Colorectal carcinoma (CRC), encompassing colon and rectum cancer, holds the alarming status of being the third most frequently diagnosed cancer (IARC, 2020). Even more concerning is its position as the second-leading cause of cancer-related deaths worldwide, responsible for nearly 2 million new cases and 1 million deaths in 2020, with a rising incidence observed in developing nations (Morgan et al., 2023). CRC typically originates in the glandular epithelial cells of the large intestine and represents a complex disorder influenced by genetic and epigenetic mutations across various signaling pathways (Akhtar et al., 2020). Extensive research efforts have been dedicated to unraveling the intricate mechanisms underlying CRC (Emam et al., 2022; Abd El Fattah et al., 2023). Signaling pathways such as Bcl-2, Wnt/β-catenin, p53, ERK/MAPK, and NF-κB pathways have been pivotal subjects of investigation. These pathways play crucial roles in processes like proliferation, invasion, progression and apoptosis inhibition in CRC cells (Koveitypour et al., 2019). Specific genes within these pathways, such as Bax, Bcl-2, APC, Wnt-1, TP53, CTNNB1, and EGFR, among others, have been identified for their significant involvement in CRC progression (Pricci et al., 2020). Mutations in these genes lead to the development of hyperactive cells characterized by increased replication and survival, ultimately resulting in the formation of benign adenomas. Over time, these adenomas can progress into carcinomas and eventually metastasize over several decades (Rawla et al., 2019).

While advancements in early detection and treatment have contributed to reduced mortality rates, the emergence of drug-resistant cancers and adverse side effects associated with current treatments emphasize the pressing need for innovative and highly efficient therapeutic agents (Giordano and Tommonaro, 2019).

Phytochemicals, including flavonoids, alkaloids, phenolics, glycosides, carotenoids, glucosinolates, anthraquinones, and nitrogenous derivatives, are present in various plant parts (Akhtar et al., 2020). These compounds have demonstrated remarkable biological properties, such as antimicrobial, anti-inflammatory, analgesic, antioxidant, antitumor, and anticancer activities, making them valuable candidates for the treatment and prevention of several chronic diseases (Garcia-Oliveira et al., 2021). Phytochemicals manifest their anticarcinogenic effects through diverse mechanisms (Das et al., 2023). Curcumin, a polyphenolic compound extracted from turmeric (Curcuma longa), has garnered considerable scientific interest since its initial extraction in 1870 (Pricci et al., 2020). Over recent decades, extensive research has explored its unique properties, encompassing antioxidant, anti-inflammatory, antiandrogenic, anti-tumor, antiproliferative, antimicrobial, hypoglycemic, and chemopreventive activities, leading to its recognition as a multifaceted therapeutic agent (Tomeh et al., 2019). Notably, its remarkably minimal toxicity allows for safe consumption, with no reported side effects, even at doses as high as 10 g per day (Mansouri et al., 2020). In the realm of anticarcinogenic effects, curcumin works through diverse mechanisms, including the regulation of several cellular pathways (Kabir et al., 2021). Numerous studies conducted over the years have evidenced that the modulation of these signaling pathways can suppress the activity of proteins and cytokines associated with cancer initiation, progression, proliferation, and tumor development. Additionally, curcumin can induce the activity of other proteins implicated in apoptosis or autophagy (Mansouri et al., 2020; Ojo et al., 2022). Curcumin has been found to downregulate the Wnt/β-catenin pathway, which is overactive in cancer and leads to abnormal cell proliferation and tumor growth. This effect could be achieved by modulating specific genes like Wnt-1, APC, CCND1, EGFR, and CTNNB1 (Vallée et al., 2019). Additionally, the p53 pathway is commonly disrupted in cancer due to TP53 gene dysregulation. Studies have shown that curcumin can regulate the expression levels of p53 pathways (Ismail et al., 2019).

