Cancer is a multifaceted and life-threatening disease that remains one of the leading causes of death globally (Hanahan, 2022). According to the World Health Organization (WHO), cancer accounted for nearly 10 million deaths in 2020, with cervical cancer (caused by HeLa cells) and colorectal cancer (modeled by HCT116 cells) representing significant proportions of this burden. Current treatment options such as chemotherapy, radiation and surgery, while effective to varying degrees are often accompanied by severe side effects and limitations in selectivity towards malignant cells (Brayet al., 2022; Bray and Parkin, 2022). These challenges have prompted a global pursuit for safer, cost-effective and targeted anticancer therapies. One promising avenue involves exploring plant-derived bioactive compounds, which exhibit significant anticancer activity due to their ability to modulate multiple cancer pathways while maintaining biocompatibility (Sunget al., 2021).

Plant secondary metabolites, commonly referred to as phytocompounds, have gained substantial attention in recent years due to their diverse biological properties, including antioxidant, anti-inflammatory and anticancer activities (de Lunaet al., 2023; Marqueset al., 2021). Polyphenols, flavonoids, terpenoids and alkaloids, among others, have been shown to induce apoptosis, suppress angiogenesis and interfere with cancer-promoting pathways such as those mediated by Nuclear Factor-Kappa B (NF-κB), Mitogen-Activated Protein Kinase (MAPK) and Vascular Endothelial Growth Factor (VEGF) (Parekhet al., 2023; Situmoranget al., 2024). For example, extracts from Asystasia gangetica, enriched with polyphenols, demonstrated remarkable cytotoxicity in in vitro studies, alongside significant interactions with cancer-related proteins in molecular docking (in silico) analyses (Pyoet al., 2024; de Lunaet al., 2023).

Modern research methodologies combining in silico and in vitro approaches offer a robust platform to evaluate the anticancer potential of phytochemicals. In silico methods, including molecular docking and dynamics simulations, facilitate the prediction of compound-protein interactions, offering insights into the binding efficiency and mechanism of action of phytochemicals against critical cancer targets like Bcl-2 and VEGFR (Samadarsiet al., 2022; Yousefet al., 2022). These computational predictions, when coupled with in vitro studies such as cell viability assays, flow cytometry and ROS production analysis, provide a comprehensive understanding of the therapeutic potential of these compounds. For instance, a study on the bioactive compounds from Annona macroprophyllata demonstrated their ability to induce apoptosis in cancer cells through Bcl-2 inhibition, validated both computationally and experimentally (Ramírez-Santoset al., 2024).

Cervical cancer, represented by HeLa cells, is predominantly caused by persistent infection with Human Papillomavirus (HPV), leading to genetic mutations and uncontrolled cell proliferation. On the other hand, colorectal cancer, modeled by HCT116 cells, is characterized by mutations in the Wnt/β-catenin signaling pathway, among others. Both cancer types are aggressive and require novel therapeutic strategies that can selectively target tumor cells. Plant-derived compounds, due to their structural diversity and multitarget potential, provide an ideal foundation for developing such therapies (Wanget al., 2020; Sharmaet al., 2021).

This study focuses on evaluating the anticancer activity of selected phytocompounds from plant extracts using in silico and in vitro techniques. By analyzing their interactions with key cancer proteins and assessing their cytotoxicity against HeLa and HCT116 cell lines, the research aims to identify potent, natural anticancer agents with translational potential for clinical applications. The integration of computational and experimental approaches in this study underscores the importance of combining technological advancements with natural product research to address the global challenge of cancer treatment.

In this study, leaves of Solanum virginianum, Hibiscus rosa-sinensis, Piper betle and Eclipta alba were gathered from the Tirunelveli district of Tamil Nadu, India, during September and October 2023. Dr. C. Babu, Head and Associate Professor of Botany at Pioneer Kumaraswamy College, Nagercoil, identified and authenticated the plant specimens. The leaves were thoroughly rinsed to eliminate dust, laid out on plain paper and air-dried in the shade at room temperature for about 10 days. The dried leaves were then ground into a fine powder, which was stored in airtight containers for future use.

To prepare the extracts, 25 g of powdered material from each plant (Solanum virginianum, Hibiscus rosa-sinensis, Piper betle and Eclipta alba) were mixed with 250 mL of ethanol and extracted using a Soxhlet apparatus until the solvent turned colorless. The solvent was then evaporated at room temperature and the extract was preserved in airtight containers for further testing.

Phytochemical analyses of the plants (Solanum virginianum, Hibiscus rosa-sinensis, Piper betle and Eclipta alba) were conducted following standard procedures established by RNS Yadav and Munin Agarwala (Yadav and Agarwala, 2011). The leaf samples were subjected to various qualitative tests to identify the presence of specific phytochemicals, including terpenoids, steroids, fatty acids, phenolic compounds, alkaloids, saponins and flavonoids.

