Cytotoxic withanolides from the leaves of Moroccan Withania frutescens

Context: Withania species are a rich source of interesting phytochemical substances (withanolides) which have shown several biological properties. Objective: To investigate the cytotoxic potential of Withania frutescens (L.) Pauquy (Solanaceae) leaf extracts and isolated active compounds against cultured tumor cell lines. Materials and methods: The crude methanol extract of W. frutescens leaves was partitioned with dichloromethane, ethyl acetate and n-butanol. MeOH extract and its fractions were tested for their cytotoxic activity against cancer cell lines (HepG2 and HT29) using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Bioassay-guided fractionation was performed for the active CH₂Cl₂ fraction employing column chromatography and preparative high-performance liquid chromatography. Structural elucidation of the isolated active compounds was carried out mainly by 1D and 2D NMR and mass spectrometry. The compounds were then tested for their cytotoxic activity. Results: The CH₂Cl₂ fraction was the most active against HT29 cell line. The fractionation procedure resulted in the isolation of 4β,17α,27-trihydroxy-1-oxo-22-R-witha-2,5,24-trienolide (1), 5β,6β-epoxy-4β,17α,27-trihydroxy-1-oxowitha-2,24-dienolide (2) and 2,3-dihydroxywithaferin A-3β-O-sulfate (3). The latter exhibited the strongest cytotoxic activity against HT29 cancer cell lines (IC₅₀ of 1.78 ± 0.09 µM) which was comparable to that of 5-fluorouracil (5-FU) used as the positive antimitotic control. Discussion and conclusion: Compounds 2 and 3 were isolated from W. frutescens for the first time. Data obtained suggest that the sulfated steroidal lactone (3) can be considered as a compound with potential application in the new anticancer drugs development field.

Keywords: Cancer cell lines; cytotoxic activity; MTT assay; steroidal lactones

Withanolides are highly oxygenated C28 steroidal lactones which have been isolated from about 25 genera of the Solanaceae (Eich, [9]; Suleiman et al., [26]; Zhang et al., [32]) including Withania, Acnistus, Datura, Jaborosa, Nicandra and Mandragora. These compounds have also been reported in Taccaceae (Yokosuka et al., [31]), Lamiaceae (Khan et al., [16]) and Myrtaceae (Vankar et al., [27]) as well as in soft coral (Ksebati & Schmitz, [17]). Several biological activities are known for this class of compounds, including antitumor, cytotoxic, cancer preventive, antifeedant, anti-inflammatory, leishmanicidal and immunomodulating effects (Anjaneyulu et al., [3]; Chen et al., [6]; Glotter, [11]; Ray & Gupta, [24]; Veleiro et al., [28]). Withania is a small genus of 10 species, which are distributed in the eastern Mediterranean region, extending to South Asia (Hepper, [15]). To date, six of these have been reported to contain withanolides: Withania somnifera (L.), W. coagulans (Stocks) Dunal, W. frutescens (L.) Pauquy, W. obtusifolia Täckh, W. aristata (Ait.) Pauquy and W. adpressa Coss (Abdeljebbar et al., [1]; Anjaneyulu et al., [3]; Chen et al., [6]; Hepper, [15]; Veleiro et al., [28]). Withania frutescens is a perennial plant that grows in Morocco, Algeria, Spain and Canary Islands (Hepper, [15]). In Morocco, this species is known as "Tirnet" and traditionally used for the treatment of dysentery (Bellakhdar, [5]). Phytochemical studies on this species have led to the isolation of calystegines (Bekkouche et al., [4]) and several withanolides (González et al., [12], [13]). Different extracts obtained from W. frutescens are reported to exhibit protective and curative action against carbon tetrachloride (CCl4)-induced hepatotoxicity (Montilla et al., [21]), and to possess antimicrobial and antioxidant activities (El Bouzidi et al., [10]). Cytostatic activity studies of Spanish W. frutescens have established significant activity of withaferin A, 14-en-withaferin A and 5-hydroxy-6-chlorowithaferin A against HeLa 229 cell lines (González et al., [14]). Continuing with our investigations of the withanolides from species of Withania growing in Morocco, we have reported the isolation of four withanolides from Withania adpressa, which exhibit an interesting anticancer activity (Abdeljebbar et al., [2]). In the present study, the bioassay-guided fractionation of the CH2Cl2 extract from the leaves of W. frutescens has led to the purification of three withanolides. Their cytotoxic activities against HT29 (colon adenocarcinoma) human cancer cells are also reported herein.

