Gracilone, a new sesquiterpene lactone from Tanacetum gracile (Tansies)

Abstract The methanolic extract of the Tanacetum gracile afforded the isolation of new sesquiterpene lactone, named gracilone (1) along with four known compounds as 14α-taraxeran-3-one (2), 14α-taraxeran-3-ol (3), apigenin (4) and β-sitosterol (5). The structure of compound 1 was elucidated on the basis of 1D, 2D NMR and MS spectroscopic analysis. Antimicrobial, antioxidant and anticancer activities of all compounds were evaluated, from which gracilone (1) showed a moderate antibacterial activity, while apigenin (4) showed comparatively more antibacterial activity against both grampositive and gramnegative tested strains.


Introduction
Tanacetum is a genus of about 200 species of flowering plants in the family, Asteraceae, native to many regions of the northern hemisphere. They are known commonly as tansies. The genus includes several strongly scented annual and perennial species. Tanacetum species are widespread in many countries of North America, Asia and Europe (Kumar & Tyagi 2013;Ghanbarian et al. 2015). Tanacetum species have been used as remedies in traditional medicine since ancient times all over the world. Many species of the genus Tanacetum, such as Tanacetum annuum, Tanacetum balsamita, Tanacetum indicum, Tanacetum nubigenum, Tanacetum santolinoides, Tanacetum parthenium and Tanacetum vulgare, are used therapeutically around the world. Some species of genus Tanacetum have been used in popular medicine such as expectorants, antiseptic vermifuges, and spasmolytics (Oksuz 1990). Traditionally, the leaves or infusions of the herb have long been used as a febrifuge, and to relieve menstrual and rheumatic pain and migraine (Kubo & Kubo 1995). Various Tanacetum species are reported to possess antimicrobial, antioxidant, anti-inflammatory and trypanocidal activity (Tabanca et al. 2007;Piras et al. 2014;Cogo et al. 2012;Maxia et al. 2015;Polatoglu et al. 2015). Anti-inflammatory activity of Tanacetum has been attributed to its parthenolide contents that impair platelet activation in human blood (Abad et al. 1995;Goren et al. 2002;Maxia et al. 2015).
Tanacetum species are bestowed with the presence of large number of chemical constituents such as terpenoids, especially sesquiterpenes and sesquiterpene lactones, triterpenoids, flavonoids and other phenolic compounds. The sesquiterpene lactones are very important group of bioactive molecules isolated predominantly from the Asteraceae family. Sesquiterpene lactones appear to have a common biosynthetic origin, and are likely to provide useful chemical characters in the tribe. As a part of our programme for the chase of bioactive constituents from high altitude medicinal plants of Kashmir Himalayas (Bhat et al. 2014a(Bhat et al. , 2014bBhat et al. 2015), we investigated different plant species from time to time. This time we again look into the bioactive fractions of Tanacetum gracile which previously led to isolation of β-thujone, ketopelonolide B and artemetin (Shawl 1993). The chromatographic isolation of the methanolic extract of the root of T. gracile led to the isolation of six compounds (1-5) including a new sesquiterpene lactone named as Gracilone (1). The structure elucidation of all the compounds was achieved by spectroscopic methods. All the isolated compounds (1-5) were evaluated for antibacterial, antioxidant and anticancer activities.
Antibacterial, antioxidant and anticancer activities of isolated compounds 1-5 were evaluated and results are shown in Tables 1-3. Gracilone (1) type compounds are of great interest to chemists due to their wide structural diversity and pharmacological properties such as antimicrobial, cytotoxic and anti-inflammatory activities (Chadwick et al. 2013). In our study, gracilone (1) showed moderate antibacterial and antioxidant activity, lower than flavonoid apigenin (4). Overall antibacterial, antioxidant and cytotoxic activities of flavonoid apigenin (4) were higher as compared to other isolated compounds.

General
Melting points were determined in glass capillary tubes using Buchi B-545 melting point apparatus. Optical rotations were measured in Rudolph Autopol IV digital polarimeter. Ultra violet (UV) spectra were recorded on Specard S100 in methanol. IR spectra were recorded on a FT-IR Spectran Two (Perkin Elmer). 1 H NMR and 13 C NMR were run on a 400 and 500 MHz Bruker instrument (Avance NMR systems) using CDCl 3 /CD 3 OD/DMSO as the solvents. The  Table 1. Antibacterial activity (diameters of growth inhibition zones, mm) of isolated compounds 1-5 and reference compound (50 μg/disc) determined by the agar disc diffusion method.
(-) Not active at the tested concentration against the specific microorganism. Mean values ± SD (in mm) of two independent experiments, including the diameter of the disc (6.0 mm).

Plant material
The 6 kg of fresh plant material was collected from a high altitude area of Drass (Kashmir, India) in the month of August 2012. The collected plant was identified and reference specimen deposited in Center for Biodiversity and Taxonomy, University of Kashmir vide voucher specimen No. 1755 KASH.

