Research Article

Phytosterol Glycosides from Olax subscorpioidea Oliv. Exhibit Cytotoxic Effects in In Vitro and In Silico Studies

Introduction

Plants have provided medicine for humans for many centuries.^1,2 A large percentage of the world’s population, especially in developing countries, depend on medicinal plants as sources of medicines.³ Secondary metabolites have been recognised as the healing principles in plants. Cancer is a leading cause of death globally.⁴ It kills about 10 million people every year across the globe.⁵ Approximately 70% of cancer-related deaths occur in low- and middle-income countries.⁶ Many cancer drugs have been developed, the majority of which have serious adverse effects.^1,7 Therefore, there is a need for anticancer drugs that are more tolerable, effective, and less toxic.

Olax subscorpioidea Oliv. belongs to the Olacaceae family that comprises shrubs, trees and woody dicotyledonous plants with significant economic and medicinal values. This family includes a range of secondary metabolites, such as flavonoids, triterpenoids, and secoiridoids, each exhibiting diverse biological activities.^8-10 The plants, distributed within about 25 genera, are widely distributed in temperate and tropical regions.⁹

Olax subscorpioidea is a shrub-like tree that reaches a height of approximately 10 metres.¹¹ The plant is traditionally used to treat arthritis, cancer, depression, diabetes mellitus, neurodegenerative disorders, oxidative stress, and swelling.^12,13 Ethnobotanical surveys showed that O. subscorpioidea is an important ingredient in traditional cancer remedies in Nigeria.^14-16 We recently reported cytotoxic triterpenoid saponins from O. subscorpioidea.¹⁷

The preliminary cytotoxic analysis of the methanol root extract of O. subscorpioidea showed antiproliferative activity against human rhabdomyosarcoma (RD) and breast cancer (MCF-7) cell lines.¹⁸ The current study, therefore, aimed at isolating and characterising bioactive compounds from O. subscorpioidea root extract. The potential of these compounds as inhibitors of the growth of human cancer cell lines was evaluated by the MTT assay. Molecular docking and ADMET (absorption, distribution, metabolism, excretion, toxicity) studies were also conducted.

Methods

General experimental procedures

The infrared spectroscopy (IR) spectrum was obtained using a Cary 630 FTIR spectrophotometer (Agilent Technologies, USA). 1D and 2D nuclear magnetic resonance (NMR) data were obtained on a Bruker Avance III NMR spectrometer at 600 MHz (Bruker, USA). The sample was dissolved in dimethyl sulfoxide (DMSO-d6) and the solvent residue served as the internal standard. Chemical shifts (δ) were measured in parts per million (ppm), and coupling constants were measured in hertz (Hz). High-performance liquid chromatography (HPLC) grade solvents from Fisher Scientific (Loughborough, United Kingdom) were used. Cell culture materials include Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, UK), L-glutamine 200 mM (100X) (Gibco, UK), antibiotic-antimycotic (100X) (Gibco, UK), TrypLE Express (1X) (Thermo Fisher Scientific, UK), Fetal bovine serum (Sigma-Aldrich, UK), Phosphate buffered-saline (Thermo Fisher Scientific, UK), and MTT (Sigma-Aldrich, UK). Vinblastine from Tocris (UK) was used as a reference drug. Absorbance measurements were performed on a Tecan Spark 10M multimode microplate reader (Switzerland).

Plant collection

The root of O. subscorpioidea was collected in April 2021 from the University of Ibadan botanical garden, Ibadan, Nigeria. The collection and identification of the plant were performed by the garden curator, Mr Michael Owolabi. A sample was deposited at the Forest Herbarium, Ibadan (FHI) with voucher number FHI:113182. The root material was cleaned, peeled, and dried in the lab. After that, it was ground into powder.