Thin-layer chromatography (TLC) was performed on silica gel 60, F254 (0.20 mm, Merck). Spots were viewed under ultraviolet light at 254 and 365 nm and stained by spraying with Godin reagent. For column chromatography, silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany) and Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden) were used. Preparative high-performance liquid chromatography was carried out on a GILSON instrument (PLC 2020 Personal Purification System, Villiers-le-Bel, France) on a C18 column (Varian microsorb, Varian, Palo Alto, CA. 5 µm, 250 × 21.4 mm) with a UV detector. 1H-NMR (400 MHz), 13C-NMR (125 MHz) and 2D-NMR spectra were recorded on a Bruker Avance DRX 400 (Bruker Corporation, Billerica, MA). The chemical shift (δ) values are reported in ppm in CDCl3 and CD3OD, and coupling constants (J) are in Hz. HREI-MS measurement were performed on a QStar Elite (Applied Biosystems SCIEX, Foster City, CA) mass spectrometer.

The leaves of W. frutescens were collected in El Hassania (20 km from Marrakech), Morocco, in March of 2009 and were identified by Prof. A. Abbad, a plant taxonomist in the Department of Biology, Faculty of Sciences Semlalia, Cadi Ayyad University, Morocco. A voucher specimen (No. mhnm.bk.kh.1) has been deposited at the herbarium of Natural History Museum of Marrakech, Morocco.

The dried, powdered leaves of W. frutescens (1.2 kg) were first defatted with hexane (4.8 L) and then extracted with methanol (4.8 L) using a Soxhlet apparatus for 72 h, for each solvent. After removing the solvent under reduced pressure, the methanol extract (MeOH, 309.1 g) was suspended into distilled water, filtered and then partitioned successively with dichloromethane (CH2Cl2, 32 g), ethyl acetate (EtOAc, 7 g) and n-butanol (n-BuOH, 41 g), respectively. The CH2Cl2 fraction (30 g) was subjected to chromatography on a silica gel column (5 × 80 cm) and eluted initially with CH2Cl2 and then with CH2Cl2/MeOH mixtures of increasing polarity. Fractions of 100 ml each were collected and monitored with TLC. Similar fractions were pooled, yielding 15 fractions (F1–F15).

Fraction F7 (6.8 g) was further chromatographed on a silica gel column (2 × 120 cm) using a CH2Cl2/MeOH gradient as eluent to afford four sub-fractions. The purification of the sub-fraction F7-2 (4.5 g) was carried out on a silica gel column (2 × 120 cm) eluted initially with CH2Cl2 and then with CH2Cl2/MeOH (9.8:0.2) to give compound 1 (370 mg). Fraction F8 (1.1 g) was chromatographed by gel filtration on Sephadex LH-20 (2 × 120 cm), eluting with CH2Cl2:MeOH (1:1), to afford three sub-fractions. The sub-fraction F8-2 (0.36 g) was fractionated over a silica gel column (2 × 120 cm) using CH2Cl2/MeOH gradient as eluent to afford four sub-fractions. Sub-fraction F8-2-1 (0.1 g) was then purified on a silica gel column (2 × 120 cm) using CH2Cl2:EtOAc:MeOH (5:8:1) as eluent to afford compound 2 (51 mg). Fraction F10 (1.4 g) was further fractionated on a silica gel column with a CH2Cl2/MeOH mixture of increasing polarity as eluents to give five sub-fractions. Sub-fraction F10-3 (0.6 g) was again loaded on a column packed with Sephadex LH-20 (2 × 120 cm) and eluted with a mixture of CH2Cl2:MeOH (1:1) to give compound 3 with impurities, this was further purified by RP-HPLC on a C18 column (250 × 21.4 mm) eluting with MeOH:H2O (from 30:70 to 50:50, flow rate 22 ml/min) to afford pure compound 3 (8 mg) (Figure 1).

Graph: Figure 1. Isolation scheme of compounds 1–3.

Data of chemical structures of compounds 1–3 are presented in Table 1.

Table 1. NMR spectroscopic data for compounds 1–3.

