H30O4 was found to be unambiguously matched with the reported data for conferol, previously isolated from Ferula pallida (Su et al., 2000).

Anticancer Activity
Natural organic compounds have extensively used as a new pharmacophore template in drug discovery. The bio-assay guided isolation of new anti-cancer agents (compounds 1 and 2) were performed from F. narthex. In the preliminary step of bio-assay guided isolation the all crude fractions were evaluated for their anti-cancer activity against PC3 cells (prostate cancer). As a result, the n-hexane fraction showed significant activity against PC 3 cancer lines with an IC 50 value of 5.434 ± 0.249 µg/mL, followed by crude MeFn extract 7.317 ± 0.535 µg/mL, and CHCl 3 fraction 9.613 ± 0.548 µg/mL ( Table 1). After fractionation of chloroform, the compounds 1 and 2 were isolated. Both the isolated compounds were also evaluated against PC 3 cancer lines by employing the same protocol that were used for the fractions Among tested pure compounds, only the compound 1 showed good anticancer activity with an IC 50 value of 14.074 ± 0.414 µg/mL. The remaining fractions and compound 2 showed no activity against PC 3 cancer cell lines and the IC 50 values of these were >30 as presented in Table 1.
As reported previously regarding the anticancer potential of Ferula, the oleo-gum resin of F. foetida was reported to prevent carcinogenesis (Saleem et al., 2001). This activity support the use of various isolated compounds from this genus for treatment of cancer along with other co-administered anticancer agents like vincristine (Unnikrishnan and Kuttan, 1990;Saleem et al., 2001). A prenylated coumarin (diversin, 1) together with four new sesquiterpene lactones (diversolides A, D, F, and G, 2-5) isolated from the roots of Ferula diversivittata were showed cancer chemo preventive activity (Iranshahi et al., 2010). Galbanic acid isolated from F. assafoetida was reported to exhibit anticancer effect (Kim et al., 2011).

Molecular Docking Simulation
PASS (Prediction of Activity Spectra) is an online tool (Lagunin et al., 2000), which predicts almost 900 types of activities based on the structure of a compound. PASS analysis ( Table 2) of the compound 1 predicts anti-cancer activity (antineoplastic) with Pa (probability to be active) value of 0.303. To evaluate the inhibitory nature of the compound 1 as anti-cancer agent, the compound 1 was docked against human histone acetyltransferase (anticancer drug target), molecular docking simulations were carried out. Molecular docking is an efficient method to get an insight into ligand-receptor interactions. Molecular docking studies were performed using Molecular Operating Environment (MOE) software (www.chemcomp.com). The 3D crystal structure of human histone acetyltransferase was downloaded from Protein Data Bank (PDB ID: 4PZS) (Oikonomakos et al., 2000). Before docking experiment, the structure of compound 1 was prepared for docking by minimizing its energy using MOE. Most macromolecular crystal structures contain little or no hydrogen coordinate data due to limited resolution and thus protonation was done prior to docking using Protonate 3D tools implemented in MOE. Protonation was followed by energy minimization up to 0.05 Gradient using Amber99 forcefield. The docking protocol predicted the same conformation as was present in the crystal structure with RMSD value close to the allowed range (Paul and Mukhopadhyay, 2004) and surrounded by the same active site residues of the enzyme. Amongst the generated docking conformations the top-ranked conformation was visualized for ligand-enzyme interaction using MOE. Analysis of the docking results showed that compound 1 fit well within the active site of histone acetyltransferase enzyme (Figure 2). From the docking conformation it was observed that oxygen atom of the compound established hydrogen bond with the active site residue Gln169. The phenyl ring of the compound formed arene-arene interaction with the active site residue His165 (Figure 2). Furthermore, several hydrophobic interactions between compound 1 and the hydrophobic residues FIGURE 2 | Docking conformation of compound 1 (generated by MOE docking software) in the active site of human histone acetyltransferase enzyme.
Frontiers in Pharmacology | www.frontiersin.org of the enzyme were also observed. The presence of hydrogen bond, arene-arene and hydrophobic interactions confirm that the compound 1 may be specific to this site. These preliminary results suggest that the compound 1 might exhibit inhibitory activity against histone acetyltransferase enzyme. These computational predictions are verified by our experimental results in the compound 1 showed good anti-cancer activity ( Table 1).

CONCLUSION
The anticancer activity showed that F. narthex can be used in the management of cancer as n-hexane fraction, crude MeFn, CHCl 3 fraction, and pure compound 1 showed good to moderate anticancer activity against PC 3 cancer lines. Moreover the activity support the use of different compounds isolated from the genus Ferula as anticancer agents.

Mathematical Formula
Standard deviation x i − x n = sample data set; ; x = mean valve of sample data; N = size of data.

AUTHOR CONTRIBUTIONS
BA and ARK gave the project idea. Ajmal Khan and SB were project supervisors. MA performed isolation of compounds and AR helped in structure elucidation. Ayesha Khan and AA performed anticancer activity. AW and AKJ carried out docking studies. Ajmal Khan was also involved in the useful discussion and participated in manuscript writing. All authors read and approved the final manuscript.