Extraction and fractionation

Approximately 600 g of powdered O. subscorpioidea root material was extracted with methanol using the Soxhlet extraction method. The extract was concentrated in vacuo using a rotary evaporator at a temperature below 40 °C (Heidolph HB Digital, Germany). The resulting extract was dried at room temperature in the laboratory and stored for later use. About 63.7 g of extract was obtained. The extract was further partitioned into n-hexane, dichloromethane (DCM), and water/methanol. Approximately 40 g of the extract was dissolved in water/methanol (20/80%) in a separating funnel and partitioned with an equal volume of n-hexane. The hexane fraction was obtained and concentrated. The extraction was done three times. Into the aqueous fraction, an equal volume of DCM was added (extraction was carried out three times) and the fraction was concentrated. The aqueous fraction was also obtained and concentrated.

Isolation of bioactive compounds

The isolation of compounds was carried out on an open glass chromatographic column packed with 60-120 mesh silica gel. The DCM fraction, 5.48 g, was re-suspended and loaded on the chromatography column (75 cm × 3.5 cm). The sample was eluted with n-hexane/ethyl acetate 70%:30% to 100% ethyl acetate, followed by ethyl acetate/methanol, 90%:10%; 80%:20%; 70%:30%; 50%:50%; 30%:70%; and 0%:100%. A total of 64 fractions were obtained (approximately, 30 mL each), which were pooled into 5 (F1–F5) according to their retention factor (Rf) pattern (F₂₅₄ silica gel 60 TLC plate, 70/20/10% n-hexane/ethyl acetate/methanol). Fraction F4 was further purified by crystallisation to obtain a white amorphous powder. The substance contained a mixture of two compounds assigned as 1 (9.8 mg). The compound showed a retention factor (Rf) of 0.6 in ethyl acetate/methanol 9.5/0.5%. Compound 1 was visualised under an ultraviolet (UV) lamp (UVITEC, Cambridge). The developed TLC plate (Figure S1) was detected by being sprayed with conc. H₂SO₄, and then left in the oven at 110℃ for about 10 min (Oven DHG-9053A, Ocean Med, England).

Cytotoxic analysis

Cells and cell culture

Human Caco-2, HeLa, and MCF-7 cancer cell lines were originally obtained from the American Type Culture Collection (ATCC) or the European Collection of Authenticated Cell Cultures (ECACC). As previously described,¹⁷ the cells were cultured in DMEM supplemented with 10% Foetal Bovine Serum (FBS), 1% 2 mM L-glutamine, and 1% penicillin-streptomycin-amphotericin B solution. They were maintained in a 37 °C incubator with a 5% CO₂ humidified atmosphere. Cells were passaged approximately twice weekly. Before the treatment, cells were seeded into 96-well microplates and incubated for 24 hours. Cell visualization (at × 10 magnification) was performed using an Olympus CKX41 inverted microscope.

Cell treatments and cell viability assessment with the MTT assay

Stock solutions were prepared in DMSO, and all further dilutions were made in full growth medium. The highest concentrations used contained no more than 0.1% DMSO, which was non-toxic to the cells. Each well containing approximately 7,500 cells, prepared as previously reported,¹⁷ was treated with the sample (concentrations: 1, 10, 50, 100, and 200 µg/mL). Each experiment was performed in triplicate and repeated three independent times (n = 3). The effects on the viability of the three human cell lines of the isolated compounds were assessed using the in vitro MTT assay.¹⁹

Molecular docking study

Ligand, protein retrieval and preparation

The ligands used in this study were the structures of stigmast-5,22-dien-3-O-β-D-glucoside and sitosterol-3-O-β-D-glucoside isolated from the methanol root extract of O. subscorpioidea. Their structures were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The 3D structures were prepared for docking using the UCSF Chimera tool (1.16).²⁰ Gasteiger charges and hydrogen atoms were added.²¹ Energy minimisation of the structures was carried out at 100 steepest descent steps.