Resveratrol (trans-3,4,5-trihydroxystilbene), a naturally occurring polyphenol belonging to the stilbene class, is synthesized by approximately 72 different plant species, including those within the spermatophytes family such as peanuts, grapes, mulberries, cranberries, blackberries, and blueberries (Gianchecchi and Fierabracci, 2020; Talib et al., 2020). Plants produce resveratrol as a natural defense mechanism in response to external stimuli, including biotic infections, physical injury, ultraviolet radiation, and excessive sunlight exposure (Vervandier-Fasseur and Latruffe, 2019). Initially identified in 1939 from the white hellebore plant (Veratrum grandiflorum), resveratrol has since garnered significant attention due to its diverse therapeutic properties, which include antioxidative, anti-proliferative, antimicrobial, anti-inflammatory, and anti-neurodegenerative effects (Chen et al., 2022). Resveratrol modulates multiple signaling pathways associated with cell growth, division, angiogenesis, apoptosis, metastasis, autophagy, and chemotherapy resistance (Tian et al., 2019). Like curcumin, resveratrol can also influence the Wnt/β-catenin signaling pathway, a crucial network that regulates cell cycle, proliferation, migration, and differentiation. Treatment with resveratrol has been demonstrated to reduce the expression of Wnt-1 and CTNNB1, genes linked to proliferation (Pashirzad et al., 2021). Moreover, resveratrol exhibited a potent pro-apoptotic impact in CRC, as evidenced by an elevated Bax/Bcl-2 ratio, heightened p53 levels, and activation of caspase-3 and -8 (Gavrilas et al., 2019). Recent studies emphasize a synergistic interaction upon combining curcumin and resveratrol, resulting in decreased survival of cancer cells (Arena et al., 2021). The synergy between curcumin and resveratrol emanates from their cytotoxic properties, inducing apoptosis and autophagy through diverse signaling pathways (Zheng et al., 2022). Even with the mentioned advantages, the therapeutic efficacy of curcumin and resveratrol is impeded by multiple limitations (Tomeh et al., 2019). Principal among these challenges are their poor solubility, hydrophobic characteristics, weakened cellular uptake, and significant first-pass effect. These factors collectively reduce bioavailability and chemical stability (Kabir et al., 2021).

Nanotechnology, a rapidly evolving research field, focuses on manipulating functional systems at a nanoscale (Wilczewska et al., 2012; Sahare et al., 2022). Bioformulation-based delivery systems have found extensive applications, including gene therapy, antibiotic delivery, vaccine administration, and protein delivery, illustrating their multifaceted utility (Kumar et al., 2021). Bioformulation-based delivery systems offer several advantages, including non-toxicity and biodegradability, and their small size enables diverse administration routes such as intravenous, nasal, oral, and intraocular, among others (Kumar et al., 2021). Furthermore, these systems enhance drug biodistribution and efficacy by specifically targeting cancer cells, thus minimizing undesirable side effects associated with off-target deliveries (Bamrungsap et al., 2012). Bioformulations also safeguard the therapeutic properties of drugs, preventing rapid elimination or degradation within the body. This preservation results in higher drug concentrations in target tissues, maximizing the therapeutic impact (Wilczewska et al., 2012).

For delivery systems, bioformulations must be non-toxic and biocompatible. In this context, BS have gained attention as promising nanocarriers due to their unique attributions, including high loading capacity, extensive surface area, and large pore size ranging from 2 to 50 nm (Lu et al., 2007; Bharti et al., 2015). Equisetum miryochaetum, a horsetail species native to South America, is crucial in this study because of its unique silica accumulation pattern. In this plant, silica is concentrated in dense thickenings, forming clusters dispersed across the plant’s surface. Cytotoxicity assessments have confirmed the biocompatibility of BS with the human body, demonstrating their biodegradability and low toxicity. Significant positive outcomes have also been observed in biodistribution and excretion evaluations.

