Chemical Profiling, In Silico and In Vitro Studies to Identify Potential CDK2 and mTOR Inhibitor From Selaginella inaequalifolia (Hook. & Grev.) Spring Ethanolic Extracts.

1
Introduction
In Asia and Africa, 80% of peoples are depending on the traditional herbals for their medicinal requirements [1]. In the modern world, plant extract is eco‐friendly in nature, and are used as the source of insecticides, pesticides, larvicidal, wound healing, antibacterial, antioxidant, anti‐inflammatory, antidiabetic, ant‐cancer, and so forth [2, 3, 4, 5, 6, 7, 8]. Pteridophytes, including ferns and horsetails, have gained attention for their cytotoxic properties, particularly in cancer research, which have been shown to inhibit cell growth and induce apoptosis in cancer cells [9, 10]. Species like Cyathea have demonstrated significant cytotoxicity against MCF‐7 cancer cell lines, suggesting their potential for anticancer therapy [11]. In vitro assays, including brine shrimp lethality and MTT assays are commonly used to evaluate the cytotoxic effects of pteridophytes genus like Phlebodium, Cyathea, Selaginella, and Pteris extracts [12, 13]. These studies highlight the promising role of pteridophytes as natural sources of cytotoxic agents with selective activity against cancer cells.
The genus Selaginella belongs to Selaginellaceae family with 700–750 species. Several species of Selaginella are used as medicines and food from pre‐historic period [14]. Selaginella is used as the traditional medication for wounds, childbirth, problems with menstruation, skin diseases, headaches, fevers, urinary tract infections, liver disease, tumors, osteoarthritis, and bone fractures etc. [15]. Various metabolites like lignans [16], terpenoids [17], triterpenoids [23], alkaloids [18], flavonoids [63] are reported from several species of Selaginella, which have shown several kinds of biological measures such as antioxidant [3, 19, 20], immunomodulatory [19], antimicrobial [21, 22, 23], antibacterial [24], antifungal [25], anticancer [26], antidiabetic [27], cytotoxicity [12]. Nine derivatives of special chemicals known as selaginellins are present in species of Selaginella contains alkynyl phenol and p‐quinone methide functional groups and isolated some compounds like selaginisoquinoline and 3 ethoxy selaginellins [28], 16 compounds are isolated including two new secondary metabolites from Selaginella doederleinii [29]. New two flavone glucosides namely, 7‐O‐(β‐glucopyranosyl (1→2)‐[β‐glucopyranosyl(1→6)]‐β‐glucopyranosyl)flavone‐3′,4′,5,7‐tetraol and 7‐O‐(β‐glucopyranosyl(1→2)‐[β‐glucopyranosyl(1→6)]‐β‐glucopyranosyl)flavone‐4′,5,7‐triol are isolated, two new biflavonoids 2,3‐dihydroflavone‐5,7,4′‐triol‐(3′→8″)‐flavone‐5″,6″,7″,4‴‐tetraol and 6‐methylflavone‐5,7,4′‐triol‐(3′→O→4‴)‐6″‐methylflavone‐5″,7″‐diol, two new lignans (7′E)‐3,5,3′,5′‐tetramethoxy‐8:4′‐oxyneolign‐7′‐ene‐4,9,9′‐triol and 3,3′‐dimethoxylign‐8′‐ene‐4,4′,9‐triol together with two known monolignans, four known lignans, and four known bioflavonoids are isolated from Selaginella moellendorfii (Wu et al. 2011) [30]. Based on the pharmacological qualities, molecular systematic and micro morphological characteristics, Selaginella is an interesting and little‐studied genus [31].
Target proteins are primarily selected based on their established role in disease pathogenesis and their regulations. For example, CDKs are critical regulators of cell cycle progression, DNA replication, and transcription, and their dysregulation is strongly implicated in cancer development [32]. Proteins that act as key nodes in disease‐related signaling pathways or metabolic networks are prioritized because inhibiting their activity can effectively disrupt pathological processes [33]. In the present study, cyclin‐dependent kinase 2 (CDK2) (PDB ID: 4GCJ) and serine/threonine‐protein kinase mTOR (PDB ID: 4JSX) are selected.
Several reports have demonstrated strong binding affinities of Selaginella‐derived compounds through molecular docking for instance, S. doederleinii extract having good binding affinity in diosgenin with GLUT1 and LDHA (−11.8 and −9.6 kcal/mol, respectively) [34], and robustaflavone with α‐glucosidase (−11.33 kcal/mol) (Gao et al. 2024) [35]. Selaginella bryopteris having a stability of amentoflavone in complex with MAPK1 and MAPK14 proteins are confirmed through molecular dynamics simulations and MM‐PBSA analysis [60]. These computational efforts exhibit notable limitations. Most existing studies focus on individual compounds rather than complete extract profiling, thereby overlooking possible synergistic effects among multiple bioactive constituents, which may be insufficient to capture long‐term stability and conformational dynamics of protein–ligand complexes. Furthermore, current research tends to emphasize isolated therapeutic targets without developing a comprehensive structure–activity relationship across different Selaginella species and their diverse phytochemical compositions. Another major shortcoming is no report on the chemical constituents and in silico studies on Selaginella inaequalifolia (Hook. & Grev.) Spring ethanolic extracts. To fill the gap, the current study is aimed to reveal the phytoprofile of S. inaequalifolia using GC–MS and predict the drug properties, toxicity, biological properties of S. inaequalifolia ethanolic extracts (SiEE) using in silico methods and in vitro toxicity assays, namely, MTT and BSLB assay.

