Phytochemical Investigation of Egyptian Spinach Leaves, a Potential Source for Antileukemic Metabolites: In Vitro and In Silico Study

Spinacia oleracea L., Amaranthaceae, leaves cultivated in Egypt demonstrated a potential antileukemic activity against the chronic myeloid leukemia, K562 cell line. Thus, the aim of this study is to carry out a phytochemical investigation of S. oleracea leaves as well as the isolation of its antileukemic phytoconstituents. Phytochemical investigation of S. oleracea leaves resulted in the isolation of seventeen known compounds. The biological study revealed that compounds hexaprenol, phytol, and 18-[(1-oxohexadecyl) oxy]-9-octadecenoic acid exhibited a remarkable antiproliferative activity against K562 cells in vitro. A mechanistic in silico study showed that hexaprenol, phytol, and 18-[(1-oxohexadecyl) oxy]-9-octadecenoic acid exhibited a strong binding affinity towards topoisomerase (docking score −12.50, −9.19, and −13.29 kcal/mol, respectively), and showed as well a strong binding affinity towards Abl kinase (docking score −11.91, −9.35, and −12.59 kcal/mol, respectively). Molecular dynamics study revealed that 18-[(1-oxohexadecyl) oxy]-9-octadecenoic acid produced stable complexes with both topoisomerase and Abl kinase with RMSD values of 1.81 and 1.85 Å, respectively. As a result of our findings, we recommend more in vivo and preclinical studies to confirm the potential benefit of spinach leaves for chronic myeloid leukemia patients.


Introduction
Chronic myeloid leukemia (CML) is ranked as the fourth predominant cancer in upper Egypt that constituted about 10.2% of all the reported cancer cases, after breast, liver, and bladder cancers that constituted about 34, 23.4, and 16.6% of cases, respectively (Ibrahim et al. 2014). Chronic myeloid leukemia is a myeloproliferative neoplasm that is characterized by uncontrolled myeloid cell divisions in the bone marrow (Shahrabi et al. 2014). Chronic myeloid leukemia arises due to genesis of the BCR-ABL oncogene as a result of the reciprocal translocation between chromosome 9 and chromosome 22 (Deininger et al. 2000). The BCR-ABL oncogene encodes a constitutively activated tyrosine kinase enzyme which activates several proliferatory signaling pathways inside the cells such as RAS, a small GTPase, mitogenactivated protein kinase, signal transducers and activator of transcription, and phosphoinositide-3-kinase pathways (Sattlermc and Griffin 2003). Targeting Abl kinase was reported as a successful strategy to treat CML. Tyrosine kinase inhibitors (TKIs) such as imatinib, ilotinib, dasatinib, bosutinib, and ponatinib are currently used to treat CML (Akard 2010; Shamroe and Comeau 2013;Sacha 2014). However, mutations in BCR-ABL and multi-drug resistance make TKIs less effective, so, there is a constant need for alternative therapeutic strategy (Ali 2016).
In a previous study to our group investigating the antileukemic potential activities of fifty-six medicinal plants grown and used in upper Egypt, the 75% hydroethanolic extract of Spinacia oleracea L., Amaranthaceae, leaves demonstrated a promising antileukemic activity and was one of the most active plants against the chronic myeloid leukemia cell line K562 (Abdelgawad et al. 2021). Moreover, few studies were available in the literature about the phytoconstituents of S. oleracea leaves cultivated in Egypt. So, this study was carried out with the aims of isolating the antileukemic metabolites from S. oleracea leaves as well as the phytochemical investigation of the plant.
Spinacia oleracea is an annual popular winter vegetable crop that is widely cultivated in Egypt where the leaves are used as fresh, canned, or frozen product (Fekry and Nawar 2017). Spinach is considered as one of the healthy vegetable crops for human consumption. It contains major vitamins such as vitamin C, A, and E, as well as minerals such as iron, calcium, potassium, manganese, and zinc (Toledo et al. 2003). Spinach is a vegetable with a high biological value due to the presence of secondary metabolites from various chemical classes such as flavonoids (Aritomi et al. 1985;Ferreres et al. 1997;Edenharder et al. 2001;Sultana and Anwar 2008), steroids (Dawidar and Amer 1973;Modlin et al. 1994), triterpenoid saponins (Mithöfer et al. 1999), phenolic acids Bunea et al. 2008), carotenoids (Metha and Belemkar 2014), and glycolipids (Wang et al. 2002). Spinach phenolic compounds exhibited a wide range of biological effects including antioxidant , anti-inflammatory (Lomnitski et al. 2000), antiproliferative (Nyska et al. 2003), anti-carcinogenic (Nyska et al. 2001), antibacterial (Parekh and Chanda 2008;Altemimi et al. 2017), hepatoprotective (Abdul-Wahab and Jalil 2012), hypolipidemic (Hetta et al. 2017), and CNS suppressant (Das and Guha 2008) properties.