The aim of this research, which constitutes the initial phase of a broader study, is to examine the synergistic therapeutic effects of Curcumin-Resveratrol co-loaded BS on Colorectal Cancer Cell lines. This work represents the first endeavor to investigate the therapeutic benefits of curcumin-resveratrol-loaded plant-based BS on CRC cells. This investigation also involves assessing the expression levels of proto-onco or tumor suppressor genes such as TP53, Bax, Wnt-1, and CTNNB1. Understanding the molecular alterations induced by the bioformulation in these key genes is crucial for establishing the groundwork for subsequent phases of the study.

CRC stands as a significant challenge for global healthcare systems, ranking as the third most frequently diagnosed cancer worldwide and representing the second leading cause of cancer-related deaths, affecting both men and women (Lewandowska et al., 2022; Osorio-Pérez et al., 2023). Consequently, there is a dedicated focus on innovative approaches to cancer treatment, exploring new strategies and solutions (Biller and Schrag, 2021). While conventional methods like chemotherapy and radiotherapy have historically been crucial in cancer treatment, their associated side effects significantly impact patients’ quality of life (Khan et al., 2019). As a result, recent scientific attention has shifted towards natural remedies, particularly investigating the potential of natural phytochemicals and biocompatible bioformulations (Berretta et al., 2020). Studies have highlighted the ability of curcumin and resveratrol to significantly inhibit proliferation and induce apoptosis in various cancer cells, establishing them as vital components in cancer therapy (Vervandier-Fasseur and Latruffe, 2019; Zheng et al., 2022).

In this study, BS was extracted from Equisetum myriochaetum and the subsequent surface modification involved the immobilization of APTES, a widely employed silane coupling agent, introducing an amine-terminated group through silanization. This procedure established a covalent bond between the surface of BS and curcumin (Majoul et al., 2015). Following this, the silanized samples were subjected to immobilization with GTA, incorporating an aldehyde group to facilitate the covalent binding of resveratrol (Ionescu, 2022). These chemical modifications enabled the immobilization of curcumin and resveratrol onto amine-terminated and aldehyde groups, respectively, leading to the creation of three distinct bioformulations: Cur-BS, Res-BS, and Cur-Res-BS.

Thorough characterization of bioformulations is essential for a comprehensive understanding of their composition, as well as their physical, optical, and morphological attributes. This study employed specific characterization techniques to scrutinize these facets, including FTIR, TGA, SEM, XRD, BET, and UV-visible spectrophotometry. FTIR was employed to assess the purity of BS extracted from Equisetum myriochaetum. Noteworthy peaks observed in the BS spectrum were identified at 451, 793, 968, 1,058, 1,641, and 3,418 cm⁻¹, corresponding to characteristic stretching and vibrational bonds between atoms. These peaks indicate the exclusive presence of BS, devoid of any detrimental elements (Shokri et al., 2009). Additionally, FTIR was used to confirm the successful immobilization of curcumin and resveratrol through APTES and GTA functionalization. Distinct peaks observed in the FTIR spectra of Cur-BS, Res-BS, and Cur-Res-BS, including those at 1,058, 1,400, 1,550, 1,600, 3,000, 3,300, 3,500, and 3,600 cm⁻¹, are attributed to the presence of specific functional groups, thus confirming the success of surface modification process (Majoul et al., 2015; Elbialy et al., 2020). XRD analysis indicated the amorphous nature of the BS samples, positively influencing their solubility. The abolition of crystalline peaks of curcumin and resveratrol makes the bioformulation compatible, and the appearance of a peak around 2ϴ angle 14.11° confirms the successful attachment of curcumin and resveratrol onto the BS. The same was also confirmed by DLS and TGA studies. Furthermore, SEM delineated the porous surface nature of the BS, a characteristic corroborated by BET analysis, which affirmed the existence of pores featuring an average diameter of 4.9175 nm and an average volume of 0.652 cc/g.