The 3D structures of the following proteins were retrieved from the Protein Data Bank (PDB) (https://www.rcsb.org/).²² Alpha-beta tubulin complexed with taxol (PDB ID: 1JFF),²³ alpha-beta tubulin complexed with colchicine (PDB ID: 1SA0),²⁴ caspase-3 (PDB ID: 1GFW),²⁵ human 17 beta-hydroxysteroid dehydrogenase-1 (PDB ID: 1FDW),²⁶ phosphoinositide 3-kinase (PDB ID: 1E8Z),²⁷ poly (ADP-ribose) polymerase-1 (PDB ID: 5DS3),²⁸ bromodomain-containing protein 4 (PDB ID: 7JKW),²⁹ epidermal growth factor receptor tyrosine kinase domain (PDB ID: 1M17),³⁰ human oestrogen receptor alpha (PDB ID: 3ERT),³¹ and polo-like kinase-1 (PDB ID: 3FC2).³² To prepare the proteins, they were individually uploaded to the workspace of Chimera. All ligands, small molecules, water, and other co-crystallised compounds were deleted. Hydrogen atoms were added to the proteins. In addition, partial charges were added by ANTECHAMBER. The structures were then minimised to 200 steepest descent steps at 10-minute intervals. The prepared proteins were saved in.pdb to be used for docking.³³

Docking

A molecular docking study was carried out to obtain information about the various binding conformations and binding energies of the compounds when interacting with the selected proteins. A site-directed docking was performed for all proteins. First, they were uploaded to the GPU of the Python Prescription Virtual Screening Tool (PyRx). They were then made into macromolecules. The active site of each protein had been pre-determined as the residues within 5 Å region of the co-crystallised ligand in Chimera. The ligands were imported to the GPU of PyRx, converted to AutoDock ligand format (.pdbqt), and the grid box set, fitting the active site which was defined by the grid box coordinates as shown in Table 1. Docking was carried out with the AutoDock Vina of PyRx.³⁴ BIOVIA Discovery Studio Visualiser was used to analyse the protein-ligand interactions.²¹

ADMET study

Drug-likeness means how much a compound’s properties resemble those of existing drugs. The physicochemical parameters and ADME properties of the isolated compounds were evaluated using a freely available web tool, SwissADME (http://www.swissadme.ch/).³⁵ Some of the parameters obtained include molecular weight, lipophilicity, hydrogen bond acceptors and donors, topological polar surface area (TPSA), enzyme inhibition, and synthetic route accessibility.

Statistical analysis

Each experiment was carried out three independent times (n = 3). Data is presented as mean of IC₅₀ ± SEM (standard error of the mean). GraphPad Prism (9.4) was used to calculate the IC₅₀. One-way ANOVA was used to determine the statistical significance of differences between means (P < 0.05 considered significant).

Results and Discussion

Cytotoxic effects of O. subscorpioidea fractions

Olax subscorpioidea is found in many countries across West and Central Africa and its traditional applications include its use to treat cancer, diabetes mellitus, neurodegenerative disorders, and swelling.^12,13 An ethnobotanical study among people in Ogun State, Nigeria, discovered that a mixture containing O. subscorpioidea root, along with other plants including Alafia barteri leaf, Anthocleista djalonensis root, Calliandra portoricensis root, Clausena anisata stem bark, Macaranga barteri stem bark, Tephrosia vogelii stem bark, Triclisia subcordata leaf, Xylopia aethiopica, and potash, is used to treat breast cancer.¹⁵ A preliminary study of the cytotoxic activity of O. subscorpioidea revealed activity against MCF-7 and RD.¹⁸ The methanol root extract was further fractionated into n-hexane, DCM, and aqueous media. The DCM fraction showed the highest activity against the two cell lines with percentage cell inhibition of 80.08% for MCF-7 and 82.01% for RD. The activity was closely followed by that of the n-hexane fractionwith percentage cell inhibition of 78.42% for MCF-7 and 79.98% for RD. The aqueous fraction showed lower activity, having cell inhibition of 8.46% for MCF-7 and 40.87% for RD.¹⁸ The current study focused on isolating bioactive compounds from the DCM fraction and evaluating their cytotoxic effects against human cancer cell lines.