• 4β,17α,27-Trihydroxy-1-oxo-22-R-witha-2,5,24-trienolide, C28H38O6 (1): White amorphous powder;  + 53 (c 0.475, pyridine); HREIMS m/z: 471.2741 ([M + H]+, 100), (Calcd for C28H39O6. 470.2668); 1H NMR [500.13 MHz, CD3OD:CDCl3 (1:1)] and 13C NMR [125,13 MHz, CD3OD:CDCl3 (1:1)].
• 5β,6β-Epoxy-4β,17α,27-trihydroxy-1-oxowitha-2,24-dienolide, C28H38O7 (2): White amorphous powder,  + 10 (c 0.475, pyridine), HREIMS m/z: 487.2690 ([M + H]+, 100), (Calcd for C28H39O7. 486.2261); 1H NMR (400.13 MHz, CD3OD) and 13C NMR (100,13 MHz, CD3OD).
• 2,3-Dihydroxywithaferin A-3β-O-sulfate, C28H40O10S (3): White crystalline solid,  + 14.5 (c 0.21, MeOH), HREIMS m/z: 568.2342 ([M + H]+, 100), (Calcd for C28H41O10S. 569.2415); 1H NMR (400.13 MHz, CDCl3) and 13C NMR (100,13 MHz, CD3OD).

Human cancer cell lines, HT29 (colon adenocarcinoma) and HepG2 (hepatocellular carcinoma), were kindly provided by Dr. J. Ciccolini (Laboratory of Pharmacokinetics and Toxicokinetics, Faculty of Pharmacy, Aix-Marseille University, France). Both cell lines were cultivated in the RPMI-1640 medium supplemented with 10% fetal bovine serum FBS, 1% of a stock solution containing 10 000 IU/ml penicillin and 10 mg/ml streptomycin and 0.16% kanamycin 50 mg/ml in an incubator at 37 °C under a humidified atmosphere containing 5% CO2. For routine passages, cultures were split 1:10 when they reached 70–90% confluence, generally every 3 days. All experiments were performed on exponentially growing cells.

Viable cell growth was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, cells were dispensed to 96-well plates at a concentration of 3000 and 10 000 cells per well for HT29 and HepG2, respectively, in duplicate plates and allowed to attach for 24 h at 37 °C. Serial dilutions of tested extracts (100, 50, 25, 12.5 and 6.25 µg/ml) or compounds (100, 10, 1, 0.1 and 0.01 µM) were added. Each experiment was performed using eight replicate wells for each drug concentration in duplicate plates. After 72 h incubation, the medium was replaced with 100 µl of MTT solution (0.5 mg/ml MTT in a fresh medium). After 2 h at 37 °C, the supernatant was discarded and the remaining cell pellet was resuspended with 200 µl of DMSO. The optical density (OD) of the formazan solution was measured at 570 nm by an automated microplate reader (Eon, BioTEK, France). 5-Fluorouracil (5-FU) was used as positive control. The percentage of viable cells was determined by comparing the average absorbance in drug-treated wells with the average absorbance in control wells. The drug concentration causing a 50% reduction in cell viability was determined as the 50% inhibitory concentration (IC50). Cell viabilities and IC50 values were determined using Gen5 software (BioTek Instruments, Inc., Winooski, VT). The results are presented as the mean ± standard deviation of three independent experiments.

The CH2Cl2 fraction obtained from a methanol extract of the leaves of W. frutescens showed significant cytotoxicity against HT29 colon adenocarcinoma cell (IC50 = 35.2 µg/ml). Further separation of this fraction by repeated chromatography over silica gel and Sephadex-LH20, as well as by RP-HPLC, resulted in the isolation of three compounds (1–3), the structures of which were elucidated by 1D and 2D NMR spectra and comparison with literature data (Figure 2).

Graph: Figure 2. Structures of the isolated withanolides (1–3).

Compound 1 was isolated as a white amorphous solid. The molecular formula was established as C28H38O6 by its HREI-MS {m/z 471.2741 ([M + H]+)}, implying the presence of 10 degrees of unsaturation. The 1H-NMR spectrum of this compound showed four methyl signals, typical of a withanolide, including three singlets at δ: 2.06, 1.43 and 0.83 and a doublet at δ: 1.05 (J = 6.4 Hz) (Pelletier et al., [23]). The proton signals at δ: 4.36 and 4.30 (d, J = 11.9 Hz) suggested the presence of C-27–CH2OH. Compound 1 showed the characteristic signals of H-2, H-3 and H-6 for a 1-oxo-2,5-dienewithanolide at δ: 5.87 (d, J = 9.9 Hz), 6.80 (dd, J = 4.6 and 9.9 Hz) and 5.89 (brd, J = 5.7 Hz), respectively. The 13C NMR spectrum exhibited 28 carbons including 4 methyl, 7 methylene, 9 methine and 8 quaternary carbons. A downfield quaternary carbon signal resonating at δ: 85.60 was assigned to the hydroxyl-bearing C-17, as supported by HMBC interactions of H-14 (δ: 1.72), H-18 (δ: 0.83), H-20 (δ: 2.25), H-21 (δ: 1.05) and H-22 (δ: 4.66). Compound 1 was established as 4β,17α,27-trihydroxy-1-oxo-22-R-witha-2,5,24-trienolide which was previously isolated from W. frutescens (González et al., [13]).

