Sterols from Cyathea Bicrenata Liebm Rhizome and their Breast Cancer Activity in vitro and in silico Evaluation

Sterols from Cyathea Bicrenata Liebm Rhizome and their Breast Cancer Activity in vitro and in silico Evaluation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sterols from Cyathea Bicrenata Liebm Rhizome and their Breast Cancer Activity in vitro and in silico Evaluation Ruth Esperanza Márquez-López, Irene Chaparro-Hernández, Ignacio Villanueva-Fierro, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6813685/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Sep, 2025 Read the published version in Bulletin of the National Research Centre → Version 1 posted 9 You are reading this latest preprint version Abstract Herbal infusions are considered as a kind of therapeutic approach that aids, and in many cases replaces, conventional pharmacological treatments. However, the identification of its compounds and their activities in human health continue to be the subject of multidisciplinary research. This research aimed to evaluate the cytotoxic effect of the sterols fraction isolated from Cyathea bicrenata Liebm ( Cyatheae ) rhizome in MCF-7 carcinoma cell line and simulate their blocking effect in the anti-apoptotic Bcl-2 protein. The sterols fraction was isolated by solid phase extraction (SPE). Mass spectrometry (LC-MS/MS) was employed to analyze the fraction containing a high concentration of sterols.). Cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay method. Binding energy and the stability of sterols trajectories on pro apoptotic Bcl-2 protein active site was carried on by in silico simulation. Six sterols were identified. The sterol-rich fraction showed a cytotoxicity (IC 50-24h) = 157.79 micrograms per milliliter (µg/ml) and IC 50-48h =120.23 µg/ml for the MCF-7 line. The sterol-rich fraction generated an apoptotic response in the MCF-7 cell line; while the control cell line (normal human epithelial cell, HMEC) exhibited a lower response. The sterols from C. bicrenata rhizome exhibited properties as an alternative pharmacological treatment of human breast cancer. Cyathea bicrenata cytotoxicity evaluation MCF-7/HMEC cell line docking simulation Bcl-2 protein Figures Figure 1 Figure 2 Background Globally, breast cancer in women remains a leading cause of disease and mortality. According to the 2020 global cancer statistics, there were approximately 2.26 million new cases (Ferlay et al., 2021 ). However, earlier diagnosis and advancements in treatment have the potential to reduce breast cancer-related deaths. The gap between developed and underdeveloped countries is a significant problem. Women from low and middle-income countries show high mortality rates while those in high-income countries show low breast cancer mortality rates (Francies et al., 2020 ). Breast cancer has been correlated with numerous endogenous and exogenous risk factors, such as age, family history, race/ethnicity, breast tissue density, pregnancy and breastfeeding, aberrant DNA mutations, prolonged estrogen exposure, and endometrial or ovarian cancer (Admoun and Mayrovitz, 2022). Significant advances have been made in tools for detecting early stages of breast cancer, as mammography, digital breast tomosynthesis, magnetic resonance imaging, and biopsies (UN-WHO, 2023; Mousumi et al., 2024 ). Despite prevention programs and improved access to therapeutic programs (pharmacological, surgical and irradiation), breast cancer remains the most common cause of death in women, with an increasing incidence in men. Therefore, recent investigations have centered on developing new treatments that are more personalized based on the cancer stage. These include endocrine therapy, chemotherapy, or small-molecule inhibitor therapy combined with chemotherapy (Kaur et al., 2022 ). However, not all patients can benefit from personalized treatment. To deal with this problem, research providers must go beyond conventional treatments and employ other approaches. Chemotherapeutic drugs play a pivotal role in cancer treatment because they have toxic effects on cancer cells but adverse