Structure determination

The DCM fraction yielded a white amorphous powder, 9.8 mg (Compound 1). Extensive NMR experiments revealed that the isolated compound was a mixture of two known sterol glycosides. In the ¹³C NMR experiment, the signals at δ_C 101.05 and 101.27, and 121.66 and 121.69 occurred as pairs each indicating a mixture of two similar compounds. The heteronuclear multiple bond correlation (HMBC) spectrum was used to verify the identity of the individual compounds in each mixture, which were identified as stigmasterol glucoside and β-sitosterol glucoside by detecting the distinct signals at positions C-22 and C-23.³⁶ Using HMBC spectrum, the chemical shifts of stigmasterol glucoside at positions C-22 and C-23 were confirmed as 138.5 and 129.3 ppm, respectively, indicating the presence of an alkene group. In contrast, β-sitosterol glucoside showed chemical shift signals for C-22 and C-23 resonating at 33.8 and 25.9 ppm, respectively, suggesting an alkyl group (Table S1). Homonuclear correlation spectroscopy (¹H-¹H COSY), heteronuclear single quantum correlation (HSQC), and HMBC spectra helped confirm all ¹H and ¹³C assignments (Figures 1a and 1b, and Figures S2–S7).

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Figure 1.

Structure of compound 1a and 1b

Figure 1.

Structure of compound 1a and 1b

Stigmast-5,22-dien-3-O-β-D-glucoside (1a), white amorphous powder. IR: 3360 cm^-1, broad (OH stretch), 2910 cm^-1 (CH stretch), and 1010 cm^-1 (CO stretch). ¹H NMR (DMSO, 600 MHz): δ_H 2.12, 2.35 (2H, C-1), 3.45 (m, 1H, C-3), 1.79 (2H, C-4), 5.33 (t, 1H, C-6), 1.91 (2H, C-7), 1.43 (1H, C-8), 0.99 m (1H, C-14), 1.79 (2H, C-16), 0.66 (s, 3H, C-18), 0.95 (s, 3H, C-19), 0.99 (d 6.48 Hz, 3H, C-21), 5.15 (m, 1H, C-22), 5.02 (m, 1H, C-23), 1.50 (1H, C-24), 1.34 (1H, C-25), 0.82 (3H, C-26), 0.78 (d 2.32 Hz, 3H, C-27), 0.77 (3H, C-29), 4.20 (d, 7.84 Hz, C-1’). ¹³C-DEPT-Q δ_C: 38.8 (C-1), 77.4 (C-3), 37.3 (C-4), 140.9 (C-5), 121.7 (C-6), 31.8 (C-7), 55.8 (C-17), 12.3 (C-18), 19.6 (C-19), 21.6 (C-21), 138.5 (C-22), 129.3 (C-23), 51.1 (C-24), 31.9 (C-25), 21.4 (C-26), 19.3 (C-27), 12.6 (C-29), 101.2 (C-1’) 73.9 (C-2’), 77.2 (C-3’), 70.5 (C-4’), 77.2 (C-5’), 61.5 (C-6’).^37-40

Sitosterol-3-O-β-D-glucoside (1b), white amorphous powder. IR: 3360 cm^-1, broad (OH stretch), 2910 cm^-1 (CH stretch), and 1010 cm^-1 (CO stretch). ¹H NMR (DMSO, 600 MHz): δ_H 3.45 m (1H, C-3), 5.33 (t, 1H, C-6), 1.91 (2H, C-7), 0.99 m (1H, C-14), 1.38 (2H, C-15), 1.78 (2H, C-16), 1.13 (1H, C-17), 0.66 (s, 3H, C-18), 0.80 (s, 3H, C-19), 1.33 (1H, C-20), 0.95 (3H, C-21), 1.00, 1.29 (2H, C-22), 1.14 (2H, C-23), 0.91 (1H, C-24), 0.90 (d, 6.22 Hz C-27), 0.81 (3H, C-29), 4.20 (d, 7.84 Hz C-1’), 3.08 (1H, C-3’), 3.04 (1H, C-5’), 3.39, 3.63 (1H, C-6’). ¹³C-DEP-Q δ_C: 36.7 (C-1), 29.0 (C-2), 77.3 (C-3), 140.9 (C-5), 121.7 (C-6), 50.1 (C-9), 36.7 (C-10), 21.1 (C-11), 37.3 (C-12), 56.6 (C-14), 25.4 (C-15), 28.3 (C-16), 55.9 (C-17), 12.2 (C-18), 20.2 (C-19), 36.0 (C-20), 19.4 (C-21), 33.8 (C-22), 25.9 (C-23), 45.6 (C-24), 29.2 (C-25), 19.4 (C-26), 19.1 (C-27), 23.1 (C-28), 12.3 (C-29), 101.1 (C-1’), 73.9 (C-2’), 77.2 (C-3’), 70.5 (C-4’), 77.2 (C-5’), 61.5 (C-6’).^38,41,42