The GC-MS analysis for the ethanolic extracts was conducted at Heber Analytical Instrumentation Facility (HAIF) at Bishop Heber College in Trichy using a SHIMADZU GC MS QP2020 system. This system included an autosampler, injector, gas chromatograph (GC-2010) and mass spectrometer, utilizing a capillary standard non-polar SHRxi-5Sil-MS column (30.0 m length, 0.25 mm diameter, 0.25 µm film thickness). The electron ionization apparatus operated at 70 eV, with a 5 µL injection volume and helium carrier gas (99.99% pure) flowing at 1.20 mL/min and a split ratio of 10. The GC oven was programmed to begin at 50ºC, held for 2 min, then ramped to 280ºC over 10 min. Data was collected in the m/z range of 50-500 with a 0.3-second scan interval, taking 21 min to complete. Percentage quantification for each identified component was calculated from the ratio of its peak area to the total peak area using Shimadzu GC-MS real-time software (Sehimet al., 2023).

The GC-MS spectra were analyzed using the National Institute of Standards and Technology (NIST) (Stein, 2012) and WILEY (Hubschmann, 2015) databases, which contain extensive patterns for spectral comparison. Each unknown compound’s spectra were compared with known profiles in these libraries to identify molecular structure, weight and formula.

Molecular docking was performed to study interactions between proteins from Colon (5FGK) and Cervical (4J96) cancer cell lines and plant compounds, using AutoDock Vina (Trott and Olson, 2010). Chemical structures of the phytochemicals were built with ChemDraw 8.0 and ChemBio3D was used for energy minimization. Ligand structures were then used as input for AutoDock Vina for docking simulations (Elmezayenet al., 2021). Receptor structures were downloaded from the Protein Data Bank (5FGK for Colon, 4J96 for Cervical) and prepared with AutoDock 4.0’s auto preparation feature. The docking grid box, defined to set simulation parameters, measured 30×30×30 grid points with 0.375-point spacing. Coordinates for Colon protein (5FGK) were -6.278197, -19.093869, and 148.253541; for Cervical protein (4J96), they were 37.442917, 11.807821, 22.762488. The AutoDock Vina algorithm identified optimal ligand-protein binding configurations, examining up to nine conformers for each ligand. PyMOL and Discovery Studio Visualizer tools analyzed the interactions, with the lowest free energy conformations selected for further examination, displaying hydrogen bonds and interacting residues in detail.

The Table 1 summarizes the qualitative phytochemical analysis of four different plant materials: Solanum virginianum, Hibiscus rosa-sinensis, Piper betle and Eclipta alba. Each plant was tested for the presence of various phytochemicals, including proteins, carbohydrates, phenols, flavonoids, saponins, steroids, terpenoids, alkaloids, glycosides and tannins. Hibiscus rosa-sinensis lacked detectable protein content. In Carbohydrates (Benedict Reagent Test): High carbohydrate presence was noted in Solanum virginianum and Eclipta alba. Hibiscus rosa-sinensis showed a low concentration, while Piper betle did not show carbohydrate presence. In Phenols (Ferric Chloride Test), Hibiscus rosa-sinensis, Piper betle and Eclipta alba showed a high phenol content, suggesting strong antioxidant potential in these plants. Solanum virginianum contained a moderate amount. In Flavonoids (Lead Acetate Test), Hibiscus rosa-sinensis and Eclipta alba showed moderate flavonoid content, whereas Solanum virginianum and Piper betle exhibited lower levels. In Saponins (Growth Test), Present at moderate levels in Hibiscus rosa-sinensis and in low concentrations in Solanum virginianum and Eclipta alba). Piper betle did not show any presence of saponins. In Steroids (Chloroform Test), Steroids were highly present in Solanum virginianum, while Hibiscus rosa-sinensis, Piper betle and Eclipta alba contained low levels. In Terpenoids (Chloroform Test), High terpenoid content was noted in Solanum virginianum and Piper betle, with moderate levels in Hibiscus rosa-sinensis and Eclipta alba. In Alkaloids (Wagner and Mayer Test), High concentrations of alkaloids were observed in Solanum virginianum and Eclipta alba, while Hibiscus rosa-sinensis and Piper betle showed moderate levels. In Glycosides (Brown Ring Test), High levels of glycosides were present in Eclipta alba, with moderate concentrations in Solanum virginianum and low levels in Hibiscus rosa-sinensis and Piper betle. In Tannins (NaOH Test), Solanum virginianum, Eclipta alba and Hibiscus rosa-sinensis all exhibited high tannin concentrations, while Piper betle showed no presence of tannins.