2
Materials and Methods
2.1
Collection of Plants
Healthy, disease‐free S. inaequalifolia (Hook. & Grev.) Spring collected from Kakachi, Kothayar, Tirunelveli Hills, Western Ghats, South India on January 26, 2021. The plants are identified by Dr. M. Johnson Curator, Centre for Plant Biotechnology Herbarium, St. Xavier's College (Autonomous) based on the “Pteridophyte Flora of the Western Ghats, South India” by Manickam and Irudayaraj [36]. Herbarium specimen is prepared at the collection site itself and the voucher specimen (CPBH 1303) is deposited in the Centre for Plant Biotechnology Herbarium (CPBH), Palayamkottai, Tamil Nadu, India.

2.2
Preparation of Extracts
The collected whole plants of S. inaequalifolia are thoroughly washed with tap water followed by distilled water. The washed whole plants of S. inaequalifolia are blotted on the blotting paper and spread out at room temperature in shade to remove the excess water contents. The shade dried whole plants of S. inaequalifolia are ground to fine powder using mechanical grinder. The powdered samples are stored in refrigerator for further use. Thirty grams of powdered materials of S. inaequalifolia are extracted with 180 mL of ethanol using Soxhlet extractor for 8 h at a temperature not exceeding the boiling point of the solvent. The SiEE are filtered using Whatman filter paper (No. 1) and then concentrated in vacuum at 40°C using rotary evaporator. The obtained residues of SiEE are stored in a freezer until further tests.

2.3
GC–MS Analysis
To reveal the chemical constituents, present in the SiEE, gas chromatography–mass spectrometry (GC–MS) analysis is performed using the Clarus 500 GC–MS (PerkinElmer). Two microliters of SiEE is injected for GC–MS analysis [37, 58]. The Clarus 500 GC used in the analysis employed a fused silica column packed with Elite‐1 (100% dimethyl poly siloxane, 30 nm × 0.25 nm ID × 1 µm df) and the compound constituents are separated using helium as carrier gas at a constant flow of 1 mL/min. Two microliters SiEE injected into the instrument is detected by the Turbo gold mass detector (PerkinElmer) with the aid of the Turbo mass 5.1 software. During the 36th minute of GC extraction process, the oven is maintained at a temperature of 110°C with 2 min holding. The injector temperature is set at 250°C (mass analyzer). The different parameters involved in the operation of Clarus 500 MS, are also standardized (inlet line temperature: 2000°C; source temperature: 2000°C). Mass spectra are taken at 70 eV; a scan interval of 0.5 s and fragments from 45 to 450 Da. The MS detection is completed in 38 min. The identified compounds biological activities are predicted using PASS.

