Metabolic Profiling, in vitro Cytotoxicity and in silico Investigation of Lycium shawii Roem. Extract

Significant progress has been made in cancer therapy, leading to a remarkable improvement in the rates of survival of breast cancer (BC) patients. However, even with these progressions, BC still stands as the primary contributor to cancer-related fatalities in women across the globe (Hortobagyi et al., 2005). In 2016, it accounted for 535,000 deaths in 195 countries, presenting considerable clinical challenges (Fitzmaurice et al., 2018), (Xiao et al., 2019), (Carpenter et al., 2019). BC can be classified into four primary molecular subtypes based on the expression of estrogen receptor (ER), epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR). Among these subtypes, triple-negative BC (TNBC) stands out as the most aggressive and fast-growing form of BC, characterized by the absence of HER2, ER, PR and receptors (Burguin et al., 2021). Consequently, standard treatments like hormone therapy and targeted drugs are ineffective, leaving limited options for TNBC treatment. Cytotoxic chemotherapy is the primary approach in this context, showing initial efficacy in earlier stages but a higher recurrence rate than other BC types (Wang et al., 2019). Managing TNBC, especially its highly metastatic variant, remains a considerable challenge due to the absence of targeted therapies. Therefore, there is an urgent need for innovative treatment modalities to save lives (Mehanna et al., 2019).

Between 1981 and 2014, over 150 drugs derived from natural products were introduced to the pharmaceutical market (Baskar et al., 2012). The extensive biodiversity of plant species offers a vast resource for the discovery of new compounds with anticancer potential and ongoing investigations continue to unveil the healing potentials of these natural extracts in the fight against cancer (Cragg et al., 2009) (Feng et al., 2023) (Manosroi et al., 2017). Lycium shawii Roem. and Schult, an indigenous plant found in the Arabian Peninsula (Ali et al., 2020). It is a thorny shrub belonging to the Solanaceae family. Locally referred to as “Awsaj,” it has a history of use in traditional medicine for treating conditions like jaundice, stomach ailments, mouth sores and coughs (Rehman et al., 2016).  Notably, L. shawii has demonstrated a wide range of beneficial properties, including anti-inflammatory, antimicrobial, antioxidant, anti-diabetic and anticancer properties (Albarrak, 2021; Lee et al., 2012; Ali et al., 2020; Usha et al., 2016; Tahraoui et al., 2007).

This study aimed to explore the cytotoxic, apoptotic and anti-migration properties of L. shawii extracts. In addition, in silico molecular docking identified potential bioactive anticancer compounds, offering valuable insights for the development of novel drugs.

Study area
 
This experiment was conducted at King Saud University, Diriyah, Kingdom of Saudi Arabia, from October 2022 to December 2023.
 
Plant material
 
The plant used in this study was collected from Irqah, Riyadh province (Saudi Arabia). A specimen of Lycium shawii Roem was deposited in the herbarium collection under the acquisition number BRC-IRQA7-23. The plant was dried using a hot air oven at 50^oC for 48 h. The aerial parts were ground in a commercial mill and used for extraction.
 
Extraction of plant material
 
Extraction of saponin
 
The dry aerial parts (25 grams) of L. shawii were refluxed in an EtOH-H₂O mixture (2:8, v/v, 0.3 L × 2) for 4 hours and subsequently sonicated for 30 minutes. The resulting extract was filtered using cheesecloth and centrifugation at 4350 × g for 3 minutes. The volume was then reduced to 200 mL using a rotavapor at 45^oC. The extract was defatted with hexane (3 × 200 mL) and then extracted with n-BuOH (3 × 200 mL). The butanol extract was subsequently evaporated using a rotavapor at 50^oC, isolating an n-BuOH-soluble fraction (520 mg).
 
Extraction of phenol
 
For 5 days, the powdered material (34 g) was allowed to macerate in 400 mL of 80% methanol while occasionally shaken. It was then filtered and concentrated under reduced pressure using a rotary evaporator at 45^oC.
 