General Experimental Procedures
Liquid chromatography analysis was conducted using an Agilent 1100 HPLC system, RP-C18 column (150 × 4.6 mm; particle size 5 μm; Luna) with column oven temperature set at 25°C and a gradient system of eluent water (A) and methanol (B) used. The gradient condition was as follows: 0-8 min (30% B), 9-11 min (40% B→80% B), 12-15 min (100% B). The flow rate of the solvent was 2 m/min, and the injection volume was 50 μl. All the analysis was carried out at wavelength of 280 nm with a run time of 16 min. HPLC-grade methanol and water solvents were used. Acetic acid was added as a modifier to achieve a final concentration of 0.1% in each solvent. GC/MS analysis was performed with an Agilent 7890B gas chromatograph. Optical activity was measured using an AA-65 series automatic polarimeter (Cambridgeshire, PE26 1NF, England). High-resolution electrospray ionization mass spectrometry (HR-ESIMS) data were acquired using a Bruker BioApex-FTMS with electrospray ionization (ESI). 1D and 2D NMR spectra were recorded on a Bruker 400-and 500-MHz spectrometer.

Plant Material
The fresh leaves of Spinacia oleracea L., Amaranthaceae, were purchased from the Sara Organic Food Farm (https:// www.sarasorganicfood.com/), Egypt, in June 2018. The plant material was authenticated as S. oleracea L. (Baladi cultivar) by Hesham Elfayoumi, lecturer at Plant Taxonomy Department, Faculty of Science, Fayoum University, and voucher specimen (FUPD-48) was kept at the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Fayoum University, Egypt.

Extraction and Isolation Procedure
The shade dried leaves (1.6 kg) were ground and extracted six times with 75% ethanol at room temperature. The combined 75% hydroethanolic extract was concentrated in vacuum to afford a crude extract (303.8 g), that was suspended in water, and fractionated successively with hexane, dichloromethane, ethyl acetate, and n-butanol to afford fractions of 31, 2.9, 7.3, and 25.3 g, respectively, and aqueous mother liquor 235 g. The obtained fractions were subjected to biological testing against leukemia K562 cell line.

Phytochemical Study of the Hexane Fraction [Fr-A]
The hexane fraction of S. oleracea leaves was saponified according to the published procedure (Finar 1973) and the fatty acid methyl ester (FAME) and unsaponifiable matter (USM) were then subjected to GC/MS analysis.

Cytotoxicity Assay
K562 cells from the American Type Culture Collection (ATCC) were plated in a clear 384-well plate at an initial density of 2500 cells/well in 40 μl of growth medium (DMEM with 10% FBS and 1% pen/strep). Next day, the test agents were added in quadruplicate at the specified concentration and the treatment continued for 48 h and the cell viability was finally assessed using WST-8 assay Cell Counting Kit from Bimake, according to manufacturer's instructions. The results were calculated by measuring the absorbance at 450 nm using a Spectra Max M5 plate reader (Molecular Devices). Cell viability was calculated in comparison to DMSO as a negative control, Taxol and doxorubicin as a positive control (Kageyama et al. 2018). The extract and fractions were screened primary at concentration of 20 μg/ml and the percentage inhibitions were calculated. Isolated compounds were screened at six concentrations (5, 10, 25, 50, 75, and 100 μg/ml) and IC 50 s were calculated.

Molecular Docking
All docking simulations were conducted using MOE 2019 software (https://www.chemcomp.com). The receptors and the ligands were prepared using the standard structure optimization protocol of the software. The receptors were obtained from the protein data bank, PDB IDs: 3QX3, 3QRJ, 1M17, 2SRC, and 6QS9 for topoisomerase, Abl Kinase, EGFR-tyrosine kinase, SRC kinase, and albumin, respectively. Then they were energy minimized under AMBER12: EHT force field. The active sites were set as where the co-crystalized ligand was bound. The docking was performed using a molecular structure of compounds isolated from S. oleracea leaves using the general protocol of MOE DOCKTITE Wizard. Triangle matcher and London dG were utilized as the placement method and scoring algorithm, respectively. The validation of docking experiments was achieved through the re-docking of the cocrystalized ligands into their corresponding active sites and then the root mean square deviation (RMSD) was calculated. The docking results were visualized, and the docking scores were reported in kcal/mol.