2.4
ADME and Toxicity Prediction
ADMET is employed to study the in silico ADMET and the toxicity properties of the identified chemical compounds of SiEE. To reveal the in silico ADME and toxicity of chemical constituents identified from SiEE, namely, 2,5‐dihydroxyacetophenone, bis(trimethylsilyl) ether, cyclopentasiloxane, decamethylcyclopentasiloxane, oxazepam DITMS, hydroquinone 1,4‐benzenediol, 1‐hexadecene, methyl 3‐hydroxyl tetradecanoate, tetradecanoic acid, 3‐hydroxy‐, methyl ester, salicylaldehyde hydrazone benzaldehyde, 2‐hydroxy‐, hydrazine, α‐d‐glucopyranose, 4‐O‐α‐d‐galactopyranosyl lactose, α, 2‐propenoic acid, 3‐(4‐fluorophenyl)‐, ethyl ester, î‐N‐formyl‐l‐lysine, pentadecanoic acid, 13‐methyl‐, methyl ester, 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid, 5,6‐dimethoxy phthalaldehydic acid, the smile format are retrieved from NCBI PubChem and submitted to the Swiss ADME (http://www.swissadme.ch/). Swiss ADME online server analyzes various parameters including physicochemical properties, medicinal chemistry, pharmacokinetics, water solubility of the identified compounds of SiEE. Similarly, toxicity factors such as acute inhalation, acute oral, acute dermal toxicity, eye irritation and corrosion, skin sensitization, skin irritation and corrosion of the identified compounds of SiEE are also predicted using online tool STOP TOX (https://stoptox.mml.unc.edu/) [38, 39].

2.5
Preparation of Ligands
The 3D SDF file format of the identified ligands of SiEE, namely, 2,5‐dihydroxyacetophenone, bis(trimethylsilyl) ether, cyclopentasiloxane, decamethyl, oxazepam ditms, hydroquinone 1,4‐benzenediol, cyclodecasiloxane, 1‐hexadecene, methyl 3‐hydroxyl tetradecanoate tetradecanoic acid, 3‐hydroxy‐, methyl ester, salicyl aldehyde hydrazone benzaldehyde, 2‐hydroxy‐, hydrazine, α‐d‐glucopyranose, 4‐O‐α‐d‐galactopyranosyl lactose, α, 2‐propenoic acid, 3‐(4‐fluorophenyl)‐, ethyl ester, î‐N‐formyl‐l‐lysine, pentadecanoic acid, 13‐methyl‐, methyl ester, 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid, and 5,6‐dimethoxyphthalaldehydic acid are downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) database for in silico docking analysis. The in silico docking analysis is performed between the identified compounds of SiEE and selected proteins, namely, CDK2 (PDB ID: 4GCJ), serine/threonine‐protein kinase mTOR (PDB ID: 4JSX).

2.6
Preparation of Proteins
The targeted proteins, namely, CDK2 (PDB ID: 4GCJ) and serine/threonine‐protein kinase mTOR (PDB ID: 4JSX) are downloaded from RCSB (Research Collaboratory for Structural Bioinformatics) Protein Data Bank (PDB) (https://www.rcsb.org/) in PDB format.

2.7
Molecular Docking Analysis
The identified compounds/ligands of SiEE are docked against the protein CDK2, serine/threonine‐protein kinase mTOR using CB‐DOCK 2 (http://183.56.231.194:8001/cb‐dock2/php/blinddock.php#job_list_load) [40, 62].

2.8
Cytotoxic Activity—MTT Cell Proliferation Assay Cell Line and Culture
To validate the in silico observation, the toxicity and cytotoxicity studies are performed for SiEE against brine shrimp and MCF cell lines. The cell line of MCF‐7 (human breast carcinoma) is obtained from National Centre for Cell Science, Pune, India and the experiments are performed in Amala Cancer Research Centre, Thrissur, Kerala, India. The cells are cultured in a growth medium (DMEM, PH 7.4), supplemented with 10% FBS and antibiotics, penicillin (100 units/mL) and streptomycin sulfate (100 µg/mL).