Fractionation
 
The methanol extract was suspended in distilled water (500 ml) and transferred into a separatory funnel followed by the addition of 200 mL of n-hexane (× 2) and shaking vigorously and then left until two layers were formed. The hexane layer was separated and kept for evaporation. The exact process was repeated using solvents of increasing polarity, namely ethyl acetate (EtOAc) and butanol (n-BuOH). Each separated fraction was concentrated under reduced pressure using a rotary evaporator at 45^oC.
 
Extraction of bound phenolic compounds
 
Alkaline hydrolysis was used to extract the bound phenolic compounds of L. shawii according to the described method (Irakli et al., 2018) with some modifications. Briefly, the 23-gram residues obtained after free phenolic compounds extraction were washed with water and dried in an oven. They were then treated with 2 N NaOH (200 mL) for 15 min using a sonicator at 60^oC. The pH was adjusted to 7 using 2 N HCl and was extracted 5 times with EtOAc (3 × 300 mL). The EtOAc fraction was concentrated individually under reduced pressure at 45oC.
 
Cell culture
 
MDA-MB-231 (metastatic breast cancer), HepG2, Huh-7 (human hepatoma cell lines) and normal HUVECs (Human umbilical vein endothelial) cell lines were sourced from the DSMZ Cell Bank (Germany). These cell lines were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with fetal bovine serum (FBS, 10%). The cultures were kept at 37^oC in a 5% CO₂ humidified atmosphere.
 
MTT cytotoxicity assay
 
The cells were seeded in 24-well culture plates (1000 μL of medium/well) at a 5 × 10⁴ cells/well density. After 24 hours, the test substances were added to the wells in triplicate at different concentrations (50 to 500 μg/mL) and 0.01% methanol as the control. Proliferation activity was assessed by quantifying mitochondrial activity through the previously reported MTT reduction method (Al-Zharani et al., 2019).
 
Reactive oxygen species (ROS)
 
Fraction 2 (IC₅₀ concentration) was added to MDA-MB-231 cells (5 × 10⁴ cells/well) and then incubated for 24 h. Subsequently, 25 µM of DCFH-DA was added and incubated for 30 minutes. As a control, methanol (0.01%) was maintained. Images were captured using a fluorescent microscope (EVOS, USA).
 
Wound-healing assay
 
The impact of Fraction 2 on cellular migration was qualitatively assessed using a wound-healing method (Al-Zharani et al., 2019). In brief, 5×10⁴ MDA-MB-231 cells/well were seeded in 6-well plates, forming a confluent monolayer. Scratching was done with a sterile pipet tip and fresh FBS-deprived medium was added. Plates were incubated for 24 hours with 200 μg/mL Fraction 2. Scratch areas were imaged at 0, 24 and 48 hours and quantified using Image J.
 
Gas chromatography-mass spectrometry (GC-MS) analysis
 
The GC-MS analysis was carried out using the method of Abd El-Kareem et al. (2016). The chemical composition fraction 1 was carried out using GC-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) using TG–5MS capillary column (30 m × 0.25 mm × 0.25 µm film thickness). The compounds were identified using NIST14 and WILEY 09 mass spectral databases. di-o-glucoside).
 
Molecular docking
 
Based on the literature, estrogen receptor alpha Y537S (ER-ALPHA (PDB ID-6CHZ), apoptosis regulator Bcl-2 (PDB:4MAN), Myeloid leukemia 1 (MCL-1) (PDB ID: 5FDO) and BCL-W (PDB ID: 2Y6W) were selected as a drug target for breast cancer  (Abdulrahman et al., 2023; Kaur et al., 2022). The X-ray crystal structure of protein targets and its co-crystallized ligands were obtained from the RCSB Protein Data Bank. The protein and the chemical structures of compounds (ligands) sourced from the PubChem database were prepared using AutoDock Tools (version 1.5.7) (Trott and Olson, 2010). After calculating the docking scores for various protein-ligand pairs, we selected the one with the most negative energy for further investigation. This chosen protein-ligand complex was subjected to more detailed scrutiny using a Protein-Ligand Interaction Profiler (PLIP) (Adasme et al., 2021) and PyMol software.
 
