Secondary Metabolites from Pterocaulon alopecuroides and their Antiproliferative Activities

Objective: To isolate secondary metabolites from the aerial parts of Pterocaulon alopecuroides, elucidate their structures and evaluate their antiproliferative activities on selected human cancer cell lines. Materials and Methods: The ethanolic extract of P. alopecuroides afforded five compounds, which were characterized using spectroscopic techniques and by comparison with data from the literature. Antiproliferative activities of all isolates were evaluated. Results: The compounds 7-(2,3-dihydroxy-3-methylbutoxy)-6-methoxycoumarin (1), 5,6-methylenedioxy-7-(2,3-dihydroxy-3-methylbutoxy) coumarin (2), Dihydrokaempferol (3), 5,7,4 ́-trihydroxy6-(α,α-dimethylallyl)dihydroflavonol (4) and 5,4 ́-dihydroxy-7-(γ,γ-dimethylallyloxy)dihydroflavonol (5) were isolated. The antiproliferative activity of all compounds was evaluated in a panel of six human solid tumor cell lines showing GI50 values for the most active compounds in the low micromolar range. Conclusion: Compound 2 is reported for first time from P. alopecuroides. Isolated coumarins show no antiproliferative activity, whilst among flavonoids compound 5 showed the best antiproliferative activity.


INTRODUCTION
The genus Pterocaulon (Asteraceae) has 18 species, of which twelve are American and six are Australian. 1 Two of them are present in the island of Hispaniola: P. alopecuroides and P. virgatum. 2 Continuing our interest in studying the flora present in Hispaniola, the phytochemical study of the species Pterocaulon alopecuroides (Lam.) DC. was carried out, which led to the isolation and structure elucidation of compounds 1-5. To the best of our knowledge, this is the first time that compound 2 is reported to be isolated from P. alopecuroides.

MATERIALS AND METHODS
General NMR spectra were recorded using a Bruker Ascend Aeon spectrometer with cryoprobe operating at 400 MHz in 1 H and 100 MHz in 13 C NMR respectively. The chemical shift (δ) values are given in ppm and coupling constants (J) are given in Hz. CDCl 3 or (CD 3 ) 2 CO were used as solvents. Column chromatography was performed on a Biotage Isolera One flash purification system (Biotage, Charlotte, North Carolina, USA) using SNAP ULTRA silica gel cartridges. Analytical and preparative TLC were developed on silica gel 60 F 254 plates (Merck KGaA, Darmstadt, Germany).

Plant Material
Aerial parts of P. alopecuroides were collected on October 2015 at Cordillera Central, Municipio Rancho Arriba, San José de Ocoa province, Dominican Republic. The plant material was identified by Teodoro Clase, botanist at Jardín Botánico Nacional "Dr. Rafael Ma. Moscoso", Santo Domingo, Dominican Republic, where a voucher specimen (JBSD 126571) has been deposited.

Extraction and Isolation
Aerial parts of P. alopecuroides were air-dried and ground to a fine powder. The ground material (105 g) was extracted with 95% EtOH using a Soxhlet apparatus. The resulting crude extract (18.5 g) was dissolved in 95% EtOH (250 mL) and treated with a 5% Pb (OAc) 2 solution (250 mL) to precipitate chlorophyll. After 24 h, the mixture was filtered, concentrated in vacuo to remove most of the EtOH and extracted successively with hexanes, Et 2 O and AcOEt (3 × 500 mL each). The Et 2 O residue (2 g) was subjected column chromatography, eluting with mixtures of hexanes-acetone with increasing polarity to afford 40 fractions. Repeated column chromatography, followed by PTLC afforded compounds 1-5.

Antiproliferative assays
The cell lines used in this study were A549 and SW1573 (lung), HBL-100 and T-47D (breast), HeLa (cervix) and WiDr (colon) and were a kind gift of Prof. Godefridus J. Peters (VUMC, The Netherlands). Compounds 1-5 were evaluated for antiproliferative activity using the protocol of the National Cancer Institute (NCI) of the USA with minor modifications. [3][4][5] Cells were seeded onto 96-well plates at cell densities of 2,500 (A549, HBL-100, HeLa and SW1573) or 5,000 (T-47D and WiDr) cells per well, depending on their doubling times. The results, expressed as GI 50 (50% growth inhibition) values after 48 h of drug exposure, were calculated according to NCI formulas.

Antiproliferative activity
All isolates were evaluated for their antiproliferative activity against the human solid tumor cell lines A549, HBL-100, HeLa, SW1573, T-47D and WiDr. The bioactivity of compounds 1-5 on the mentioned cell lines was expressed as GI 50 . The results (Table 1) shows that coumarins 1-2 are inactive whilst flavonoids 3-5 display growth inhibition. The most active compound of the series is flavonoid 5, which show GI 50 values in the range 16-20 μM.

CONCLUSION
In summary, we have reported the isolation of five secondary metabolites from the aerial parts of Pterocaulon alopecuroides. Compound 2 is reported for first time as isolated from this species. The study of the antiproliferative activity against selected human solid tumor cell lines showed that the isolated coumarins (1, 2) were not active, while the isolated flavonoids (3)(4)(5) were more active, being compound 5 the most active of all the ones tested with GI 50 values ranging from 16 to 20 μM.

ACKNOWLEDGEMENT
This project has been partially supported by the program FONDOCYT of the Ministerio de Educación Superior, Ciencia y Tecnología (MESCYT), Dominican Republic (Grant 2014-1D4-02). QAC thanks Dr. Ernesto Abel-Santos (University of Las Vegas, Nevada, USA) for his help in this project. aValues are given in μM and are means of two to three experiments. Figure 1: Compounds 1-5 isolated from the aerial parts of Pterocaulon alopecuroides.