2.9
MTT Assay
The cytotoxicity of SiEE against human breast carcinoma (MCF‐7) is determined by the MTT (3‐[4,5‐methylthiazol‐2‐yl]‐2,5‐diphenyl‐tetrazolium bromide) assay [41]. The cells are seeded into wells of microtiter plate (96 well) at 3 × 103 cells per well with 100 µL of DMEM growth medium. It is then incubated for 24 h at 37°C under 5% CO2 in a humidified atmosphere. Later, the medium is removed and fresh growth medium containing different test doses of SiEE (12.5, 25, 50, 100, and 200 µg/mL) are added. Five wells are included in each concentration. After 3 days of incubation at 37°C under 5% CO2, the medium is removed. Twenty microliters of 5 mg/mL MTT (pH 4.7) is added per well and cultivated for another 4 h, the supernatant fluid is removed. One hundred microliters of DMSO is added per well and shaken for 15 min. The absorbance at 570 nm is measured with a UV‐spectrophotometer, using wells without cells as blanks. All the experiments are performed in triplicates. The absorbance of untreated cells is considered 100%. The IC50 value is determined graphically (Scatter Regression) by using MS EXCEL. The conventional anticancer drug, adriamycin is used as a positive control. The inhibition of cell growth is calculated as percent anticancer activity using the following formula:

2.10
Brine Shrimp Lethality Bioassay
Cytotoxic activity of SiEE is evaluated using brine shrimp lethality bioassay method [42]. About 1 g of Artemia salina cysts is aerated in 1 L capacity glass jar containing filtered seawater. The air stone is placed in the bottom of the jar to ensure complete hydration of the cysts. After 24 h incubation at room temperature (25°C–29°C), newly hatched free‐swimming nauplii are harvested from the bottom outlet. As the cysts capsules are floated on the surface, this collection method ensured pure harvest of nauplii. The freshly hatched free‐swimming nauplii are used for the bioassay. Thirty clean test tubes are taken, of which 25 tubes are used for the samples with five different concentrations, namely, 100, 200, 300, 400, and 500 mg/mL and five tubes for control. With the help of a Pasteur pipette, 20 nauplii are transferred to each tube containing various concentrations of SiEE. Five replicates are made for each concentration and a control DMSO is also maintained. The standard plumbagin is used as positive control. The setup is allowed to remain for 24 h under constant illumination. After 24 h, the dead nauplii are counted with a hand lens. Using the recorded observations, LC50, LC90, and chi square values are also calculated.

2.11
Statistical Analysis
For the cytotoxicity analysis using MCF cell lines, the IC50 value is determined graphically (scatter diagram) by using MS EXCEL, the concentration of SiEE and adriamycin is taken in the X‐axis and the percentage of growth inhibition is taken in the Y‐axis. For the toxicity analysis using BSLB, LC50, LC90, and chi square values are calculated using SPSS.

3
Results
A total of 27 compounds are identified from SiEE with varied retention time (RT) and their structures are identified based on mass spectrometry (Figure 1). The identified compound bioactivities are predicted based on online PASS prediction (Table 1).
Out of 27 compounds identified from SiEE, 15 compounds followed the Lipinski's five rule, namely, molecular weight, lipophilicity (LogP), number of hydrogen bond donors and acceptors and molar refractivity [43] and other 12 identified compounds do not meet the Lipinski's rule. Among the 27 identified compounds, 15 compounds physicochemical properties confirmed their suitability for oral drug.

