Bioactive Withanolides from Withania obtusifolia

Seven withanolides were isolated from the leaves of Withania obtusifolia. Of these, one was new [obtusifonolide (1)], five were new to the species [sitoindoside IX (2), 6α-chloro-5β-hydroxy withaferin A (3), isowithanone (4), 2,3-dihydro-3-ethoxywithaferin A (5), and daturataturin A (6)], and one was reported previously from W. obtusifolia [withaferin A (7)]. The structures were elucidated using a set of spectroscopic and spectrometric techniques. Compounds (1–7) were evaluated for cytotoxicity against a human cancer cell panel and for antimicrobial activity in an array of bacteria and fungi. Compound 7 showed cytotoxic activity against the MDA-MB-435 (human melanoma) and SW-620 (human colon cancer) cell lines with IC50 values of 1.7 and 0.3 μM, respectively. The in vitro activity of 7 on 17β-hydroxysteroid dehydrogenase and 5αreductase was also investigated.


Introduction
The withanolides are a group of naturally occurring polyoxygenated C-28 ergostane-type steroids (Chen et al., 2011, Lavie et al., 1965. A common feature among most of them is the oxidation at C-1, C-22, and C-26 (Chen et al., 2011). Withanolides occur largely, but not exclusively, in genera belonging to the plant family Solanaceae, including Withania, Lycium, Datura, Dunalia, Acnistus, Jaborosa, Nicandra, and Physalis (Misico et al., 2011). Withaferin A, isolated in 1965, was the first withanolide to be characterized from Withania somnifera (Lavie et al., 1965) and from Acnistus arborescens (Kupchan et al., 1965). It showed in vitro and in vivo cytotoxic activity against an array of tumor cells (Glotter, 1991, Samadi et al., 2010. Withanolides can be divided into two types, those with a δ-lactone or δ-lactol, resulting from appropriate oxidation of C-22 and C-26, and those with a γ-lactone or γ-lactol involving C-23 and C-26; most of the withanolides belong to the former type (Chen et al., 2011, Glotter, 1991, Misico et al., 2011. Biogenetic transformations of the steroidal skeleton and the side chain have diversified the structures of withanolides (Chen et al., 2011, Glotter, 1991, Misico et al., 2011. Withanolides have attracted attention due to their wide range of biological activities, including antitumor, anti-inflammatory, antifeedant, antimicrobial, cytotoxic, immunomodulating, and cancer chemopreventive activities (Chen et al., 2011, Glotter, 1991, Misico et al., 2011. Moreover, recent studies have suggested that withanolides may also act as growth regulators due to their common biosynthetic origin with brassinosteroids, a well-known class of growth regulators (Sangwan et al., 2008).
As part of an ongoing project to explore medicinal plants of Jordan for anticancer leads (Alali et al., 2005, Alali et al., 2008, Alali et al., 2010, seven withanolides (1-7), of which one was new, were isolated and characterized from an ethanolic extract of the leaves of Withania obtusifolia.

Results and discussion
Dried leaves of W. obtusifolia were extracted with EtOH and partitioned with organic solvents to yield five fractions (F01-F05). Fraction F05 was purified using silica gel column chromatography to yield 241 sub-fractions, and similar ones were combined into 11 pools. Pools P08 and P10 were subjected to gel filtration on Sephadex LH-20 to yield a total of 7 and 5 subfractions, respectively. Sub-fractions P08-5 and P10-3 were found to be rich in withanolides as evidenced by thin layer chromatography (TLC), and were purified further using preparative and semipreparative reversed-phase high performance liquid chromatography (RP-HPLC) to yield seven compounds (1-7) with >95% purity as evidenced by ultra-performance liquid chromatography (UPLC) (Fig. S1, Supplementary data).
The biological effects of withaferin A (7) on the activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) and 5α-reductase (5α-R) were investigated. These enzymes have an important role in the biosynthesis of 5α-dihydrotestosterone (DHT) and pathologies associated with the prostate gland (i.e. hyperplasia and prostate cancer) (Bonkhoff et al., 1996, Geissler et al., 1994, Marberger, 2006, Thomas et al., 2005. Type 5 17β-HSD catalyzes the conversion of androstenedione into testosterone (Peltoketo et al., 1999). On the other hand, 5α-R converts testosterone into the more potent androgen, dihydrotestosterone. The hyperplasia of the prostate gland and prostate cancer has been associated with high levels of serum DHT (Bonkhoff et al., 1996, Marberger, 2006, Thomas et al., 2005. Since different plant-derived 5α-colestane molecules have been identified as selective inhibitors or anabolic agents with minimal or no androgenic side effects (Esposito et al., 2011), it was of interest to study the in vitro effect of withaferin A on the enzymes 17β-HSD and 5α-R. Withaferin A (7) stimulated the activity of 17β-HSD and 5α-R enzymes with half maximal effective concentration (EC50) values of 63 ± 8.7 and 20 ± 6.5 nM, respectively (Fig. 3).