The occurrence of β-sitosterol and stigmasterol or their glycosides together in the same plant is common. Similarly, the isolation of the two compounds or their glycosides together as a mixture is also frequent. Kamal et al³⁶ isolated a mixture of β-sitosterol and stigmasterol from Vitex pinnata L. Ekhuemelo et al⁴³ isolated a mixture of sitosterol, stigmasterol and cycloeucalenol from Erythrophleum suaveolens (Guill. & Perr.) Brenan. Here are a few other plants from which a mixture of the compounds was reported: Baccaurea macrocarpa Miq. Mull. Arg,⁴⁴ Cassia sieberiana DC.,⁴⁵ Premna herbacea Roxb.,⁴⁶ and Ricinus communis L.⁴⁷ Elfita et al⁴⁸ isolated a mixture of their glycosides from Garcinia griffithii T. Anderson. These compounds also exhibit a variety of biological activities.

Cytotoxic effects of the isolated compounds

The cytotoxic potencies of the isolated compounds, as indicated by the IC₅₀ values, are presented in Table 2. Compound 1, a mixture of stigmast-5,22-dien-3-O-β-D-glucoside (1a) and sitosterol-3-O-β-D-glucoside (1b), showed cytotoxicity against the HeLa and MCF-7 cell lines. Its most potent cytotoxicity was against the HeLa cell line, with an IC₅₀ value of 37.0 ± 4.51 µM, although it also exhibited mild cytotoxicity against the MCF-7 cell line, with an IC₅₀ value of 137.07 ± 19.43 µg/mL (Table 2). Ikpefan et al⁴⁹ reported that stigmast-5,22-dien-3-O-β-D-glucoside was sensitive to MCF-7 and NCI-H460 cell lines with GI₅₀ values of 40.83 ± 0.1 and 58.83 ± 11.2 µM, respectively, using the sulforhodamine B (SRB) cytotoxicity assay. Quradha et al⁴² reported that sitosterol-3-O-β-D-glucoside had moderate activity against the HeLa cell line, with a cell inhibition of 48%, and a weak effect on the 3T3 cell line (mouse fibroblast), with a cell inhibition of 39%. This compound isolated from the aerial part of Cynara cardunculus var. altilis was also shown to have an IC₅₀ value of 53.6 µg/mL against the hepatocellular carcinoma (HepG2) cell line.⁵⁰ A study investigating the effects of sitosterol-3-O-β-D-glucoside on HepG2 and SMMC-7721 cells, via the Wnt/β-catenin signalling pathway, found that it inhibited cell migration and invasion in HepG2 cells through this signalling pathway.⁵¹ Another study revealed that sitosterol-3-O-β-D-glucoside exhibited its cytotoxic effects through the induction of apoptosis and activation of caspase-3 and -9 in the hepatocellular (Huh7 and HepG2) cancer cell lines.⁵²