Molecular Dynamics
To conduct the required molecular dynamics (MD) simulations, Groningen Machine for Chemical Simulations (GROMACS) 5.1.1 software was employed (Abraham et al. 2015). To validate the retrieved binding modes from the docking study, two MD simulation experiments were conducted. The two simulation experiments were performed on the most active compound 3 in complex with Abl kinase and topoisomerase. GROMOS96 force field was implemented to generate the ligand topology using the GlycoBioChem PRODRG2 Server (Schüttelkopf and Van Aalten 2004). Later on, complex topology was generated through joining both ligand and enzymes. As already published in the literature, the typical scheme for enzyme-ligand simulations by GROMACS was applied, starting with system solvation using a single point charge (SPC) water model and ending with neutralization by adding the suitable number of counter ions (El Hassab et al. 2020, 2021, 2022a, 2022b. The two solvated neutralized systems were energy minimized under GROMOS9643a1 force field using the steepest descent minimization algorithm with a maximum of 50,000 steps and 10 kJ/mol force under. All the systems were equilibrated to the used temperature (310 K) and pressure (1 atm) using NPT (isothermal-isobaric ensemble) for 2 ns preceded by NVT (canonical ensemble) for 1 ns. To compute the long-range electrostatic values, the particle mesh Ewald (PME) method with a 12-Å cut-off and 12-Å Fourier spacing was implemented. All the systems were subjected to a production stage of 50 ns. Every two consecutive steps were separated by 2 fs and the structural coordinates were saved every 20 ps. The V-rescale weak coupling method (modified Berendsen thermostat) and the Parrinello-Rahman method were used to regulate the temperature (310 K) and the pressure (1 atm) throughout the simulation (Parrinello and Rahman 1981;Berendsen et al. 1984). The root mean square deviation (RMSD) of the entire system was calculated from the generated trajectories from the production step.

Results and Discussion
Spinacia oleracea was selected based on the results of our previous screening of certain Egyptian leafy vegetables for antileukemic activity. The total 75% ethanolic extract of S. oleracea leaves exhibited a strong antiproliferative activity against K562 cell with 88.9% percentage of inhibition at a concentration of 10 mg/ml. The hexane, dichloromethane, ethyl acetate, n-butanol, and the aqueous fractions were tested at a concentration of 20 μg/ml and percentage inhibitions were calculated as 23, 19, 19, 20, and 18%, respectively, compared to doxorubicin and Taxol (86 and 79% at 10 μM, respectively). Based on the biological screening results as well as TLC screening of the bioactive fractions, the hexane and ethyl acetate fractions were selected for further phytochemical study with the aim of isolating the bioactive compounds.

Biological Study
The antileukemic activity of the major constituent of the saponifiable matter of hexane fraction, the linolenic acid, was previously reported in many research articles (Beaulieu et al. 2011;Ge et al. 2009;Harada et al. 2002;Jóźwiak et al. 2020;Liu and Leung 2014;Mainou-Fowler et al. 2001;Moloudizargari et al. 2018;Valencia-Serna et al. 2013). Methyl oleate (Saab et al. 2011) and methyl linolenate (Ge et al. 2009) were also reported for their in vitro antileukemic activity against K562 cells. Thus, the activity of saponifiable matter may be attributed to the presence of those compounds. The compounds isolated from USM, hexaprenol (1), phytol (2), and 18-[(1-oxohexadecyl) oxy]-9-octadecenoic acid (3) (NMR spectrum Figs. S5-S7, Supplementary materials), exhibited antiproliferative activity against K562 cells with IC 50 of 44.89, 33.28, and 70.58 μg/ml, respectively, compared to doxorubicin and Taxol with IC 50 of 11.41 and 1.70 μg/ml, respectively. The antileukemic activity of compound 2 against K562 cells was previously reported (Anuchapreeda et al. 2020) while this is the first report of the activity of compounds 1 and 3 against K562 cells. The compounds isolated from the ethyl acetate fraction showed no antiproliferative activity against K562 cell line. However, some of these compounds have several applications that could be beneficial in the management of CML disease. For example, vitexin-2′′-Orhamnoside (16), which is a major compound in the EtOAc fraction, reported for its antioxidant and anti-apoptotic activities (Wei et al. 2014). Margaritene (15) also was reported for its antioxidant activity (Lou et al. 2015). Additionally, 22,23dihydrospinasterol (10) and spinasterol (11) were recently reported for their moderate antioxidant activity (Ahmed et al. 2022).
In a clinical study conducted on 47 CML patients, the oxidative stress was reported to be associated with the pathophysiology of CML (Ahmad et al. 2008). Thus, the use of antioxidants could be beneficial for CML patients. Spinach leaves extract was reported for its antioxidant activity in several clinical studies (Cao et al. 1998;Castenmiller et al. 1999;Pool-Zobel et al. 1997;Porrini et al. 2002). Therefore, the antioxidant activity of the spinach extract or its isolated compounds in addition to the antileukemic activities of the isolated compounds might work in symphony to improve CML disease, but this still needs further in vivo and preclinical investigations.

