Phytochemical Profiling and Cytotoxic Potential of Senecio flavus n-Hexane Extract using in vitro and in silico Approaches

Liver cancer is an aggressive type of cancer with a high fatality rate (Siegel et al., 2018). Chemotherapy offers limited benefits to a minority of patients, often leading to drug-related toxicity or inefficacy over time. Although surgical interventions are available, the prognosis remains unfavourable due to frequent relapses (Liang et al., 2021). Treatment with sorafenib is widely used in late-stage cases and presents significant challenges. Less than a third of patients experience any benefit from this therapy resistance to the drug typically develops within six months of starting treatment (El-Serag et al., 2008).

Moreover, prolonged use of chemotherapeutic agents like sorafenib is associated with toxicity issues and decreased effectiveness. Consequently, existing chemotherapy has failed to yield substantial improvements for liver cancer patients. Therefore, there is a need for extensive research to identify more effective approaches for managing this disease (Anwanwan et al., 2020). Similarly, the widespread misuse of antibiotics resulted in increased antimicrobial resistance, rendering many antibiotics ineffective (Vaou et al., 2021). The World Health Organization (WHO, 2023) considers this alarming trend a critical issue, highlighting the urgent need for novel antimicrobial agents. These new agents must be capable of reducing antibiotic usage and combating the development of resistance.

Traditional plants are being assessed as an alternate therapeutic approach to overcome the limits of anticancer and antimicrobial current treatments (Dehghani and Saeidi 2023; Alghamdi et al., 2024; Moatasem et al., 2023). Senecio flavus (Decne.) is a member of the Asteraceae family and is found in Afghanistan, Algeria, Spain, Chad, Egypt, Iran, Mauritania, Morocco, Namibia, Pakistan, Palestine, Sinai, Spain, Sudan, Tunisia (https://www.gbif.org/species/3107479). However, there is a significant gap in scientific research on S. flavus and its potential therapeutic efficacy.

GC-MS is extensively employed to identify bioactive phytocompounds. It is a highly effective, fast accurate method for detecting various chemicals such as nitro compounds, amino acids, alkaloids, alcohols, organic acids, steroids esters long-chain hydrocarbons (Al-Rubaye et al., 2017). Similarly, computer-based tools have emerged as advanced strategies in drug discovery, particularly in screening medicinal plants for bioactive compounds. Molecular docking, characterized by its efficiency and cost-effectiveness, is a valuable approach for developing and testing pharmaceuticals. This technique provides insights into drug-receptor interactions, enabling predictions on how drug models will bind to target proteins, ultimately leading to reliable binding at specific ligand binding sites (Pagadala et al., 2017).

Therefore, this study evaluated the antimicrobial and cytotoxic activities of various extraction methods of S. flavus, identified potential bioactive components using Gas Chromatography-Mass Spectrometry (GC/MS) employed in-silico methods for further analysis.
 

Study area
 
This study was carried out at King Saud University, Diriyah, Saudi Arabia, from March 2022 to February 2024

Collection of the plant

The Senecio flavus (Decne.) was collected from Riyadh, King Saud University campus. The plant was authenticated in the Department of Botany (voucher specimen: KSU-RYD-091) and was preserved in the Bioproduct Research Chair at King Saud University. The aerial part of the S. flavus was washed with distilled water, shade-dried, pulverized stored in an airtight container (Fig 1).



Extraction and fractionation
 
Seventy-two grams of powdered material was macerated in 500 mL of 70% methanol for 3 days at 25°C with occasional shaking. Subsequently, the mixture was filtered through a muslin cloth the resulting extract was evaporated at 45°C using a rotary evaporator. The concentrate was then suspended in 500 mL of distilled water and subjected to liquid-liquid extraction (2 × 200 mL) using hexane (F1), ethyl acetate (F2) n-butanol (F3), respectively. The aqueous layer was discarded each separated fraction was concentrated individually using a rotary evaporator (Sadiq et al. 2014).
 