4
ADME
The physicochemical and pharmacokinetic profiling of the compound library revealed a diverse range of molecular characteristics and drug‐like behaviors. The physicochemical properties have shown balanced molecular weight distribution, moderate hydrogen bonding capacity (HBD/HBA), and topological polar surface area (TPSA) values, with most compounds exhibiting acceptable numbers of rotatable bonds. Lipophilicity analysis (Consensus LogP) indicated that 59.1% of compounds fell within the optimal drug‐like range (LogP 1–5), though notable outliers such as docosanoic acid triglyceride (LogP 21.43) reflected algorithmic variability. Water solubility classification placed compounds into six solubility categories, identifying highly soluble molecules like N‐formyl‐l‐lysine (28,400 mg/mL) and α‐d‐glucopyranose, 4‐O‐α‐d‐galactopyranosyl lactose, α (696.0 mg/mL), as well as essentially insoluble ones like docosanoic acid triglyceride (8.56 × 10−32 mg/mL). Pharmacokinetic predictions showed 54.5% of compounds with high gastrointestinal (GI) absorption and 45.5% capable of crossing the blood–brain barrier, with bioavailability scores ranging from 0.17 to 0.85 of which the highest score is observed for 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid (0.85). Drug‐likeness analysis across five rule sets (Lipinski, Ghose, Veber, Egan, and Muegge) are categorized into three tiers: Tier 1 (optimal, 31.8% with 0–1 violations), Tier 2 (moderate, 31.8% with 2–4 violations), and Tier 3 (poor, 36.4% with ≥5 violations). The integrated assessment identified 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid and 2,5‐dihydroxyacetophenone bis(trimethylsilyl) ether as lead candidates for further development (Tables 2, 3, 4, 5, 6; Figure 2). Among them, nine compounds fully met all the criteria, including 2,5‐dihydroxyacetophenone, cyclopentasiloxane, Oxazepam ditms, hydroquinone, methyl 3‐hydroxytetradecanoate, salicylaldehyde hydrazone, î‐N‐formyl‐l‐lysine, pentadecanoic acid, and 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid. These compounds showed good potential as drug‐like molecules. On the other hand, three compounds—cyclodecasiloxane, cyclooctasiloxane, and α‐d‐glucopyranose failed to meet two or more rules of Lipinski, mostly due to their size and high number of hydrogen bond donors or acceptors, which may reduce their chances of being absorbed well in the body. A few other compounds like 1‐hexadecene, 2‐propenoic acid (ethyl ester), and 5,6‐dimethoxyphthalaldehydic acid had one or two rule violations, meaning they may still have potential but might face some issues with absorption or bioavailability. Overall, most of the compounds studied show promise for further drug development, especially those met Lipinski's criteria (Tables 2, 3, 4, 5, 6; Figure 2).
Figure 2 visuals interpretation of individual radar plots revealed three clear tiers of compound performance based on ADME parameters. Tier 1 (optimal candidates), represented by large and regular hexagons, included compounds such as octaethylene glycol mono dodecyl ether, 2,5‐dihydroxyacetophenone bis(trimethylsilyl) ether, cyclopentasiloxane decamethyl, hydroquinone (1,4‐benzenediol), and salicylaldehyde hydrazone, all shown polygons extending uniformly toward the outer edges, indicating strong and balanced ADME profiles across all six parameters. Tier 2 (moderate candidates), shown as medium‐sized polygons with slight indentations, comprised compounds like Oxazepam ditms, cyclooctasiloxane hexadecamethyl, methyl 3‐hydroxytetradecanoate, 4‐d‐glucopyranose derivatives, and 2‐propenoic acid, 3‐(4‐fluorophenyl)‐ethyl ester, each displaying good overall performance but with specific weaknesses such as reduced solubility or excessive lipophilicity. Tier 3 (poor candidates) featured small, irregular polygons with deep indentations, including docosanoic acid, 1,2,3‐propanetryl ester, 1‐hexadecene, cyclodecasiloxane eicosamethyl, pentadecanoic acid, 13‐methyl‐, methyl ester, 1‐(+)‐ascorbic acid 2,6‐dihexadecanoate, and octadecanoic acid, 9,10‐dichloro‐, methyl ester, all showing pronounced ADME limitations such as high molecular weight, low solubility, or poor bioavailability. Interestingly, 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid stood out within this group as an exception, forming one of the largest and most regular polygons, reflecting an excellent and well‐balanced ADME profile overall.
Figure 2 shows the selected bioactive compounds identified through GC–MS, showing their 2D chemical structures, molecular docking interactions, and pharmacokinetic profiles. The accompanying radar plots represent drug‐likeness parameters such as lipophilicity, molecular weight, hydrogen bonding, and solubility, indicating their compliance with pharmacokinetic criteria [53]. Overall, the figure summarizes the structural diversity, binding potential, and ADMET properties of the analyzed compounds.

