Bioactive metabolites from the leaves of Withania adpressa

Abstract Context:Withania (Solanaceae) species are known to be a rich source of withanolides, which have shown several biological properties. Objective: To identify the compounds responsible for Withania adpressa Coss. antioxidant activity and further test them for their NF-κB inhibition and antiproliferative activity in multiple myeloma cells. Materials and methods: Compounds were obtained from the EtOAc extract of W. adpressa leaves. Structure elucidation was carried out mainly by 1D- and 2D-NMR, and mass spectrometry. Isolated compounds were tested in a dose-response for their in vitro NF-κB inhibition and antiproliferative activity in multiple myeloma cells after 5 and 72 h treatment, respectively. Results: The fractionation resulted in the isolation of a new glycowithanolide named wadpressine (5) together with withanolide F, withaferin A, coagulin L, and nicotiflorin. The latter showed a moderate ability to scavenge free radicals in DPPH (IC50 = 35.3 µM) and NO (IC50 = 41.3 µM) assays. Withanolide F and withaferin A exhibited low µM antiproliferative activity against both multiple myeloma cancer stem cells and RPMI 8226 cells. Furthermore, they inhibited NF-κB activity with IC50 values of 1.2 and 0.047 µM, respectively. The other compounds showed a moderate inhibition of cell proliferation in RPMI 8226 cells, but were inactive against cancer stem cells and did not inhibit NF-κB activity. Discussion and conclusions: One new glycowithanolide and four known compounds were isolated. Biological evaluation data gave further insight on the antitumor potential of withanolides for refractory cancers.


Introduction
The genus Withania (Solanaceae) consists of eight species (The Plant List 2013) occurring predominantly in North Africa and from the Mediterranean basin to India (Hepper 1991). This genus is known for elaborating withanolides, which are steroidal lactones characterized by a C28 basic skeleton. Since the isolation of withaferin A (Lavie et al. 1965), more than 750 withanolides with various functional groups have been isolated, largely but not exclusively, from about 25 genera of Solanaceae (Hang et al. 2012). In recent years, withanolides have attracted a significant attention from numerous researchers owing to their structural features and their multiple bioactivities such as cytotoxic (Cordero et al. 2009;Hang et al. 2012), anti-inflammatory , immunomodulatory (Mesaik et al. 2006), anticholinesterase (Choudhary et al. 2005), and antioxidant (Bhattacharya et al. 2001) properties.
The crude extracts of W. somnifera and W. frutescens were reported to have in vitro and in vivo antioxidant activities (Bhattacharya et al. 2001;El Bouzidi et al. 2011). The compounds responsible for the antioxidant activity included glycowithanolides (Bhattacharya et al. 1997). Interest has increased in naturally occurring antioxidants since they may be used to protect humans from oxidative stress damage and to lower the incidence of various cancers. Furthermore, Withania spp. extracts and several withanolides isolated from them have shown potential for their antitumor activity (Mathur et al. 2006).
To investigate the potential of W. adpressa leaves for cancer chemoprevention and therapy, the EtOAc extract was selected for bioactivity-guided fractionation based on its antioxidant activity. Four known compounds (1-4, Figure 1) and a new glycowithanolide, named wadpressine (5), were isolated. To further investigate their potential to control carcinogenesis, pure compounds were tested for their ability to inhibit NF-jB, a transcription factor involved in various aspects of the carcinogenesis process such as inflammation, cell survival, proliferation, migration, and angiogenesis (Aggarwal 2004). Finally, these compounds were tested against two multiple myeloma (MM) cell lines. MM is a hematological cancer in which the over activation of the NF-jB pathway is particularly crucial (Demchenko and Kuehl 2010).