Saponin extraction
 
To extract the sample, 20 grams were placed in a glass flask and mixed with 500 mL of 15% aqueous ethanol. The mixture was heated using a hot water bath with continuous stirring at 55°C for 4 hours. Afterwards, the extract was rotary evaporated to 50 mL at 50°C and then transferred into a 500 mL separatory funnel. To prepare the extract, petroleum ether (2×200 mL) was added and vigorously shaken. The resulting aqueous layer was retained, whereas the ether layer was discarded. This purification procedure was repeated n-butanol (2×200 mL) was added. The combined n-butanol extract underwent washing with 100 mL of 5% aqueous NaCl, repeated twice. Subsequently, the remaining solution was evaporated (Edeoga et al., 2005).

Cell culture

The HepG2 and Huh-7 liver cancer cell lines were obtained from the German cell culture collection (DSMZ) and maintained as monolayers in 75 cm² flasks (Nest, China) using Dulbecco’s Modified Eagle’s Medium (DMEM). The DMEM media (Invitrogen, USA) were supplemented with 10% fetal bovine serum (Invitrogen, USA). The cells were cultured under at 37°C in a humidified atmosphere with 5% CO₂.

Cytotoxicity assay
 
The cells (25,000 cells/well) were seeded in 48-well plates (Nest, China) and incubated for 24 hours at 37°C in a humidified atmosphere of 5% CO₂. Following this incubation period, the cells were treated with varying extract concentrations (500-25 mg/mL) for 24 hours. Subsequently, 50 mL of MTT solution (Invitrogen, USA) was added to each well the plates were incubated at 37°C for 2 h. The optical densities were measured at a wavelength of 490 nm using a multi-well plate reader (ChroMate, USA). The results are presented as a percentage of the control and were used to calculate the inhibitory concentration (IC50) values using OriginPro 8.5. The IC50 value represents the extract concentration required to reduce the cell number to 50% of the untreated control. Each extract was tested in triplicate (Khasawneh et al., 2022).
 
Analysis of cell morphology
 
As reported earlier, human liver cancer cells (Huh-7) were seeded in 48-well plates and incubated for 24 hours to facilitate cell attachment and growth. Post-incubation, the cells were exposed to S. flavus extract at a concentration of 150 μg/mL, with the hexane extract prepared in DMSO ensuring that the final DMSO concentration in each well did not exceed 0.1%. The plates were incubated for an additional 24 h under identical conditions (37°C, 5% CO₂). Following the 24-hour treatment period, the cell morphologies in treated and untreated control wells were observed and recorded using an inverted microscope (EVOS, USA).
 
Fluorescence staining of cells
 
The cells were seeded, treated with the hexane extract (150 μg/mL) incubated for 24 h as described in the previous section (Analysis of Cell Morphology). After incubation, the cells were stained with a 42,6-diamidino-2-phenylindole (DAPI) solution at a 5 μg/mL concentration. Similarly, for Acridine Orange/Ethidium Bromide (AO/EB) staining (Sigma-Aldrich, USA), the cells were stained with a solution containing 100 μg/mL AO and 100 μg/mL EB in PBS for 2 minutes. The stained cells were then processed according to the method reported by (Khasawneh et al., 2022). Images were captured using an EVOS fluorescent microscope.
 
Microbial strains and culture
 
The microbial strains required for the study were sourced from the Microbiology Department at King Saud University in Riyadh, Saudi Arabia. Six of the selected microbial species were bacterial strains; one was a fungal strain investigated for its antifungal properties. The bacterial strains, including Acinetobacter baumannii (ATCC®BAA-747), Staphylococcus epidermidis (clinical isolate), Bacillus subtilis 16406, Staphylococcus aureus (ATCC®29213) Listeria monocytogenes (ATCC®7644) were subcultured on Nutrient Agar (NA) media. These cultures were grown at 37°C for 24 hours. Meanwhile, the fungal strain C. albican (ATCC®90028) was subcultured in potato dextrose agar.
 