Molecular docking study

Molecular docking is a crucial computational tool in predicting binding conformations and binding energies of small molecule drugs (ligands) to receptor proteins, which enables high-throughput virtual screening.⁵³ In this study, ten cancer-related proteins were selected for the docking study. The compounds docked well with all the proteins. Stigmast-5,22-dien-3-O-β-D-glucoside consistently showed stronger interactions than sitosterol-3-O-β-D-glucoside with some of the proteins. The best interactions, with the least negative binding energies, were observed with alpha-beta tubulin complexed with colchicine (PDB ID: 1SA0). Compounds 1a and 1b showed binding energy of –10.3 and –10.0 kcal/mol, respectively. For compound 1a, this was followed by epidermal growth factor receptor tyrosine kinase domain (EGFR) (PDB ID: 1M17) and 17β-hydroxysteroid dehydrogenase-1 (17β-HSD1) (PDB ID: 1FDW) with a binding energy of –9.3 kcal/mol. Poly (ADP-ribose) polymerase-1 (PARP-1) (PDB ID: 5DS3) had a binding energy of –9.2 kcal/mol for compound 1a. For compound 1b, EGFR ranked second with a binding energy of –9.3 kcal/mol, while PARP-1 had a binding energy of –9.2 kcal/mol (Table 3). The two compounds showed the least binding interactions with bromodomain-containing protein 4 (BRD4) (PDB ID: 7JKW) and caspase-3 (PDB ID: 1GFW).

The compounds show similar conformations at the colchicine-binding site of alpha-beta tubulin (Figures 2A and 3E). αβ-Tubulin heterodimers are microtubule proteins that are crucial for the mitotic phase of cell division. Classical inhibitors of the β-tubulin subunit act by promoting or disrupting microtubule assemblage during mitosis by binding to the colchicine-, vinca-, or taxol-binding sites.⁵⁴ The isolated compounds form a bridge between the α- and β-subunits of tubulin. The compounds were orientated in such a way that the polar glycosidic tail pointed toward the α-chain, while the non-polar head formed hydrophobic interactions with the β-chain. The compounds were observed to stabilise at the colchicine-binding pocket with crucial amino acid residues such as threonine (THR) 179 and cysteine (CYS) 241, the residues that have been implicated in the binding of colchicine-like compounds to the protein.⁵⁴ Similarly, colchicine hydrogen bonds with the CYS241 residue of β-tubulin and shows hydrophobic interaction.⁵⁵

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Figure 2.

Interacting residues of (A) Alpha-beta tubulin, (B) EGFR, (C) 17β-HSD1, and (D) PARP-1 with compound 1a. Green: Conventional hydrogen bonding. Lilac: Alkyl/pi-alkyl interaction. Blue: Carbon hydrogen bonding. Purple: Pi-sigma bonding. Light green: van der Waals forces

Figure 2.

Interacting residues of (A) Alpha-beta tubulin, (B) EGFR, (C) 17β-HSD1, and (D) PARP-1 with compound 1a. Green: Conventional hydrogen bonding. Lilac: Alkyl/pi-alkyl interaction. Blue: Carbon hydrogen bonding. Purple: Pi-sigma bonding. Light green: van der Waals forces

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Figure 3.

Interacting residues of (E) Alpha-beta tubulin, (F) EGFR, (G) 17β-HSD1, and (H) PARP-1 with compound 1b. Green: Conventional hydrogen bonding. Lilac: Alkyl/pi-alkyl interaction. Blue: Carbon hydrogen bonding. Purple: Pi-sigma bonding. Light green: van der Waals forces

Figure 3.

Interacting residues of (E) Alpha-beta tubulin, (F) EGFR, (G) 17β-HSD1, and (H) PARP-1 with compound 1b. Green: Conventional hydrogen bonding. Lilac: Alkyl/pi-alkyl interaction. Blue: Carbon hydrogen bonding. Purple: Pi-sigma bonding. Light green: van der Waals forces

17β-HSD1 converts weakly active oestrone to a pharmacologically more active estrogen, 17β-oestradiol, in the breast tissue using nicotinamide adenine dinucleotide (NADH) or its phosphate (NADPH) as a cofactor. 17β-oestradiol is crucial for the growth of breast cancer cells.⁵⁶ The two compounds interacted with important 17β-HSD1 residues involved in its catalytic functions, which include tyrosine (TYR) 155 and lysine (LYS) 159 (Figures 2C and 3G).⁵⁷ EGFR residues interacted with the compounds via alkyl, van der Waals, and hydrogen bonds (Figures 2B and 3F). The sugar moiety was used to interact with the protein via hydrogen bonding. In PARP-1, compound 1a formed a π-sigma bond with the protein in addition to alkyl, π-alkyl, van der Waals, and hydrogen bondings (Figures 2D and 3H). Since most of the selected proteins are expressed in the tested cancer cell lines, it can be suggested that the in silico study supports the in vitro study.