Statistical analysis
 
All experiments were conducted in triplicate and the significance of the findings was determined using a t-test. The results are presented as mean ± SD, with a p-value less than 0.05 considered significant.

Upon extracting 34 grams of L. shawii powder with 85% ethanol, the fractions obtained were as follows: 370 mg from Hex (Fraction 1), 205 mg from EtOAc (Fraction 2) and 490 mg from n-BuOH (Fraction 3), while 23 grams of bound phenol extraction yielded 220 mg for EtOAc (Fraction 4) and 940 mg for n-BuOH (Fraction 5), with 520 mg for saponin (Fraction 6).

Our findings indicated that MDA-MB-231 (IC₅₀: 407.3 μg/mL) and HUVECs (IC₅₀: 500 μg/mL) cell proliferation was inhibited only with Fraction 2. However, the extract did not exhibit activity against HepG2 and Huh-7 liver cancer cell lines. Fig 1 illustrates the cytotoxic properties of L. shawii against these cell lines. The most potent anticancer effect of the extract was observed against MDA-MB-231 cancer cells. Fraction 2 induced notable morphological alterations in MDA-MB-231 cells, characterized by cytoplasmic shrinkage, contraction and detachment, leading to the complete loss of cellular integrity. In contrast, untreated cells displayed normal cellular morphologies (Fig 2 A and b). Similar results were observed in a previous study documenting that costunolide (IC₅₀: 32) and aloe emodin (IC₅₀ 38 µM) isolated from L. shawii stem extract exhibited substantial apoptotic potential against oral squamous cell carcinoma OSCC cells. Notable cellular morphological alterations and gene and protein expression (BAK, caspase 3, 6 and 9) indicated the presence of apoptosis in treated cells.





In cancer, the invasion and migration of cells are pivotal factors contributing to recurrence and metastasis. Effectively inhibiting cell migration is essential for successful cancer therapy, as metastasis significantly impacts survival rates, reducing them to approximately 50% (Irani, 2016). As illustrated in Fig 3A and 3B, fraction 2 significantly hindered the time-dependent migration of MDA-MB-231 cells. In the absence of treatment, the cells exhibited wound closure, reaching up to 93.8% and 96.6% at 24 and 48 hours, respectively. The scratch closures at IC₅₀ doses were smaller, measuring up to 17.2% at 24 hours and 35.8% at 48 h, reflecting the potential of the extract in reducing cell migration. Several in vitro studies have provided evidence that phytochemical compounds derived from L. shawii can impede the invasion and migration of various cell lines (Choi et al., 2013; Xiao and Guo, 2009; Shams et al., 2023). 



To assess the impact of oxidative stress on the cytotoxicity of fraction 2, cell lines were exposed to its IC₅₀ concentration determined through cytotoxicity assays. Fraction 2 significantly elevated cellular ROS levels in MDA-MB-231 cells compared to the 0.01% MeOH-treated cells (control) (Fig 2 C and D).

Docking is essential for systematically exploring large chemical libraries and it remains a key tool in rational drug design and drug repurposing strategies (Friesner et al., 2004) (Huang and Zou, 2007). The GC-MS analysis revealed the presence of fifty-one compounds within fraction 2 of L. shawii (Table 1). Out of fifty-one phytoconstituents, it was observed that stigmast-5-en-3-ol, (3α,24S) and lup-20(29)-ene-3,28-diol, (3α), exhibited the most favourable interactions with 6CHZ, featuring binding energies of -10.0 and -9.9 kcal/mol, respectively (Table 1). Stigmast-5-en-3-ol, (3α,24S)-showed 15 hydrophobic interactions with Leu 346 (3x), Ala 350, Leu 384, Leu 387, Met 421, GLU 423, 424 ILE (3x), LYS 520, HIS 524 and LEU 525 (Table 1). The 2D and 3D interactions are shown in Fig 4 A and B. Similarly, the best-docked secondary metabolite with BCL-2 was LUP-20(29)-ENE-3,28-DIOL (-9.5 kcal/mol). In the binding site of BCL-2 lup-20(29)-ene-3,28-diol, (3α), interacted through 8 hydrophobic interactions with the following residues: 127A PHE (2x), 131A VAL, 132A GLU, 173A TRP, 176A GLU and 177A TYR (2x) The 2D and 3D interactions are shown in Fig 4 B.