Antimicrobial assay of extracts
 
The antimicrobial activity of the extracts was evaluated using the agar well diffusion method on Nutrient Agar (HiMedia, India) for bacterial strains and Potato Dextrose Agar (HiMedia, India) for fungal strains. The test organisms were inoculated into broth media the turbidity was adjusted to an OD600 of 0.01 to standardize the inoculum density. Plant extracts were prepared in methanol (MeOH) at 50 mg/mL. Using a sterile cork borer (6 mm), wells were created in the inoculated media plates 25 μL of each extract was pipetted into the wells. The plates were allowed to sit at room temperature (25°C) for 30 minutes to facilitate diffusion of the extracts into the agar. Subsequently, the plates were incubated at 37°C for 24 h for bacterial cultures and at 30°C for 24 hours for fungal cultures. Antibiotic tetracycline discs (30 mg) from Sigma Aldrich, Germany, were used as positive controls, while methanol (MeOH) was the negative control. Following incubation, the plates were examined for clear inhibition zones around the wells, indicating antimicrobial activity. The diameter of the inhibition zones was measured in millimetres using a digital calliper.

Gas chromatography-mass spectrometry (GC-MS) analysis

The GC-MS analysis followed the protocol outlined by (Abd El-Kareem et al., 2016). The chemical composition of hexane extract was assessed using a GC-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) equipped with a TG–5MS capillary column (30 m × 0.25 mm × 0.25 µm film thickness). Compounds were identified by referencing the NIST14 and WILEY 09 mass spectral databases.

Molecular docking

X-ray structures of sixteen target receptors (3FV7), (3TD4), (3ZNT), (4FUV), (4JAS), (4JF4), (4KOX), (4KOV), (4OHO), (4QDI), (4X55), (Y0A), (5BUF), (5W1B), (6FJY) and (6GIE) were downloaded from the Protein Data Bank (PDB) (https://www.rcsb.org). Thirty-four phyto-compounds were obtained from PubChem as structure data files (SDF) and converted to ‘.pdb’ format using Open Babel 2.3.2. Autodock 4.2 was then utilized to generate the ‘pdbqt’ file of the ligands. The protein structures were prepared for docking using AutoDockTool-1.5.7 (Huey et al., 2007), calculating Gasteiger charges, removing water molecules adding polar hydrogens and site-specific docking grid coordinates (Morris et al., 2009). Molecular docking procedures were carried out using AutoDock Vina 1.1.2. The resulting conformations of the ligand-receptor complexes were ranked based on their binding energy. 

This study evaluated the yield of extracts from S. flavus using various extraction techniques. The saponin fraction yielded the highest percentage, comprising 4.42% of the total extract. This was followed by the n-butanol extract at 0.79%, the n-hexane extract at 0.54% the ethyl acetate extract at 0.36%.

Two liver cancer cell lines were chosen to assess the cytotoxicity using the MTT assay. The results revealed that only the hexane extract exhibited cytotoxic effects on the tested cell lines, whereas the other extracts showed more than 96% viability at the highest concentration tested (500 µg/mL). The IC50 values at 24 h post-treatment in HepG2 and HuH-7 cells were 241.2 and 187.8 mg/mL, respectively. These findings indicate that the hexane extract possesses higher cytotoxicity in HuH-7 cells than in HepG2 cells (Fig 2).   In addition, S. flavus extracts were studied for their potential to induce apoptosis in liver cancer cells. Apoptosis is defined by morphological characteristics such as detachment, cell shrinkage, cell rounding nuclear chromatin condensation (Doonan and Cotter 2008). Our findings revealed that treatment with hexane extract led to noticeable morphological changes. In Fig 3A and 4A, the cells in the control wells exhibited attachment to the surface, reaching 95-100% confluence and maintaining their typical shape.



Similarly, upon staining with DAPI, as shown in Fig 3A and 4A for control samples, cell nuclei appeared uniform in shape and size. In contrast, the nuclei of samples treated with the hexane extract, as depicted in Fig 3B, 3C, 4B 4C, showed signs of shrinkage, condensation fragmentation in areas with abnormal cells. These changes strongly support that n-hexane extract activated apoptotic deaths in liver cancer cells. AO-EB dual staining was carried out to support our findings. We observed that the viable control HuH-7 cells had evenly distributed green fluorescence (AO stain), normal nuclear morphology no red fluorescence (Fig 5A). In contrast, cell viability significantly decreased in n-hexane extract-treated HuH-7 cells. However, the number of apoptotic cells increased, as is evident from the bright green and yellow-orange staining resulting from chromatin condensation and membrane integrity loss. Apoptosis induction is considered one of the most effective strategies for chemotherapy when developing new anticancer agents (Surh 2003). Therefore, identifying candidate anticancer agents that can induce apoptosis in cancer cells is crucial in the search for new and effective anticancer treatments.