Molecular Docking
Multitarget therapies are crucial in the field of complex diseases, such as cancers and inflammatory and thrombotic diseases which can be affected by several cellular pathways (Skok et al. 2019). Targeting two different pathways involved in the development of a disease can represent logic solution for this challenge and can reduce the potential for the development of resistance (Skok et al. 2019).
Recent research showed a correlation between CML cell line (K562) and several target proteins whose inhibition leads to antiproliferative effect in this cell line. Four target proteins are named: human topoisomerase II beta in complex with DNA and etoposide (PDB ID-3QX3), epidermal growth factor receptor complexed with erlotinib (PDB ID-1M17), human ABL1 kinase (PDB ID-3QRJ), and SRC kinase (PDB ID-2SRC) were reported as docking targets in the K562 cell line (James et al. 2017;Zamakshshari et al. 2019). The active isolated compounds were docked into the active site of the selected targets and the resulted docking scores are reported in Table 3. Hexaprenol (1), phytol (2), and 18-[(1oxohexadecyl) oxy]-9-octadecenoic acid (3) showed a strong binding affinity towards all the selected targets (Table 3). The best docking results were obtained with topoisomerase and Abl kinase enzymes and in turn their docking results were selected for further analysis. As depicted in Fig. 1, compound 1 showed a strong binding pattern towards the topoisomerase enzyme through forming several interactions with DT9, DG10, DC11, and DC14 in the DNA in addition to one interaction with Arg820. Similarly, compound 2 formed six interactions with DA12, DG13, Gly776, and Ala779, while compound 3 interacted with DG13, Lys505, and Arg820. The interaction pattern and docking scores of the three isolated compounds with topoisomerases is very similar to the crystal reference etoposide (Table 3, Fig. 1). Moreover, the three isolated compounds achieved acceptable docking scores with Abl kinase, having binding scores of −11.91, −9.35, and −12.59 kcal/mol for compounds 1, 2, and 3, respectively (Table 3). Moreover, compound 1 formed four interactions with Phe359 and His361, while compound 2 formed four interactions with Met290, Asn358, and Ile360 (Fig. 2). At last, compound 3 interacted with Lys271, Met290, and Phe382 (Fig. 2).
Molecular docking provided a mechanistic information on the possible antiproliferative activity against K562 cells through binding with Abl kinase and topoisomerase.
The in silico-based safety analysis of the antileukemic compounds was tested by measuring their binding affinity to albumin (PDB ID-6QS9) (Behzadi et al. 2019). The affinity of cytotoxic compounds to bind to albumin has a great impact on their pharmacodynamics and pharmacokinetic properties; for example, the anticancer agent chlorambucil is 99% bound to albumin and yet has a short half-life of 1.3 ± 0.90 h (Sparreboom and Loos 2004). In accordance with this (1) (3) etoposide Fig. 1 The 2D interaction diagrams of compounds (1, 2, and 3) and etoposide with topoisomerase information, compounds 1 and 2 are safer in silico and with greater half-life than compound 3 (Table 3).

Molecular Dynamics
Molecular dynamics (MD) simulation has been an inevitable technique in studies involving in silico drug discovery. MD provides many important parameters, data, and figures necessary in various computational and molecular modelling studies. One of the most common applications of the MD is the precise determination of the binding stability between a ligand and its target. Therefore, it was logistic to take the advantage of the MD to further endorse our docking results. Two MD simulation experiments were conducted on compound 3 bound to Abl kinase and topoisomerase. Interestingly, the calculated RMSD for compound 3 with Abl kinase and topoisomerase reached 1.85 and 1.81 Å respectively at their maximum deviations (Fig. 3). The ability of compound 3 to produce such a lower RMSD value is a powerful indicator of its ability to produce stable complexes with Abl kinase and topoisomerase. The MD results supported the docking results and highlighted the ability of the isolated compounds as antileukemic agents.

Conclusions
The hexane fraction of Egyptian Spinach leaves as well as its isolated compounds, hexaprenol (1), phytol (2), and 18-[(1oxohexadecyl) oxy]-9-octadecenoic acid (3), showed 1 2 3 rebastinib Fig. 2 The 2D interaction diagrams of compounds (1, 2, and 3) and rebastinib with Abl kinase remarkable antiproliferative activity against leukemia K562 cell line. The molecular docking study revealed that this activity is supposed to be through targeting Abl kinase and topoisomerase, and this still needs to be proved by in vitro assay of these compounds against the mentioned targets. As a result of our findings, we recommend more in vivo and preclinical investigations to confirm the potential benefit of spinach leaves for CML patients.

Declarations
Conflict of Interest The authors declare no competing interests.
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