5
STOP TOX
The STOP TOX toxicity analysis of the 22 compounds identified from SiEE assessed six major toxicity endpoints acute inhalation (ACI), acute oral (ACO), acute dermal (ACD), eye irritation and corrosion (EIC), skin sensitization (SS), and skin irritation and corrosion (SIC) to evaluate pharmaceutical safety and development potential. The results revealed a complex relationship between ADME characteristics and toxicity behavior, where several compounds with strong drug‐like profiles exhibited certain toxicity concerns, while some poorly bioavailable molecules showed minimal hazards. Among the Tier 1 ADME candidates, 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid emerged as the most promising compound, displaying nontoxic behavior across all endpoints except for mild eye irritation. 2,5‐Dihydroxyacetophenone bis(trimethylsilyl) ether also showed favorable toxicity results with only minor dermal sensitivity, and N‐formyl‐l‐lysine demonstrated an excellent safety profile with minimal irritation potential. In contrast, salicylaldehyde hydrazone and hydroquinone, despite good ADME properties, showed multiple toxicity alerts including oral, dermal, and eye irritation concerns, likely requiring further validation or formulation adjustments. 2‐Propenoic acid 3‐(4‐fluorophenyl) ethyl ester exhibited oral and inhalation toxicity, possibly due to reactive intermediates. Interestingly, some Tier 3 ADME compounds such as docosanoic acid triglyceride and 1‐hexadecene showed low toxicity but remain pharmaceutically unsuitable due to poor solubility and bioavailability. Molecular toxicity mapping further visualized these risks, where green areas indicated safe regions and red zones marked toxic structural features. Based on integrated ADME–toxicity profiling, 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid, 2,5‐dihydroxyacetophenone bis(trimethylsilyl) ether, and N‐formyl‐l‐lysine are identified as Priority 1 lead candidates; salicylaldehyde hydrazone and hydroquinone as Priority 2 (requiring further assessment); and compounds such as oxazepam ditms and other poorly soluble lipids as Priority 3 (deprioritized). Overall, these findings highlight that both favorable ADME and low toxicity are essential for drug development potential, with select compounds demonstrating strong promise for subsequent bioactivity screening and pharmacokinetic validation (Figure 3A, 3B, 3C; Table 7).
FIGURE 3(A–C) Toxic properties of the identified compounds of Selaginella inaequalifolia ethanolic extracts. Green areas show parts of the molecule that have lower toxicity (safe features), while red areas show the parts that increase toxicity (risky features). Darker colors mean a stronger effect, and lighter, fuzzy edges mean the model is less certain there. A sharp red dot highlights a specific atom or bond with a high toxicity risk.

5.1
Docking Analysis of Ligand Against CDK2 (PDB ID: 4GCJ)
A high binding affinity is observed between Pentadecanoic acid, 13‐methyl‐, methyl ester; 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid, salicylaldehyde hydrazone, 2‐propenoic acid, 3‐(4‐fluorophenyl)‐, ethyl ester; and Oxazepam ditmsa gainst CDK2. Among these, pentadecanoic acid, 13‐methyl‐, methyl ester exhibited the strongest binding affinity with a Vina score of −11.0 kcal/mol, followed by 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid (−8.9 kcal/mol) and salicylaldehyde hydrazone (−8.2 kcal/mol). Oxazepam ditms also showed a significant interaction with a Vina score of −7.0 kcal/mol, forming strong contacts with key residues in the active site (Table 8). The identified compounds with Vina scores of −5 and above are included in the Table 8. The most common and prominent interacting residues among these compounds included GLU8, GLY13, THR14, TYR15, ASP127, PHE146, GLY147, LEU148, ARG150, and PHE152, which play crucial roles in kinase activity regulation (Figure 4; Table 8). These findings suggested that these compounds may serve as potential CDK2 inhibitors (Table 8).