The most challenging aspect of microbial infections is multidrug resistance (MDR), a critical concern where conventional antibiotic treatments are often ineffective. The search for novel and potent antibacterial agents targeting MDR-A. baumannii is of utmost importance (Rafailidis et al., 2024). In current study, only the saponin fraction exhibited antimicrobial activity, as evidenced by inhibition zones with diameter values of 19 mm, 27 mm, 13 mm, 17 mm 12 mm against Acinetobacter baumannii 1100 (MDR), A. baumannii (ATCC®BAA-747), S. epidermidis, S. aureus L. monocytogenes, respectively (Table1). However, none of the extracts showed activity against C. albicans. Other researchers have reported similar results using different plant extracts. Researchers found that the oil extracted from basil, clove cinnamon exhibited potent bactericidal activity against MDR-A. baumannii, with minimum bactericidal concentrations (MBC90) of 2,1 and 0.5 mg/mL, respectively (Intorasoot et al. 2017). Our results also revealed that the saponin fraction was not toxic to the cell lines tested up to 1 mg/mL, indicating the safe use of the extract on human liver cells. However, further experiments are needed to assess its toxicity. Makhafola et al. (2012) emphasized that natural products should not be considered safe until they undergo cellular toxicity tests. It is crucial to assess the cytotoxicity using multiple cell lines because relying on just one cell line to establish the safety and efficacy of an extract can be misleading. The sensitivity of cell lines is expected to vary due to differences in metabolic activities and uptake capabilities (Froscio et al., 2009).



A GC-MS analysis was conducted to determine the quantitative analysis of hexane extract of S. flavus. A total of 34 bioactive phyto-compounds were identified using GC-MS analysis (Table 2). The major identified compound was cis-13-Octadecenoic acid (69.1%) followed by hexadecanoic acid (4.34%). The GC-MS results of the hexane extract from S. flavus revealed the existence of potential antitumor and antimicrobial composites. For example, 9-octadecenoic acid (z)-, methyl ester and 9-Octadecenoic acid (Z)- have also been reported to have antioxidant, anticancer antimicrobial potential (Duke, 1994) (Diab et al., 2021). However, 9,12-octadecadienoic acid methyl ester has also been reported to have antitumor and antioxidant properties (Reza et al., 2021).



The selected drug-like molecules of S. flavus were docked with sixteen different target receptors, namely 3FV7, 3TD4, 3ZNT, 4FUV, 4JAS, 4JF4, 4KOX, 4KOV, 4OHO, 4QDI, 4X55, 4Y0A, 5BUF, 5W1B, 6FJY and 6GIE (Fig 5). Compounds 2 and 29 were the best docked to 4JAS and 5W1B with ÄG of -9.7 kcal/mol for both compounds.

The findings from this study reveal that hexane extract showed promising cytotoxic and apoptotic effects against HepG2 and Huh-7. The saponin fraction exhibited antimicrobial activity against A. baumannii, indicating its potential as an antimicrobial agent. GC-MS analysis revealed a range of bioactive compounds in the hexane extract, with (R)-3,5,8a-Trimethyl-7,8,8a,9-tetrahydronaphtho [2,3-b] furan-4(6H)-one and Tricyclo [20.8.0.0 (7,16)] triacontane, 1(22),7(16)-diepoxy- showing favourable interactions in molecular docking studies. Further research is needed to explore its potential for medical applications.

The present study was supported by Researchers Supporting Project number (RSP2024R112), King Saud University, Riyadh, Saudi Arabia.

Funding

This project was funded by Researchers Supporting Project number (RSP2024R112), King Saud University, Riyadh, Saudi Arabia.

Disclaimers

The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

Informed consent

All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.