reactions to healthy cells, resulting in undesirable side effects (UN-WHO, 2023). Therefore, plants' secondary metabolites have been of great interest in recent years due to their cytotoxic activity against cancer cells and low toxicity to non-cancerous cells (Kaur et al., 2022 ). Plant sterols are a group of metabolites with effects on cell homeostasis. Plant cells synthesize a complex array of sterol mixtures, the most abundant are β -sitosterol, campesterol, and stigmasterol (Du et al., 2022 ). in vitro and in vivo studies have shown that sterols possess anticancer potential by inhibiting cancer cell viability and proliferation, regulating cell cycle, and promoting cell apoptosis (Evtyugin et al., 2023 ; Wang et al., 2023). Furthermore, they have little influence on the proliferation of normal cells. For this reason, sterols have been shown to possess interesting properties and are considered potential drug targets for various types of cancer. Apoptosis is essential for holding a fine and delicate balance in healthy cells. However, alteration of apoptosis may be a responsible factor not only for cancer development and progression but also for cancer resistance to treatments (Qian et al., 2022 ). BCL-2 protein family includes pro-apoptotic and anti-apoptotic proteins that influence the intracellular signaling chains related to the apoptosis pharmacoloical therapy (Singh et al., 2019 ). The anti-apoptotic protein Bcl-2 plays a key role in cancer cell survival by inhibiting apoptosis (Singh et al., 2019 ; Czabotar and García-Saez, 2023). Therefore, its inactivation by small molecules, triggers the apoptotic mechanism of cancer cells (Zhou et al., 2023 ). In human cells, six BCL-2 proteins (Bcl-xL, Bcl-2, MCL1, A1, and Bcl-B) have been reported with anti-apoptotic effects (Oltersdorf et al., 2005 ). The human Bcl-2 protein shares four BH domains and adopts similar globular structures; the helical bundle surrounding a central hydrophobic core helix (helix α5) forming a critical interface/groove for interactions between the pro-apoptotic/anti-apoptotic ligand and the protein receptor (Oltersdorf et al., 2005 ). Its have been reported the effect of some molecules against human cancer cell (inhibition the Bcl-2 anti apoptotic behavior). ABT-737, ABT263—navitoclax, ABT-199- venetoclax, S55746, APG-2575. Those molecules bind strongly to BH3 domain in the Bcl-2 protein and inactivate it (Zhou et al., 2023 ). Several species of the Cyathea genus are traditionally used for therapeutic purposes in many countries, included the breast cancer; although the biological effects of its secondary metabolites have not been studied. Chaparro-Hernández et al. ( 2022 ) reported a review of several bioactive compounds for the Cyathea genus like triterpenes, sterols, saponins, and flavonoids. This study aimed to assess the biological effect of sterols present in the rhizome of Cyathea bicrenata on breast cancer cells (MCF-7).Additionally, molecular docking approaches were used to evaluate the binding energy and stability of sterol trajectories on the active site of Bcl-2 protein. Methods Reagents and equipment All the reagents for isolate the sterol fractions, for cellular evaluation and mass spectrometry were acquired from Sigma Aldrich México and JT Baker México. The C8/SAX SPE cartridges were acquired from Finetech Ltd. The MCF-7 (human breast carcinoma) and HMEC (human mammary epithelial) cell lines were sourced from the Faculty of Medicine at the Universidad Autónoma Benito Juárez de Oaxaca, located in Oaxaca, Mexico." Plant material Cyathea bicrenata was gathered in Oaxaca, Mexico (17.523913, -96.509311; 25670 above sea level). The species was identified and documented at the herbarium of the Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional, Unidad Oaxaca—Instituto Politécnico Nacional (OAX-FLO-129-0402). Plant extract procedure The rhizome of Cyathea bicrenata was cleaned with 1% (volume-volume; v/v) of sodium hypochlorite solution, then thoroughly rinsed with distilled water. The rhizome of Cyathea bicrenata was dried to a constant weight (relative humidity < 