Table 2 summarizes the key phytocompounds found in the ethanolic extracts of Solanum virginianum, Eclipta alba, Hibiscus rosa-sinensis and Piper betle, as identified by GC-MS analysis. Each plant extract contained various compounds, categorized by retention time, percentage peak area, compound name, molecular formula, molecular weight and phytochemical classification. For Solanum virginianum, prominent compounds include Stearic acid (6.12%), Hexadecanoic acid (4.12%) and Phytol (5.78%), indicating a high concentration of fatty acids and diterpenoids. The plant also contains ester derivatives like Diethyl 2-(p-tolyl) malonate and Ethyl oleate, suggesting potential anti-inflammatory properties. In Eclipta alba, the main components were n-Hexadecanoic acid (26.98%) and 9-octadecynoic acid (26.95%), both fatty acids recognized for their anti-inflammatory and antimicrobial effects. Other noteworthy compounds include Stigmasterol and Stigmasta-3,5-dien-7-one, which are steroids with cholesterol-lowering and possible anticancer benefits. Additionally, the presence of Phytol (0.703%) as a diterpenoid and Skimmien (1.702%) as a triterpenoid supports the plant’s therapeutic potential. The primary compounds in Hibiscus rosa-sinensis include Hexadecanoic acid (22.65%) and Neophytadiene (5.28%), both known for anti-inflammatory and antimicrobial properties. Phytol (13.72%) and Loliolide, a benzofuran derivative, contribute antioxidant effects, making this plant suitable for skin-related applications. For Piper betle, notable compounds were Diethyl phthalate (6.78%) and Neophytadiene (7.42%), indicating antimicrobial and antioxidant activity. Additional compounds like Eugenol and Caryophyllene, both known for strong anti-inflammatory and analgesic effects, align with the traditional use of Piper betle in oral care and as an anti-infective treatment (Figure 1).

Figure 1:
GC-MS chromatogram of Solanum virginianum, Eclipta alba, Hibiscus rosa sinensis and Piper betle.

Table 3 provides an overview of the binding energy values for various phytocompounds from Solanum virginianum, Eclipta alba, Hibiscus rosa-sinensis and Piper betle when docked with proteins associated with cervical and colon cancer cell lines. The Table includes each compound’s binding interactions (hydrogen, electrostatic and hydrophobic) and binding energy values in kcal/mol, where more negative values indicate stronger binding affinities. Compounds from all four plants displayed a range of binding affinities with the cancer proteins, with energies between -5.0 and -11.4 kcal/mol. Notably, Eclipta alba’s Stigmasterol and Skimmien had the strongest affinities, showing values up to -10.1 kcal/mol against cervical cancer proteins and -11.4 kcal/mol against colon cancer proteins, suggesting their potential efficacy in targeting cancer-related proteins. Frequent hydrogen bonding with amino acids such as LYS517, ASP173 and PHE97 was observed, which are crucial for stabilizing protein-ligand complexes (Figure 2). Hydrophobic interactions, particularly with residues like VAL35, ALA50 and TYR566, also played a significant role in enhancing the compounds’ affinities for cancer proteins. In Solanum virginianum, Diethyl 2-(p-tolyl) malonate and Stearic acid showed moderate affinities (-6.9 and -5.7 kcal/mol, respectively) with cervical cancer proteins, indicating potential for anticancer activity via protein inhibition. In Eclipta alba, Furan-2-carbohydrazide demonstrated strong binding (-8.2 kcal/mol) with colon cancer proteins, likely due to its hydrogen bonding and electrostatic interactions.

Figure 2:
2D and 3D structure of stigmasterol (A and B) and Skimmien (C and D) interaction with HeLa and HCT116 cell line.

Hibiscus rosa-sinensis exhibited moderate binding in compounds such as 4-Allyl 2,6-dimethoxy phenol (-7.4 kcal/mol) with cervical cancer proteins, aided by hydrogen bonding and hydrophobic effects. In Piper betle, Eugenol and Caryophyllene showed affinities of -5.9 and -7.4 kcal/mol, respectively, with cervical cancer proteins, supporting their potential as complementary agents in cancer therapy. The reference drug Camptothecin demonstrated binding energies of -9.5 kcal/mol (cervical cancer) and -10.2 kcal/mol (colon cancer). Several plant compounds, including Stigmasterol, Skimmien and Stigmasta-3,5-dien-7-one, achieved or exceeded these values, suggesting they may serve as natural alternatives or complements to conventional cancer treatments.