Extraction and isolation
The whole plant material was chopped, shade dried and ground to a fine powder. The powdered plant material (1.9 kg) was extracted by maceration with methanol. The solvent was Table 2. Antioxidant activity of all isolated compounds (1-5).
a DPPH free radical scavenging activity of ascorbic acid was considered to be 100%. The free radical scavenging activity of test compounds was calculated in comparison with ascorbic acid. b Ferric reducing ability of ascorbic acid (100 µM) was considered to be 100%, whereas FRAP value for other compounds was calculated in comparison with ascorbic acid. Data in the FRAP and DPPH assays are mean ± SD of three independent experiments.  evaporated in a rotary evaporator at 40 °C to get 100 g (5%) of crude extract. This crude methanol extract obtained was dissolved in 500 ml of dichloromethane (DCM) and was subsequently followed by n-butanol and ethylacetate to obtain 9 g of DCM extract, 40 g of n-butanol extract and 10 g ethylacetate extract. All the extracts were dissolved in minimum amounts of respective solvents and adsorbed on silica gel to form slurry. The dried slurry of DCM extract was loaded on column and eluted isocratically with hexane: ethylacetate (8:2, v/v) to get two compounds 1 (20 mg) and 2 (65 mg). About 30.0 g of the n-butanol extract was subjected to column chromatography over silica gel to afford compound 3 (7 mg), using isocratic solvent system comprising DCM-EtOAc (9:1, v/v) as eluent. Ethylacetate extract (20 g) was subjected to column chromatography over silica gel and eluted with 100% DCM to afford compound 5 (34 mg). Similarly the methanol extract (40 g) was loaded on column and eluted with DCM: MeOH (8:2, v/v) to afford compound 4 (13 mg).

Bacterial strains
Six bacterial strains, two gram-positive and four gram-negative, were used for the evaluation of antibacterial activity. These strains include Pseudomonas aeruginosa MTCC-1688, Bacillus subtilis MTCC-441, Proteus vulgaris MTCC-321 and three clinical strains Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli were obtained from the department of microbiology, Sheri-Kashmir Institute of Medical Sciences (SKIMS), Soura, Kashmir. All strains were stored at 4 °C on Mueller Hinton agar slants, subcultured every month and checked for purity. The strains were activated by subculture at 37 °C for 24 h with fresh agar plate prior to antibacterial testing.

Antibacterial assay
Disc diffusion method, according to the Clinical and laboratory Standards Institute (ClSI) (Wikler 2006) was employed for the determination of antibacterial activity of all the isolates (1-5). The Mueller Hinton agar (MHA) medium was prepared and sterilised at 121 °C for 20 min in the autoclave, and 20 ml of this medium was poured into each 12 cm sterile Petri dishes under aseptic conditions and allowed to settle. Inocula was prepared by emulsifying the 24-h bacterial culture in 3 ml sterile saline following McFarland turbidity to obtain a concentration of 10 8 cell/ml and was standardised by adjusting the optical density to 0.1 at 600 nm. About 100 μl of this cell suspension with 10 6 bacteria /ml was placed in Petri dishes and dispersed over agar. Stock solutions of all the isolates were prepared in DMSO at concentrations of 10 mg/ml and filter sterilised using a 0.20-μm Millipore filter. Susceptibility discs (6 mm in diameter) were impregnated with 30 μl of pure compounds to give final concentration of 50 μg/disc. A disc prepared in the same way with only DMSO was used as negative control. The discs were gently placed at equidistant on the top of the agar plates inoculated with the test bacterial strains B. subtilis MTCC-441, P. aeruginosa MTCC-1688, P. vulgaris MTCC-321, K. pneumoniae, S. aureus and E. coli. Isolated compounds and reference drugs were allowed to diffuse for 1 h and the plates were incubated at 37 °C for 24 h in inverted position. The results were obtained by measuring the zone of growth inhibition (mm) surrounding the discs. Streptomycin was used as positive reference standard for antibacterial activity. Each assay test was run in duplicates. Diameters of inhibition zone more than 10 mm were recorded as active.

Ferric reducing-antioxidant power assay
The ferric reducing-antioxidant power (FRAP) assay was performed in a 96-well microplate using as previously described (Bharate et al. 2014). Briefly, the FRAP reagent was prepared by mixing 10 ml of 300 mM acetate buffer with 1 ml of 10 mM 2,4,6-tripyridyl-S-triazine (TPTZ) in 40 mM of hydrochloric acid and 1 ml of 20 mM FeCl 3 ·6H 2 O. The freshly prepared FRAP reagent (195 μl) was added to all the wells of the 96-well plate. Test compounds dissolved in methanol (5 μl) were added to the final concentration of 50 and 100 μM. Ascorbic acid (100 μM) was used as a positive control. The absorbance was read at 593 nm after 30 min incubation in the dark.

DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay
The DPPH assay was carried out in a 96-well microplate. Briefly, 190 μl of each test compound dissolved in methanol was added into each well of the 96-well plate with a final concentration of 50 and 100 μM. Ascorbic acid (100 μM) was used as a positive control. The reaction was started by adding 10-μl methanolic solution of DPPH (100 μM) to all the samples. After 30 min incubation, while shaking in the dark, absorbance was recorded at 517 nm. The DPPH free radical scavenging activity was calculated as percentage inhibition using the following formula: where A c is the absorbance of the control and A s is the absorbance of the test sample.

Anticancer activity using MTT (3-(4, 5-Dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide) reagent
All the isolated molecules were subjected to MTT cytotoxicity screening against a panel of four different human cancer cell lines viz. pancreatic (MIAPACA), lung (A549), breast (MCF-7) and leukemia (Hl-60). Suspension cells were seeded in 96-well plates at a density of 1.5 × 10 4 /200 μl of the medium, while as all the adherent cell lines were seeded at a density of 6 × 10 3 /100 μl of the medium. All the cells were treated with 10-100 μM of each compound for 48 h. MTT dye (2.5 mg/ml PBS) was added three hours before termination of the experiment and incubated at 37 °C for 3 h. The plates were centrifuged and MTT-formazan crystals were dissolved in 150 μl of DMSO and agitated on orbital shaker for 2-3 min. The absorbance was measured at 570 nm with reference wavelength 620 nm (Malik et al. 2007). Cell growth was calculated by comparing the absorbance of treated verses untreated cells. IC 50 values were calculated using GraphPad Prism 5.0 software.