General experimental procedures
UHPLC was performed on an Ultimate 3000 UPLC System (ThermoFisher Scientific, Waltham, MA) with an Acquity BEH C 18 column (50 Â 2.1 mm i.d.; 1.7 lm, Waters, Milford, MA) using an optimized gradient (MeCN and H 2 O both containing 0.1% formic acid) of 5-98% MeCN in 4 min and followed by a washing step with 98% MeCN for 2 min. HRMS spectra were obtained on a Q Exactive Plus Hybrid quadripole-orbitrap mass spectrometer (ThermoFisher Scientific) using electrospray in positive and negative modes. The spray voltage was set at 4.0 and 2.5 kV, and the sheath gas flow rate (N 2 ) at 47.5 and 50 units, respectively, the capillary temperature at 255 C; the S lens RF level at 50 and the probe heater temperature at 412.  Merck,Darmstadt,Germany) were used for column chromatography. Fractions were monitored by thin layer chromatography (TLC) on precoated silica gel 60F 254 (0.20 mm film thickness, Merck). Spots were visualized by exposure to UV light and/or by spraying with Godin or sulphuric vanillin reagents followed by heating at 105 C.

Plant material
The leaves of W. adpressa were collected at the flowering stage in Taznaght (South of Morocco) in April 2015 and authenticated by Dr Aziz Abbad, Department of Biology, Cadi Ayyad University. A voucher specimen (N Mar 4223) was deposited at the Botany Department Herbarium, Faculty of Sciences-Semlalia, Cadi Ayyad University, Marrakech, Morocco.

Extraction
Air-dried powdered leaves of W. adpressa (2 kg) were defatted with hexane and then extracted with methanol using a Soxhlet apparatus for 72 h for each solvent. After removing the solvent under reduced pressure, the methanol extract (168 g) was suspended into distilled water, filtered and then partitioned successively with dichloromethane, ethyl acetate, and n-butanol to obtain 28, 15, and 48 g of crude extracts, respectively.

Biological assays
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay The DPPH radical scavenging activity was carried out as described previously (Şahin et al. 2004) with slight modifications. Samples (1-200 mg/mL) were added to a 60-lM methanol solution of DPPH. The absorbance of the solution was measured at 517 nm after a 30-min incubation period at room temperature in the dark.

NF-jB inhibition assay
NF-jB inhibitory activity was assessed using a HEK293/NF-jBluc cell line (Panomics, Fremont, CA) as previously described (Ndongo et al. 2017). Briefly, cells were cultured at 37 C and 5% CO 2 atmosphere in high glucose Dulbecco's modified Eagle's medium (ThermoFisher Scientific) with 100 IU/mL penicillin, 100 lg/mL streptomycin, 100 lg/mL hygromycin B (ThermoFisher Scientific) and 10% foetal bovine serum (Biowest, Nuaill e, France). The day before the assay, cells were treated for 1 h with 2.5 lM of Cell Tracker Green CMFDA (ThermoFisher Scientific), a fluorescent dye used to quantify cell viability, in FBS-free medium. Then, 10 4 cells/well were seeded in 96-well plates and incubated overnight. After that, cells were treated with either the vehicle control (0.5% DMSO in culture medium) or the samples and stimulated for 5 h with 20 ng/mL of TNF-a (Sigma-Aldrich, Saint Louis, MO). Finally, cells were lysed with reporter lysis buffer (Promega, Madison, WI) and fluorescence signals were read on a Cytation 3 multimode plate reader (Biotek, Winooski, VT). Luciferase assay reagent (Promega) was then added to each well using the auto-injector and the luminescence signals were read. The fluorescence signal was used to normalize the luminescence signal for each well to limit the influence of cell toxicity in luminescence signal intensity. Relative NF-jB activity was quantified by comparing the normalized luminescence signal of vehicle treated nonstimulated cells with the one of vehicle treated stimulated cells. All pure compounds were first screened at 20 lM. Compounds able to inhibit more than 50% of TNF-a induced NF-jB activity at this concentration were considered as active and were selected for calculation of their IC 50 . Nonlinear regression (with sigmoidal dose response) was used to calculate the IC 50 values using GraphPad Prism. Each compound was tested in duplicate and three independent experiments were performed using parthenolide as positive control.