General experimental procedures
Optical rotations and UV spectra were acquired on a Rudolph Autopol III automatic polarimeter and a Varian Cary 100 Bio UV-Vis spectrophotometer. NMR experiments were conducted in either CDCl3 or methanol-d4 with TMSi as a reference via a Bruker NMR spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C and a JEOL ECA-500 NMR spectrometer operating at 500 MHz for 1 H and 125 MHz for 13 C. Low-resolution ESIMS data were measured with PE Sciex API 3200 mass spectrometer, while high-resolution ESIMS was performed on a Thermo LTQ Orbitrap XL mass spectrometer. UPLC was carried out on a Waters Acquity system with data collected and analyzed using Empower software. HPLC was carried out using either a Varian Prostar HPLC system equipped with ProStar 210 pumps and a Prostar 335 photodiode array detector (PDA), with data collected and analyzed using Galaxie Chromatography Workstation software (version 1.9.3.2) or on a Lachrom Merck-Hitachi, equipped with a quaternary gradient L-7150 pump, L-7455 diode-array detector, L-7200 autosampler, and D-7000 interface. For preparative HPLC, a Phenomenex Gemini-NX C18 (4 μm; 250 mm × 21.2 mm) column was used at a 21 ml/min flow rate, or a Hibar Merck prepacked column RT 250-25, Lichrosorb RP-18 (7 μm) at a flow rate of 10 ml/min. For the semi-preparative HPLC, a Phenomenex Gemini-NX C18 (4 μm; 250 mm × 10 mm) column was used at a 4.72 ml/min flow rate. For UPLC analysis, a Waters BEH C18 column (1.7 μm; 50 mm × 2.1 mm) was used with a 0.6 ml/min flow rate. Column chromatography was performed using silica gel 60 (0.06-0.2 mm; 70-230 mesh) and Sephadex ® LH-20. For thin layer chromatography, silica gel 60 with gypsum and pigment addition for UV or silica gel 60 with 15% calcium sulfate and fluorescent indicator were used. TLC spots were visualized using a UV lamp at 254 nm (Vilber Lourmat, 4 W-254 nm tube). All other reagents and solvents were obtained from either Fisher Scientific or Sigma-Aldrich and were used without further purification.

Measurement of antimicrobial activity
The minimal inhibitory concentrations (MICs) of the isolated compounds were measured by broth microdilution in 96-well microtitre plates. A 2-fold dilution series of the compounds was prepared in 96-well microtitre plates in a 50 μL volume of 1/10-strength BHIB + S and the dilution series was inoculated with 50 μL of each cell suspension. The resulting inoculated dilution series were incubated at either 30 or 37 °C (same as growth temperature) and growth, as turbidity, scored visually and recorded on the fourth day. MIC of each compound was measured in triplicate and was defined as the lowest concentration of drug resulting in a complete absence of turbidity compared with the drug-free control.
3.7. In vitro human prostatic 17β-hydroxysteroid dehydrogenase and 5α-reductase assays of withaferin A The in vitro 17β-HSD and 5α-R activity assays were carried out using the membrane fraction obtained from human prostate homogenates, as described previously (Cabeza et al., 2009, Cabeza et al., 2011, Hirosumi et al., 1995.