10%). Afterward, it is ground and stored at 25 Celsius degree (°C). Five grams of dry rhizome were macerated in 25 milliliters (ml) of hexane for 24 hours (h) at 25°C and sonicated for 30 minutes (min). The resulting mixture was centrifuged at 3000 revolutions per minute (rpm) for 2 min. The supernatant was collected and filtered through Whatman #4 paper to eliminate residual particulate matter. The fern extract was concentrated using a nitrogen stream and subsequently stored at 25°C in darkness. Purification of sterols The purification of the fern extract was carried out according to the modified methodology of Jenner and Brown (Jenner and Brown, 2017 ). The C8/SAX SPE cartridge was pre-conditioned by eluting 2 ml methanol, followed by 2 ml of formic acid solution (40 millimolar, mM—pH 4.5). The fern extract was re-suspended in 2 ml hexane and eluted through the SPE cartridge. Finally, cartridge was washed with 2 ml of a 40% (v/v) methanol—formic acid solution (40 mM—pH 4.5). The SPE cartridge was dried in nitrogen flow for 1–2 min. The pre-conditioning and washing eluates were discarded. The sterol fraction was eluted by passing 2 ml hexane through the loaded-dried cartridge. The solution, called the sterol fraction, was concentrated using a nitrogen stream and subsequently stored at 25°C in darkness. Characterization of compounds by mass spectrometry The chromatography was carried out according to the modified methodology of Korne et al. ( 2006 ). The sterol fraction was re-suspended in 1 ml of hexane and eluted by polyvinylidene difluoride (PDVF) filter (0.25 µm) before being introduced to the chromatograph. The liquid chromatographic was performed using a gradient method with two mobile phases: phase A, consisting of a water:methanol (85:15, v/v) mixture, and phase B, composed of methanol:acetone:hexane (2:2:1, v/v/v), at a flow rate of 0.4 milliliter per minute mL/min. The chromatographic gradient methodology was as follows: 0–0.1 min, 100% A; 0.1–0.3 min, 65% A; 0.3–7.3 min, 65% A; 7.3–7.4 min, 45% A; 7.1–17.4 min, 45% A; 17.4–17.5 min, 0% A; 17.5–32.5 min, 0% A. Separation was performed using a C18 column (50mm x 4.6mm x 2.6µm). Negative mode electro spray ionization was employed, with a mass range of 50 to 3000 mass/charge relation (m/z). Mass spectrometry was conducted under the following conditions:sheath gas temperature was set at 250°C with a flow rate of 11 liter per minute (l/min), nitrogen gas temperature at 300°C with a flow rate of 5 l/min, and a nebulizer gas pressure was maintained at 310.2 kilo Pascal (kPa).The capillary voltage was adjusted to 3.5 kilo volt (kV), while the nozzle voltage was set to 500 volt (V).. A 50 micro liter (µL) aliquot of sterol fraction was introduced into mass spectrometer. Compound identification was achievedby comparing the mass fragmentation spectra pattern and the mass of the molecular ion with the spectral libraries of the NIST20 software (NIST 2020). Diagnostic ions were used to confirm compound identification. Cell culture conditions For the growth of the MCF-7 tumor line, Roswell Park culture medium was used, to which the following was added: glutamine (2 mM), sodium pyruvate (1 mM), no essential amino acids (0.1 mM), sodium bicarbonate (1.5 gram per liter, g/l), 10% (v/v) fetal calf serum, 50 micro gram per milliliter (µg/ml) of gentamycin and 50 µg/ml amphotericin-B. Human mammary epithelial cells (HMEC) were cultured in serum-free mammary epithelium growth medium (MEGM) containing sodium pyruvate (0.11grm per liter, g/l), L-glutamine (1.461 g/l), dextrose (1.0 g/l), 10% (v/v) fetal calf serum, 50 µg/ml of gentamycin and 50 µg/m amphotericin-B. Both celular lines were cultured at 37°C in a humidified 5% CO 2 atmosphere, and conserved in a logarithmic growth phase. Cell growth (by Neubauer chamber), division, and morphology (inverse microscopy) were monitored periodically. in vitro cytotoxic activity assay The citotoxic assay were carried out acording the modified method of Razak et al. ( 2019 ). MCF-7 and HMEC cells, at a concentration of 5x10 6 cells per milliliter (cells/ml), were cultivated in 0.2 