The assessment of encapsulation efficiency is crucial to ascertain the successful immobilization of therapeutic agents within the inert silica host (Kankala et al., 2022). Our investigation revealed that approximately 45% of curcumin and 64% of resveratrol were effectively loaded within the pores of the Cur-Res-BS bioformulation. It is pertinent to acknowledge the diverse encapsulation efficiencies observed across different nanocarriers in prior research endeavors. For instance, solid lipid nanoparticles (SLN) exhibited encapsulation efficiencies of 74.42% for curcumin and 79.84% for resveratrol (Gumireddy, 2017), while zein-based solid nanocarriers demonstrated 54% encapsulation for curcumin and 71% for resveratrol (Leena et al., 2022). Additionally, co-loaded conjugated polymer achieved an impressive efficiency of 91.3% for curcumin and 83.2% for resveratrol (Zheng et al., 2022). Although our study did not attain the anticipated high encapsulation efficiency, it is crucial to recognize that similar challenges have been encountered by fellow researchers in bioformulation studies. Several factors could underlie the observed reduction in encapsulation efficiency. Firstly, the swift release of therapeutic cargo during the loading process could be attributed to weak interactions between the surface of BS and the guest molecules (Kankala et al., 2022). Additionally, it has been observed that a significant portion of phytochemical compounds may adhere to the external surface of BS rather than being restricted within the interior pores. This phenomenon is linked to the obstruction of silica pore gates, resulting in a decline in encapsulation efficiency (Asgari et al., 2021). Furthermore, in-depth characterization techniques and meticulous analyses are imperative to gain a comprehensive understanding of the encapsulation process, identifying potential avenues for enhancements in future studies.

APTES modification introduces amino groups onto the surface of BS, which can interact with drugs and affect their release profiles. For example, in the case of mesoporous silica nps (MSNs), the APTES-modified surface can significantly increase drug loading and decrease the rate of drug delivery (Ahmadi et al., 2014). Drug release studies have shown that APTES-modified pSi can release hydrophobic drugs like CPT continuously over extended periods, demonstrating the material’s stability and suitability for sustained drug delivery (Zhang et al., 2019). Glutaraldehyde is used to cross-link pectin-based delivery vehicles for the encapsulation of resveratrol; the cross-linking process is essential for creating nps that can specifically deliver resveratrol to the colon (Chung et al., 2020). The concentration of glutaraldehyde is a critical factor that affects both the cross-linking process and the encapsulation efficiency of resveratrol. The drug release profile at both pHs exhibits a slow and sustained release, where both the phytochemicals were released at a steady state. In the initial hours, resveratrol was released first, and thereafter release of curcumin was observed, which is in accordance with the immobilization protocol followed where the inner layer is supposed to be formed of curcumin and the outer layer is composed of resveratrol. The porous structure of the BS allows for the controlled release of curcumin, which can be designed to occur over extended periods. This gradual pattern of release could be advantageous as it can maintain the therapeutic window for a longer time period.

In recent studies, researchers have discovered that the combination of therapeutic agents such as curcumin and resveratrol enhances cytotoxicity and augments the effects of chemotherapy. However, to overcome the limitations associated with these compounds, including weak chemical stability, low solubility, and poor bioavailability, co-loaded bioformulations have been explored (Selvam et al., 2019). In our current study, we explored the cytotoxic effects of the Cur-Res-BS bioformulation on colorectal cancer cells, conducting viability assays on both HCT-116 and Caco-2 cell lines. The results demonstrated a significant reduction in cell viability, indicating a dose-dependent relationship. Specifically, the cytotoxic effects of Cur-Res-BS were observed in HCT-116 cells within the concentration range of 100–1,000 μg/mL, with an IC₅₀ value of 380.84 μg/mL.

To the best of our knowledge, none of the studies have been carried out so far to demonstrate the cytotoxic effects of curcumin or resveratrol-loaded BS on colorectal cancer cells. Predominantly, existing studies on curcumin OR resveratrol-loaded BS focus on their cytotoxic impact on breast cancer cells, although a subset of analogous studies provides a comparative framework. Colloidal BS has been documented to enhance the biological efficacy of resveratrol on HT-29 colon cancer cells, exhibiting dose-dependent reductions and a 36% decline in cell viability at a concentration of 400 μM (Summerlin et al., 2016). Furthermore, investigations involving 5-fluorouracil, a widely used clinical chemotherapeutic agent, and curcumin-loaded BS revealed an IC₅₀ of 21.3 μg/mL in Hep-2 laryngeal squamous cancer cells, emphasizing heightened synergistic effects (Wang et al., 2020). Conversely, examinations into the synergistic effect of curcumin and resveratrol in conjugated polymer nps on hepatocellular carcinoma cells reported an IC₅₀ of 18.30 μM (Zheng et al., 2022).