Drug-likeness study

In 1997, Christopher Lipinski from Pfizer introduced the “Rule of 5.” It suggests that orally administered drugs are less likely to be absorbed or permeate well if they have a molecular weight exceeding 500 g/mol, more than 5 hydrogen bond donors, more than 10 hydrogen bond acceptors, or a CLog P greater than 5.⁵⁸ Although the “Rule of Five” is not sacrosanct because many blockbuster drugs in the clinic fail it, it has greatly helped in lead optimisation and drug design. In drug development, a drug candidate is assessed for its absorption, distribution, metabolism and elimination.³⁵ Table 4 shows the physicochemical parameters and ADMET descriptors for the isolated compounds 1a and 1b as virtually estimated by the SwissADME software. The compounds have molecular weights greater than 500 g/mol, which violate the Rule of 5. However, other parameters (Log P, hydrogen bond acceptor and hydrogen bond donor) are within acceptable ranges. The compounds are also not inhibitors of metabolic enzymes.

The bioavailability radar helps in the initial evaluation of how drug-like a molecule is, using the following physicochemical properties: Lipophilicity (LIPO), Flexibility (FLEX), Insaturation (INSATU), Insolubility (INSOLU), Polarity (POLAR), and Size (SIZE) (Figure 4). Lipophilicity is measured by Log P, which ranges from −0.7 to + 5.0. Size is assessed by molecular weight ( ≤ 500 g/mol). Polarity is indicated by TPSA ( ≤ 140 Ų). Insolubility is represented by log S. Insaturation is evaluated based on the fraction of carbon atoms in sp³ hybridization. Flexibility is measured by the number of rotatable bonds ( ≤ 9).⁵⁹ The bioavailability radar in Figure 4 shows that the bioavailability rating of the two compounds was slightly within the suitable physicochemical space for oral bioavailability (www.swissadme.ch).

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Figure 4.

Bioavailability radar for the isolated compounds 1a (A) and 1b (B). LIPO: Lipophilicity. FLEX: Flexibility. INSATU: Insaturation. INSOLU: Insolubility. POLAR: Polarity. SIZE: Size

Figure 4.

Bioavailability radar for the isolated compounds 1a (A) and 1b (B). LIPO: Lipophilicity. FLEX: Flexibility. INSATU: Insaturation. INSOLU: Insolubility. POLAR: Polarity. SIZE: Size

Conclusion

Medicinal plants are a productive source of drugs, including anticancer agents. In this study, stigmast-5,22-dien-3-O-β-D-glucoside and sitosterol-3-O-β-D-glucoside were isolated from the methanol root extract of O. subscorpioidea. Their structures were established by various spectroscopic data analyses (FTIR (Figure S8), 1D & 2D NMR). The results of the MTT assay show that the compounds were cytotoxic against human cervical (HeLa) and breast (MCF-7) cancer cell lines, with IC₅₀ values of 37.0 ± 4.51 and 137.07 ± 19.43 µg/mL, respectively. The results of the docking analysis of the isolated compounds showed they had strong binding affinities for the colchicine-binding site of alpha-beta tubulin, epidermal growth factor receptor kinase, poly (ADP-ribose) polymerse-1, and 17β-hydroxysteroid dehydrogenase-1. To the best of our knowledge, this is the first report of the isolation of these compounds from O. subscorpioidea. These isolated compounds may, therefore, contribute to the cytotoxic effects of O. subscorpioidea.

Competing Interests

The authors declare no conflicts of interests.

Ethical Approval

Not applicable.

Supplementary Files

References

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