5.2
Docking Analysis of Ligand Against Serine/Threonine‐Protein Kinase mTOR (PDB ID: 4JSX)
A notable binding affinity is observed between several compounds against the target protein serine/threonine‐protein kinase mTOR (PDB: 4JSX) (Figure 5; Table 8). The identified compounds with Vina scores of −5 and above are included in the Table 8. Pentadecanoic acid, 13‐methyl‐, methyl ester exhibited the highest affinity with a Vina score of −11.4, followed by α‐d‐glucopyranose, 4‐O‐α‐d‐galactopyranosyllactose with a Vina score of −8.5, and salicylaldehyde hydrazone, benzaldehyde (2‐hydroxy‐, hydrazine) with a score of −8 (Figure 5; Table 8). Other compounds such as Oxazepam ditms and 2,5‐dihydroxyacetophenone, bis(trimethylsilyl) ether had Vina scores of −7.4 and −6.9, respectively (Table 8). 4‐Cyclohepta‐2,4,6‐trienyl‐benzoic acid and 2‐propenoic acid, 3‐(4‐fluorophenyl)‐, ethyl ester showed moderate binding affinity with scores of −7.6 and −6.8 (Table 8). Compounds like methyl 3‐hydroxytetradecanoate tetradecanoic acid 3‐hydroxy‐methyl ester showed the Vina scores of −5.8, while cyclopentasiloxane decamethyl and 1‐hexadecene displayed weaker binding with scores of −5.9 and −5.2 (Table 8). Other compounds like cyclodecasiloxane eicosamethyl and hydroquinone 1,4‐benzenediol had the lowest binding affinities with Vina scores of −5 and −5.5, respectively. The key amino acid residues in the protein involved in these interactions included THR2279, MET2281, GLN2282, GLU49, and CYS135, showing a range of binding sites across the protein's different chains (A, B, C, D) (Table 8).
The toxicity of SiEE is evaluated using the brine shrimp lethality assay, and a dose dependent toxicity is recorded. The LC50 value is determined to be 274.26 mg/mL, with 95% confidence limits ranging from 239.09 mg/mL (lower) to 307.44 mg/mL (upper). In addition, the LC90​ value is recorded as 520.59 mg/mL, indicating the concentration required to achieve 90% lethality. The chi‐square (χ
2) value of 2.300 suggested a good fit for the statistical model, confirming the reliability of the data. These findings indicated that S. inaequalifolia possesses moderate cytotoxic potential; this effect may result from its bioactive compounds, emphasizing the importance of further pharmacological research.
Figure 6 showed the effect of S. inaequalifolia on cell line growth inhibition against MCF‐7 Cell lines at different concentrations. The IC50 values of SiEE was 81.57 µg/mL and standard adiramycin was 38.58 µg/mL (Figure 6).