Despite the relatively elevated concentrations employed in our study compared to analogous bioformulations assessed across diverse cancer cell types, it is imperative to underscore that the principal aim was to establish their efficacy and assess the expression of target genes as preliminary data. For ensuing research endeavors, exploration of lower concentrations is warranted to delineate the minimal thresholds eliciting maximal effects. This prudent approach will contribute to a more exhaustive comprehension of the nuanced responses of colorectal cancer cells to the proposed bioformulations. Conversely, the impact of Cur-Res-BS on Caco-2 cells was observed within the same concentration range. However, the cytotoxic impact observed in Caco-2 cells was not as pronounced as in HCT-116 cells. It is noteworthy that the IC₅₀ value for Caco-2 cells was notably higher, measured at 1,002.42 μg/mL. This higher value indicates the need for elevated concentrations to achieve the IC₅₀ dose response in this particular cell line. The discrepancy in the cytotoxic effects of Cur-Res-BS between HCT-116 and Caco-2 cells is remarkable. Further research is essential to comprehensively understand the bioformulation’s impact on Caco-2 cells. Several factors could contribute to the divergent outcomes in these two CRC cell lines. Genetic variations among cancer cell lines can influence the metabolism rates of compounds, the expression levels of detoxifying enzymes, and the efficiency of the multidrug resistance system in expelling cytotoxic agents, as demonstrated in prior studies (Santana-Gálvez et al., 2020). Furthermore, earlier research has indicated distinct responses of Caco-2 monolayers to resveratrol treatment, where cell growth inhibition was observed only when the treatment was administered apically, leaving the basolateral membrane unaffected (Polycarpou, 2013). Additionally, when subjected to identical treatments, HCT-116 and Caco-2 cells exhibited diverse responses, indicating the involvement of distinct mechanisms in these separate cell lines (Santana-Gálvez et al., 2020).