Table 4 compares the IC₅₀ values of phytocompounds from Solanum virginianum, Hibiscus rosa-sinensis, Piper betle and Eclipta alba with the reference drug camptothecin against two cancer cell lines: HeLa (cervical cancer) and HCT116 (colon cancer). Among the plant extracts, Eclipta alba showed the lowest IC₅₀ values, with 30 µg/mL for HeLa and 24 µg/mL for HCT116, indicating the highest potency. Solanum virginianum displayed IC₅₀ values of 69 µg/mL (HeLa) and 59 µg/mL (HCT116), demonstrating moderate activity. Hibiscus rosa-sinensis had IC₅₀ values of 86 µg/mL (HeLa) and 72 µg/mL (HCT116), the highest values among the extracts, suggesting lower potency. Piper betle exhibited IC₅₀ values of 75 µg/mL (HeLa) and 66 µg/mL (HCT116), reflecting moderate efficacy (Figures 2 and 3). The reference drug camptothecin showed IC₅₀ values of 32 µg/mL (HeLa) and 28 µg/mL (HCT116), indicating greater effectiveness than all plant extracts except Eclipta alba (Figure 4). Eclipta alba demonstrated notable promise as an anti-cancer agent, showing IC₅₀ values comparable to the reference drug camptothecin, positioning it as a strong candidate for further research in cervical and colon cancer treatment. In contrast, other extracts such as Hibiscus rosa-sinensis and Piper betle exhibited lower potency, suggesting more limited effectiveness.

Figure 3:
Viability of HeLa cell line treated with Eclipta alba extract in MTT assay A. 10 µg/mL shows 69.48% viability, B. 20 µg/mL extract shows 61.16% viability, C. 40 µg/mL shows 53.01% viability, D. 80 µg/mL shows 35.98% viability, E. 160 µg/mL shows 15.93% F. vehicle control shows 53.64% viability.

Figure 4:
Viability of HCT116 cell line treated with Eclipta alba extract in MTT assay A. 10 µg/mL shows 66.22% viability, B. 20 µg/mL extract shows 43.57% viability, C. 40 µg/mL shows 31.81% viability, D. 80 µg/mL shows 20.55% viability, E. 160 µg/mL shows 15.03% F. vehicle control shows 28.32% viability.

The qualitative analysis reveals that each plant material possesses unique phytochemical profiles, which can be related to potential therapeutic benefits. For example: Eclipta alba exhibited high levels of protein, carbohydrate, alkaloid, glycoside and tannins, making it a good candidate for antioxidant and anti-inflammatory studies. Solanum virginianum showed diverse phytochemical presence, particularly in steroids, terpenoids and alkaloids, suggesting potential antimicrobial and anti-inflammatory properties. Hibiscus rosa-sinensis contained notable levels of phenols, flavonoids and tannins, supporting its known antioxidant and skin-soothing effects. Piper betle showed a strong presence of terpenoids and phenols, which may contribute to its known anti-microbial properties. This analysis suggests the potential for using these plants in therapeutic formulations, highlighting the importance of each plant’s unique phytochemical profile for different health applications.

The GC-MS analysis demonstrates that each plant has a distinct phytochemical profile with potential therapeutic applications. Solanum virginianum and Eclipta alba are notably rich in fatty acids and sterols, which may account for their anti-inflammatory and cholesterol-lowering properties. Hibiscus rosa-sinensis contains a wealth of antioxidants and anti-inflammatory compounds, suggesting its suitability for skincare applications. Piper betle features a variety of essential oils and anti-inflammatory agents, aligning with its traditional uses in oral and respiratory health. These findings underscore the medicinal potential of these plants and emphasize the need for further research to explore and confirm the therapeutic effects of these bioactive compounds.

This docking analysis highlights the ability of plant-based compounds, particularly from Eclipta alba and Solanum virginianum, to interact with cancer-related proteins through hydrogen bonding and hydrophobic interactions, with binding affinities close to those of Camptothecin. These findings point to compounds like Stigmasterol and Skimmien as promising candidates for further investigation in developing plant-derived therapies for cervical and colon cancer, laying a foundation for future in vitro and in vivo studies to confirm their efficacy.

Overall, HCT116 cells (colon cancer) exhibited slightly lower IC₅₀ values across all extracts than HeLa cells, indicating a higher sensitivity to these phytocompounds. These findings highlight Eclipta alba’s potential for future cancer studies, while Solanum virginianum, Hibiscus rosa-sinensis and Piper betle may serve as valuable options in combination therapies or as complementary treatments.

The phytochemical analysis, docking studies and IC₅₀ assays highlight Eclipta alba as having the most promising anti-cancer potential, showing the highest cytotoxicity and binding affinity among the extracts tested. Solanum virginianum, Hibiscus rosa-sinensis and Piper betle also exhibited moderate activity, with compounds like stigmasterol and skimmien achieving binding affinities close to those of camptothecin. These findings indicate that Eclipta alba is a strong candidate as a natural anti-cancer agent, meriting further in vivo research to assess its effectiveness. The distinct phytochemical profiles of each extract suggest potential for developing multi-targeted therapeutic approaches, using these plants individually or in combination with established anti-cancer drugs.