Compound 2 was isolated as a white amorphous solid with spectral data closely resembling those of compound 1. The HREI-MS established the molecular formula C28H38O7 [m/z 487.2690 ([M + H]+)]. The main difference was the presence of an epoxy group linked at C-5/6, on the basis of NMR signals at δ: 3.16 (brs, H-6), 61.20 (C-6) and 64.51 (C-5) and confirmed by HMBC analysis. Compound 2 was established as 5β, 6β-epoxy-4β,17α,27-trihydroxy-1-oxowitha-2,24-dienolide, a withanolide previously isolated from W. somnifera and W. aristata (Choudhary et al., [7]; Llanos et al., [18]), but reported here for the first time in W. frutescens.

Compound 3 was isolated as a white crystalline solid, and assigned as C28H40O10S by HREI-MS [m/z 568.2342 ([M + H]+)]. The 1H-NMR spectrum of this compound resembled that of compound 2 but the OH at C-17 was absent while of a low-field multiplet at δ: 4.83 of the C-3 signal indicated linkage of this carbon atom to a sulfate ester. Stereochemistry, α-orientation was determined for C-3 by comparing the NMR data with those reported in the literature (Xu et al., [29]). On this basis compound 3 was determined to be 2,3-dihydrowithaferin A-3β-O-sulfate. This is the first report of this compound from W. frutescens and it was recently isolated from aeroponically grown W. somnifera (Xu et al., [29], [30]). The presence of a sulfate group in steroids is quite rare and reported so far only from Datura metel (Shingu et al., [25]) and W. somnifera (Misra et al., [20]).

In this study, the methanol extract of W. frutescens leaves and its fractions were evaluated for their in vitro cytotoxicity against HepG2 (hepatocellular carcinoma) and HT29 (colon adenocarcinoma) cancer cell lines using the MTT colorimetric method with 5-FU as a positive control. Only dichloromethane fraction of the leaf extract displayed a marked cytotoxic activity against the HT29 cell line, with percentage of cell viability at a single concentration of 100 µg/ml of 12% and an IC50 value of 35.2 µg/ml (Table 2). This result encouraged us to investigate further the isolated compounds from this fraction. Thus, the three isolated compounds (1–3) were screened for their cytotoxic activity against HT29 cancer cell lines (Table 2). Compound 3 with a sulfate ester at C3 showed the most potent cytotoxicity with an IC50 of 1.78 ± 0.09 µM which was similar to the activity of the positive control, 5-FU. Withanolides 1 and 2 exhibited moderate activity with IC50 values of 13.18 ± 1.11 and 25.13 ± 3.93 µM, respectively. In terms of the structure–activity relationship, it was previously proposed that the presence of an α,β-unsaturated ketone and a 5β,6β-epoxy system is necessary for the antiproliferative activity of withanolides (Zhang et al., [32]). In addition, it was reported that the presence of a 4β-hydroxy group also leads to enhanced antiproliferative activity (Nakano et al., [22]). The high activity observed for compound 3 was likely due to its good aqueous solubility and its spontaneous conversion in the cell culture media to the corresponding withaferin A which contains a 2,3-unsaturated ketone moiety (Xu et al., [29]). Interestingly, in contrast to previous observations (Cordero et al., [8]; Machin et al., [19]), the replacement of the 5,6-epoxy functionality with a 5,6-unsaturated group (compound 1) led to an increase in activity.

Table 2. Cytotoxicity of extracts and compounds 1–3 isolated from W. frutescens leaves against two human cancer cell lines.

2 Nt: Not tested. aEach value is expressed as a mean ± SD of three independent determinations; IC50 are in µg/ml for extracts and in µM for isolated compounds. bPositive control.

In conclusion, three withanolides (compounds 1–3) were isolated from the leaves of W. frutescens. Withanolides have been reported previously from the leaves of W. frutescens; however, this is the first report of compounds 2 and 3. Data obtained suggest that 2,3-dihydroxywithaferin A-3β-O-sulfate (3) is a compound of potential interest for the development of new anticancer drugs.

Authors declare no conflict of interest. This work was supported in part by the intergovernmental program of France–Morocco FSP-ARESM (Priority Solidarity Fund on the reform of higher Moroccan education). We also thank the National Center for Scientific and Technical Research (CNRST) of Morocco for providing a research grant (no. E3/006).