Additional cytotoxicity assays were conducted to assess the efficacy and safety of the bioformulations. Initially, the cytotoxicity assay of free curcumin, ranging from 100 to 1,000 μg/mL, resulted in high cell viability on both cell lines, with figures reaching 69.8% for HCT-116% and 65.62% for Caco-2 cells at 1,000 μg/mL. Similarly, the assay of free resveratrol yielded a cell viability of 70.67% for HCT-116% and 73.09% for Caco-2 cells. These outcomes indicate that the bioformulations exhibit a greater cytotoxic effect than the free phytochemicals on both cell lines at equivalent concentrations and conditions. The increased effectiveness of bioformulations in combating cancer, when compared to free phytochemical compounds, can be attributed to several factors. After initial interaction through ligand-receptor binding or non-specific interactions, the nps are typically internalized via endocytosis. The exact pathway of endocytosis can depend on the nanoparticle properties and cell type but often involves clathrin-mediated endocytosis, caveolin-mediated endocytosis, or macropinocytosis. The clathrin-mediated endocytotic pathway internalizes poly(lactic-co-glycolic acid), D,L-polylactide, poly(ethylene glycolco-lactide), and silica (SiO₂)-based nanomaterials (Foroozandeh and Aziz, 2018). The average diameter, PDI, and size stability of nanocarrier formulations are among the parameters that determine whether or not they are appropriate for drug administration. The internalization process of the larger nps (≥165 nm) is through endocytosis but requires longer time and energy in comparison to the smaller nps (≤113 nm) (Xue et al., 2018). Cancer tissues often have leaky vasculature and poor lymphatic drainage, allowing larger particles to preferentially accumulate within the tumor tissue—a phenomenon known as the EPR effect. Tumor cells allow the entrance/exit of nanocarriers of a certain size (≤150 nm) through fenestrated capillaries (Danaei et al., 2018). It has also been reported that nps with sizes up to a few micrometers can also undergo endocytosis (Zhang et al., 2009). Though the PDI value of BS is high in our study, which indicates the existence of particles of different sizes, however, the size range is between ∼77 and ∼222 nm and as reported, particle size less than 200 nm can mediate their entrance through clathrin-mediated endocytosis while particle size above 200 nm are internalized through caveolae-mediated endocytosis. The process of macropinocytosis allows bacteria, viruses, apoptotic and necrotic cells, and antigen presentation to be swallowed. Micron-sized nps that are unable to enter into cells by the majority of other pathways can be internalized by this pathway (Foroozandeh and Aziz, 2018; Manzanares and Ceña, 2020). According to the values of zeta potential, the surface charges of BS changed from −37.8 to 2.3 mV when resveratrol was added and 2.7 mV when curcumin was added. A zeta potential value of 2.4 mV was obtained when both the phytochemicals were added to the BS, which implies that the addition of these phytochemicals imparts a positive charge to BS (Mohebian et al., 2023). As reported, positively charged nps are easily taken up by the negatively charged plasma membrane, leading to the disruption of the membrane integrity and can induce cell death. Additionally, positively charged nps have been demonstrated to produce a high level of intracellular ROS compared to neutral or negatively charged nps (Bhattacharjee et al., 2010). Furthermore, the enhanced impact of Cur-Res-BS, as opposed to free compounds, arises from the effectiveness of the bioformulation’s therapeutic window. This therapeutic window, which delineates the duration during which the treatment remains effective, is a crucial parameter for understanding the sustained efficacy of administered substances. In this context, a significant advantage of BS-based drug administration lies in the gradual release of drugs into cells, ensuring a prolonged period of effectiveness (Rosenholm et al., 2011).

Moreover, the results of the cell viability assay of BS on both cell lines showed minimal cytotoxicity, with cell viability measured at 97.92% at 1,000 μg/mL for HCT-116 cells and 97.34% at 1,000 μg/mL for Caco-2 cells. The disparity between these results and the cytotoxicity of the BS on the same cell lines is significant. These findings confirm the biocompatibility of the drug-free BS with both cell lines, as there is no substantial reduction in cell viability, indicating no safety concerns associated with the treatment in cellular terms. Previous studies have indicated that BS at 100 μg/mL also maintained viability above 80%, emphasizing the lack of apparent cytotoxicity on SW620 cells, indicating their biocompatibility (Liu et al., 2018). Additionally, it has been reported that drug-free BS do not exhibit obvious cytotoxicity to SW480 and NCM460 cells, asserting their biocompatibility and non-toxicity to cells, notably, only at extremely high concentrations of BS, around 25 mg/mL, exhibit cytotoxicity (Li et al., 2017). Furthermore, in the study conducted by Summerlin et al., 2016, it was observed that BS, when employed as a control, displayed negligible cytotoxicity in HT-29 and LS174T colon cancer cell lines. These findings are consistent with earlier research, providing further evidence of the non-toxic characteristics associated with BS.

The assessment of the cytotoxicity of the Cur-Res-BS formulation on the HEK-293 cell line revealed minimal toxicity, as evidenced by significantly elevated IC50 values of 6,766.94 μg/mL after 24 h of exposure and 8,625 μg/mL after 48 h of exposure, indicating low toxicity on healthy cells. The SI serves as a measure of the specificity of the bioformulation towards cancer cells compared to normal cells, with a higher SI value corresponding to greater selectivity (Veschi et al., 2020). The resulting SI of 17.77 signifies a substantial level of selectivity towards the HCT-116 cell line.