6
Discussion
The amalgamation of GC–MS–mass spectrometry elevated the GC–MS as one of the highly efficient tool for the separation and identification of active principles from the crude extracts. In the present study, GCMS is employed to reveal the phytoprofile and chemical constituents of SiEE. Previous studies have employed GC–MS for the qualitative and quantitative profiling of volatile, semi‐volatile, and nonpolar compounds and bioactive principles (Pteridium aquilinum—[44], Asplenium aethiopicum—[58], Dryopteris hirtipes—[45], Pteris togoensis—[46], and Dicranopteris linearis—[47]). In the present study, the GC–MS analysis revealed the existence of the various bioactive principles in SiEE. PASS is employed to predict the biological properties of the crude extracts derived compounds and fraction of the crude extracts. Previous predictions using the PASS have identified the potential bioactivities of various medicinal plants, including Vincetoxicum subramanii [61], Psidium guajava [48], Ficus benghalensis, and Ficus krishnae [59], Albizia lebbeck [49], and Andrographis paniculata [50]. The PASS analysis suggested the medicinal properties of the S. inaequalifolia. The ADME and toxicity profiles of bioactive compounds from SiEE are assessed and revealed that several compounds have favorable drug‐likeness characteristics. 2,5‐Dihydroxyacetophenone, bis(trimethylsilyl) ether, and cyclopentasiloxane, decamethyl showed good solubility and GI absorption, while methyl 3‐hydroxy‐tetradecanoate exhibited high absorption despite CYP2D6 inhibition. However, 2,5‐dihydroxyacetophenone and methyl 3‐hydroxy‐tetradecanoate may cause drug interactions due to enzyme inhibition [51]. Oxazepam ditms and hydroquinone exhibited poor solubility or toxicity, particularly in dermal and inhalation tests. IARC [56, 57] reported that Oxazepam ditms are carcinogenic to human.
CDK2 (PDB: 4GCJ) showed the strongest binding (Vina score: −11.0 kcal/mol) with pentadecanoic acid, 13‐methyl‐, methyl ester. Other compounds like 4‐cyclohepta‐2,4,6‐trienyl‐benzoic acid and salicylaldehyde hydrazone also demonstrated significant interactions. Pentadecanoic acid, 13‐methyl‐, methyl ester as a strong mTOR (PDB: 4JSX) binder with a Vina score of −11.4 kcal/mol, comparable to known inhibitors like rapamycin and Torin1 [52]. The molecular docking studies results showed that the pentadecanoic acid, 13‐methyl‐, methyl ester as potential inhibitors of CDK2 and mTOR. Hence the outcome of the present study revealed the potential of pentadecanoic acid, 13‐methyl‐, methyl ester as CDK2 and mTOR inhibitor, warranting further investigation through molecular dynamics simulations and in vitro validation.
The median lethal concentration (LC50) is a crucial parameter in BSLB, representing the concentration required to kill 50% of brine shrimp larvae. Generally, an LC50 value below 1000 µg/mL indicates significant cytotoxic potential [64]. For instance, Woodfordia fruticosa exhibited moderate cytotoxicity with LC50 of 763.34 µg/mL [54], while Plectranthus barbatus root extracts demonstrated high cytotoxicity with an LC50 of 40.07 µg/mL (Lawi et al. 2018) [55]. These findings suggest that different plant species, and even different parts of the same plant, can exhibit significantly diverse cytotoxic profiles. The observed LC50 value below 1000 µg/mL indicates that SiEE possess significant cytotoxic potential [64]. The chi‐square (χ2) value of 2.300 suggests a reliable dose‐response relationship. Compared to the aforementioned medicinal plants, SiEE exhibit extremely low cytotoxicity, making them a safer alternative for pharmacological applications that require minimal toxicity.
Cytotoxic properties of the studied S. inaequalifolia might be due to the toxic compounds present in the extracts. S. inaequalifolia is found to be most effective with LC50 value 274.26 mg/mL. Cytotoxic potentials of SiEE are examined against MCF‐7 breast cancer cell lines. The cell growth inhibition was directly proportional to the concentration of the SiEE. The IC50 values of SiEE was 81.57 µg/mL and standard adiaramycin was 38.58 µg/mL (Figure 6). Typically cytotoxic activity showed IC50 values in the range of < 1 to 100 µg/mL The crude SiEE showed a good growth inhibition with IC50 values 81.57 µg/mL. It leads to identification of novel antitumor and anticancer agents in the SiEE.

7
Conclusion
This study highlights the efficiency of GC–MS in identifying bioactive compounds from SiEE, revealed several phytochemicals with potential pharmacological applications. The ADME and toxicity profile analysis identified pentadecanoic acid, 13‐methyl‐, methyl ester as a strong CDK2 and mTOR inhibitor, suggesting anticancer potential. The brine shrimp lethality assay (LC50: 274.26 mg/mL) indicated low cytotoxicity, while MCF‐7 breast cancer cell line studies (IC50: 81.57 µg/mL) showed promising anticancer activity. These findings support S. inaequalifolia as a potential source of therapeutic agents, warranting further molecular and clinical investigations. Further studies on the isolated active principles may bring out a plant based anticancer agents.

Conflicts of Interest
The authors declare no conflicts of interest.