Stigmast-5-en-3-ol, (3α,24S)- and Lup-20(29)-ene-3,28-diol, (3α)- interact with 6CHZ, presenting a binding energy of -10.0 and -9.9 kcal/mol respectively. Notably, their binding affinity was equal to or greater than the control compounds (-9.9 kcal/mol). ER-α is associated with both hormone-dependent and hormone-independent tumours. This dual role of ER-α is notable, as it has been reported to play a part in both cancer suppression and cancer progression (Liu et al., 2020). Given that 60-70% of breast cancers in women are ER-α positive, the current therapy primarily relies on tamoxifen, which helps control ER-α-induced breast cancer progression. However, the prolonged use of tamoxifen may lead to resistance in breast cancer patients. Consequently, there is a pressing need to explore novel natural drugs and understand ER-α signalling to enhance breast cancer therapy (Xue et al., 2019).

The capacity to avoid apoptosis is a critical characteristic of cancer cells. Therefore, they frequently disrupt the apoptotic pathway to ensure the tumour cells survival by upregulating the expression of anti-apoptotic proteins within the Bcl-2 family. These proteins include Bcl2-A1, Mcl-1, Bcl-2, Bcl-w and Bcl-xL (Williams et al., 2019). In various cancer types, the elevated levels of anti-apoptotic proteins, such as Mcl-1, Bcl-2 and Bcl-xL, have been established to not only confer resistance to chemotherapy but also promote tumour initiation and progression through the microRNAs regulation and transcription factors (Valentini et al., 2022; Choi et al., 2016). The VINA scoring function predicted the ability of lup-20(29)-ene-3,28-diol, (3α) and stigmasta-5,22-dien-3-ol, acetate, (3α), to bind to Bcl-2 and Mcl-1 with similar affinities and more effectively than BCL-W.

Various studies have proved that Stigmast-5-en-3-ol can induce apoptotic cancer cell death, including MCF-7 (BC),  U937 and HL-60 leukemia cell lines with IC₅₀ values of 45.17, 37.82 and 8.294 μg/ml, respectively (Fernando et al., 2018; Moon et al., 2008). Similarly, it has been documented that the apoptotic effects induced by Stigmast-5-en-3-ol are linked to an elevation in Bax, Caspase-9, PARP cleavage and p53, while concurrently reducing Bcl-xl levels (Fernando et al., 2018). Likewise, Lup-20(29)-ene-3β,11β-diol displayed cytotoxic potential against HeLa cell lines (IC₅₀: 28.5 μM) (Nguyen et al., 2017).

The study examined the Fraction 2 of L. shawii for its impact on breast cancer cell lines and results revealed significant inhibition of cell proliferation in MDA-MB-231(IC₅₀:407.3 μg/mL) and HUVECs (IC₅₀:500 μg/mL). Molecular docking highlighted the favourable binding of specific compounds to targets associated with breast cancer. While promising, further in vivo investigations are crucial for validating these findings and exploring potential drug discovery and nutraceutical development applications.

The authors express their sincere appreciation to the Researchers Supporting Project number (RSP2024R112), King Saud University, Riyadh, Saudi Arabia.

N. Abutaha designed the study, R. Alghamdi and N. Abutaha conducted experiments. F.A. Almekhlafi, M.A. Wadaan helped in writing the manuscript and conducted data analyses.

All the data is available within the manuscript.

The authors declare no conflicts of interest.