The process of cancer development unfolds through a series of sequential mutational events as the disease progresses (Ruiz-Manriquez et al., 2022). Crucial pathways like Wnt/β-catenin, p53, and Bcl-2 play pivotal roles in regulating various biological processes, including cell differentiation, proliferation, angiogenesis, apoptosis, and survival (Koveitypour et al., 2019). The human intestine, a highly dynamic organ, possesses remarkable regenerative capabilities, allowing it to replace its entire 7-meter-long cell lining weekly. This regenerative mechanism is primarily triggered by various stresses, including microbiological, chemical, and mechanical factors originating from digestion and evacuation processes (Beumer and Clevers, 2020). Numerous signaling pathways actively participate in the self-renewal, differentiation, and proliferation of cells. Considering the crucial function these pathways serve in cell renewal, it is expected that any changes in their elements can trigger pathological occurrences (Powell et al., 2011). It is essential to understand these pathways’ fundamental functions in both normal physiological states and under pathological conditions. Exploring the impact of naturally occurring phytochemicals that modulate these pathways in CRC is crucial to advancing our understanding and developing potential therapeutic interventions (Oliveira et al., 2022).

When genetic or epigenetic abnormalities induce mutations in the canonical Wnt/β-catenin signaling pathway, it leads to abnormal proliferation and tumor growth, particularly in CRC (Pashirzad et al., 2021). Elevated levels of CTNNB1 and Wnt-1 are detected in CRC patients compared to normal tissues (Siddiq et al., 2017). Previous research has indicated that curcumin and resveratrol exert a potent inhibitory effect on colorectal cancer cell proliferation by suppressing the Wnt/β-catenin signaling pathway (Cheng et al., 2019). Our results demonstrate a decrease in Wnt-1 expression in HCT-116 of 0.21-fold after 24 h and 0.59-fold after 48 h (p < 0.05). However, the interaction between the bioformulation and time was not statistically significant (p > 0.05), therefore, more evidence is needed to prove that the effect of the bioformulation increases over time. Additionally, downregulation of CTNNB1 expression in HCT-116 cells of 0.85-fold after 24 h and 0.34-fold after 48 h was observed (p < 0.05). Moreover, Bax, a crucial member of the Bcl-2 family and a regulator of the intrinsic apoptotic pathway, plays a pivotal role in inducing cell death (Kowalczyk et al., 2017). Furthermore, the TP53 gene, frequently mutated in various cancers, including CRC, serves as a vital tumor suppressor gene (Slattery et al., 2019). Studies with the HCT-116 cell line have reported that curcumin-induced upregulation of Bax inhibits apoptosis (Moragoda et al., 2001). Additionally, the application of resveratrol has been reported to increase the expression of the tumor suppressor protein p53, which is crucial in regulating the cell cycle and apoptosis (Li et al., 2019). Exposure to Cur-Res-BS treatment resulted in a significant upregulation of Bax expression of 6.1-fold after 24 h and 1.99-fold after 48 h (p < 0.05). Nevertheless, the statistical analysis indicated that there was no significant interaction observed between the bioformulation and time (p > 0.05). Thus, further empirical evidence is required to substantiate that the impact of the bioformulation effect intensifies over time. Moreover, a significant upregulation of TP53 expression levels of 7.87-fold after 24 h and 1.77-fold after 48 h, compared to the control group, was observed (p < 0.05).

In summary, this study marks the first attempt to explore the therapeutic effects of curcumin-resveratrol-loaded BS on CRC cells. Our results revealed a significant reduction of cancer cell viability following the administration of Cur-Res-BS when compared to the individual bioformulation, indicating a synergistic impact of the nanoformulated phytochemicals. Moreover, it was also observed that the anticarcinogenic effect of current bioformulations is much higher than the free phytochemical compounds, which implies a significant advantage of BS-based drug administration. Furthermore, comprehending the molecular changes brought about by the bioformulation in the pivotal genes is essential for laying the foundation for further stages of the investigation. Since the outcome of this study is promising, further research into the intricate mechanisms and prerequisites essential for the optimal functionality of this treatment is vital, including the associated risks and limitations. Last but not least, the development of an in vivo targeted delivery system of the current bioformulation would justify its real strength, and the current research would pave the way for that.