Antiproliferative activity
Human multiple myeloma cancer stem cells (MM-CSCs) derived from the bone marrow of a MM patient, were obtained from Celprogen (Torrance, CA). MM-CSCs were used between passages 4 and 12 for experiments. The tumour plasma cells RPMI 8226 were obtained from LGC standards (Middlesex, United Kingdom). RPMI 8226 cells were used between passages 10 and 25 for experiments. The two cell lines were cultured at 37 C and 5% CO 2 atmosphere in RPMI 1640 culture medium supplemented with 10% foetal bovine serum (Biowest), 100 IU/mL penicillin and 250 lg/mL streptomycin. MTT and XTT assays were used to evaluate the antiproliferative activity of the pure compounds in MM-CSCs and RPMI 8226 cells, respectively (Issa and Cuendet 2017). MM-CSCs were seeded in 96 well plates at a density of 5,000 cells per well and allowed to adhere for 24 h, whereas RPMI 8226 cells were plated at a density of 15,000 cells per well and treated immediately. MM-CSCs and RPMI 8226 cells were treated with increasing concentrations (0-20 mM) of compounds for 72 h. Twenty microlitres of MTT solution (5 mg/ mL) or 50 lL XTT solution (1 mg/mL) were added in each well and incubated for 2 h (MTT assay) or 4 h (XTT assay). The media and MTT solution were aspirated and the cells containing formazan were solubilized in 100 mL DMSO. Absorbance was measured at 590 nm (MTT assay) or 450 nm (XTT assay). The percentage of cell viability was calculated as the absorbance of each well was divided by that of the vehicle control wells (0.5% DMSO in culture medium) and multiplied by 100. IC 50 values were calculated using GraphPad Prism. Each compound was tested in triplicate and three independent experiments were performed using bortezomib as positive control.
Compounds 1-4 were identified as withanolide F (Abdeljebbar et al. 2007), withaferin A (Lavie et al. 1965;Erazo et al. 2008), coagulin L (Atta-ur-Rahman et al. 1998), and nicotiflorin (De Sousa et al. 2014), respectively. Their structure elucidation was performed by high resolution mass spectrometry, 1Dand 2D-NMR experiments and comparison with spectroscopic data reported in the literature. These compounds are described for the first time in this species except for withanolide F (1), which was previously isolated from the leaves of this species (Abdeljebbar et al. 2007). Withaferin A (2) was previously isolated from W. somnifera, W. coagulans, W. aristata, and W. frutescens (Gonzalez et al. 1982;Neogi et al. 1988;Llanos et al. 2010) and coagulin L (3) from W. coagulans (Atta-ur-Rahman et al. 1998). It is noteworthy that this is the first report on the isolation of nicotiflorin (4) from a Withania species.
Compound 5 was isolated as a white amorphous powder. HRESIMS showed a pseudo molecular ion peak at m/z 797.3853 [M þ H] þ (calcd. 797.3864), indicating a molecular formula of C 40 H 60 O 16 . IR spectrum revealed the presence of a hydroxyl (3,420 cm À1 ), an a,b-unsaturated d-lactone moiety (1715 cm À1 ) and a carboxyl (1690 cm À1 ). 1 H-and 13 C-NMR data are given in Table 1. The structure of compound 5 was partly deduced from that of coagulin L (3). The comparison of their 13 C-NMR chemical shifts (Table S1) showed that 5 contains an additional sugar unit in position 4 0 compared to 3 (8 ppm deviation). The planar structure of compound 3 was determined by the analysis of 1 H, 13 C, COSY, HSQC and HMBC spectra and found to be identical to the one of coagulin L (Atta-ur-Rahman et al. 1998). The comparison of 13 C-NMR chemical shifts of 2,3-dihydro-3bhydroxywithanolide F (the aglycone of coagulin L) (Zhang and Timmermann 2016) or of the aglycone part of tetra-acetylated coagulin L (Atta-ur-Rahman et al. 1998), with those of compound 3, let us to conclude the identity of the latter was coagulin L, even though chemical shift comparison was biased by the use of different NMR solvents (CDCl 3 and CD 3 OD) ( Table 1). The absence of reported stereoisomers of coagulin L provides further support for our conclusion. The sugar unit in 5 is bound to the aglycone in position 3, as shown by the H-1 0 /C-3 correlation. A series of COSY correlations, starting from H-1 0 , revealed the chemical shifts of H-2 0 to H-5 0 , all of these being in axial position, as deduced from the high value of their coupling constants. The HSQC spectrum indicated the chemical shift of H-6 0 a and H-6 0 b as those of a methylene group in a primary alcohol functional group whose connection with C-5 0 was proven by the HMBC spectrum. The anomeric configuration of this glucose unit is b, as indicated by the high H-1 0 /H-2 0 coupling constant (7.8 Hz).
The anomeric position 1 00 of the sugar unit present in 5 was characterized by 1 H and 13 C chemical shifts clearly identified in the HSQC spectrum. The H-1 00 signal, hidden under the strong HOD signal of the solvent, correlated with the one of H-2 00 in the TOCSY spectrum. The COSY spectrum sequentially correlated with the signal of H-2 00 to those of H-3 00 to H-6 00 . The latter appeared as a shielded doublet of a methyl group. The small value of the H-1 00 /H-2 00 and H-2 00 /H-3 00 coupling constants and the high value of the H-3 00 /H-4 00 and H-4 00 /H-5 00 coupling constants are compatible with a rhamnose unit in a configuration. The H-1 00 /C-4 0 HMBC correlation confirmed the binding of this sugar to position 4 0 of the glucose unit. Therefore, the structure of compound 5 was determined as (14R,17S,20R,22R)-3b-O-(a-Lrhamnopyranosyl-(1!4)-b-D-glucopyranosyl)-14,17,20-trihydroxy-1-oxo-witha-5,24-dienolide and named wadpressine. The common characteristic of coagulin L (3) and wadpressine (5) is the presence of a 5-en-1-one structure that had only been reported in withanolides from Withania coagulans (Attaur-Rahman et al. 1998) and Ajuga parviflora Benth. (Lamiaceae) (Khan et al. 1999).

NF-jB inhibitory activity
More recently, it has become clear that an over activation of the NF-jB pathway plays a critical role in cancer development and progression (Aggarwal and Sung 2011). Hence, each compound was also tested for its NF-jB inhibition. Only withanolide F (1) and withaferin A (2), an already described NF-jB inhibitor (Heyninck et al. 2014), inhibited TNF-a-induced NF-jB activity with IC 50 values of 1.2 and 0.05 lM, respectively (Table 2).

Antiproliferative activity
MM remains an incurable malignancy, with a median survival of only 5 years, despite all the treatment advances (Fonseca et al. 2017). The presence of CSCs in MM is considered to contribute to disease relapse through their drug-resistant nature. Targeting MM-CSCs is therefore a promising strategy for MM treatment. The effect of pure compounds on cell growth in MM-CSCs and RPMI 8226 cells was investigated using MTT and XTT assays (Table 3). Withaferin A (2) exhibited the strongest activity with IC 50 values of 0.33 and 0.17 mM in MM-CSCs and RPMI 8226 cells, respectively. Similar results were reported for withaferin A in a previous study (Issa and Cuendet 2017).
Withanolide F (1) also showed activity against both cell lines (Table 3). Previous results have shown that withanolide F strongly inhibited the growth of several cancer cell lines Abdeljebbar et al. 2009). Compounds 3-5 were only active against RPMI 8226 cells. Interestingly, the two compounds able to inhibit NF-jB activity were also the ones active on MM-CSCs, highlighting the potential interest of NF-jB inhibitors for the treatment of refractory cancers.

Disclosure statement
No potential conflict of interest was reported by the authors.