ml of HepG2 growth medium in a humidified atmosphere at 37°C for 24 h. Cells were exposed to escalating concentrations of the sterol fraction ranging from 0 to 200 µg/mL, and viability was assessed at 24 and 48 hours by measuring absorbance at 570 nm. A 0.75% dimethyl sulfoxide (DMSO) solution served as the negative control. in silico simulation. Docking evaluation To assess interactions with the sterol profile, the crystallographic structure of Bcl-2 (PDB ID: 4IEH) was retrieved from the Protein Data Bank repository maintained by the Research Collaboratory for Structural Bioinformatics (Protein Data Bank, 2023 ) sterols and C 42 H 44 ClN 7 O 6 S 3 (Blc-2 inhibitor) molecule structures were acquire from Chemical Information database (Pubchem, 2023) The Avogadro ver 1.2.0 software (Avogadro, 2022) was used to pretreat the evaluated ligands (adjust physical structure). USCF Chimera ver. 1.17.3 software (Sanner et al, 2011) was used to pretreat the Bcl-2 protein. AutoDockTools ver. 1.5.7 software (NBCR, 2011) was employed to do in silico evaluation. Docking outcomes from the in silico analysis were reported based on the lowest (most negative) scoring function values, representing Gibbs free energy. The molecular dynamics trajectory of different ligands was evaluated using program HEX software (HEX, 2023). The docked protein target and ligand complexes underwent a molecular dynamic simulation (RMSF). Each docked structure was subjected to 50000 minimization steps, 600 picosecond (ps) of heating (using the Langevin thermostat), and 1.75 nanosecond (ns) of density equilibration (constant pressure at 101 kPa). The water model used was TIP3P, and the protein force fields were constructed using antechamber and parmchk. in silico simulation. Pharmacokinetics properties simulation SwissADME software (SwissADME, 2017) was used to evaluate the drug interactions profile of the sterol fraction. Statistical analysis and experimental design All in vitro experiments involving the sterol fraction were carried out in triplicate to validate consistency of the results. The significance of concentration and time of evaluation on cytotoxicity were evaluated by ANOVA ( p = 0.05 ). Statistica software 7.0 (Statsoft, 2004) was used to treat data. The concentration of the mean lethal dose (IC 50 ) was calculated using a linear regression model. The molecular docking (calculation of free Gibbs energy, ΔG°; and coupling region on the protein) was performed in triplicate, and mean energy and its standard deviation are reported. For compound identification by mass spectrometry, the process was performed in duplicate. Results Sterol profile The sterol profile of the Cyathea bicrenata rhizome was composed of six molecules (Table 1 ). Some identified sterols, such as ergosterol, β -sitosterol, campesterol, lupeol, and daucosterol, have already been reported in Cyathea nilgirensis, Cyathea gigantea, Cyathea crinita, Cyathea latebrosa , and Cyathea spinulosa (Talukdar et al., 2010 ; Janakiraman et al., 2023 ). As far as the authors are aware, brassicasterol, fucosterol, stigmasterol, and ergosterol have not been previously identified in any Cyathea species. Cytotoxicity assay The sterol fraction significantly inhibited the proliferation of both MCF-7 and HMEC cells (p < 0.05). For the MCF-7 cells, the linear model (R 2 adj = 0.86 p < 0.05) indicated an IC 50 of 157.79 ± 8.33 µg/ml at 24 h and 120.23 ± 6.37 µg/ml at 48 h. For the HMEC cells, the linear model (R 2 adj = 0.90 p < 0.05) indicated an IC 50 of 3239.84 ± 8.40 µg/ml at 24 h and 706.06 ± 10.93 µg/ml at 48 h. Docking evaluation The native ligand for Blc-2 (C 42 H 44 ClN 7 O 6 S 3 /ABT-737) showed the higher binding energy, -8.9 kilocalories per mol, kcal/mol (Table 2 ). Among the sterols tested, brassicasterol showed the strongest binding affinity, the highest with binding energy of -8.6 kcal/mol. Table 2 in sillico interaction parameters of ligands and Bcl-2 protein. Ligand Energy value (kcal/mol) Amino acid residue interactions C 42 H 44 ClN 7 O 6 S 3 (natural ligand) -8.9 ± 0.25 ALA59,PHE63,PHE71, MET74,LEU96,ASN102,GLY104,VAL107,ALA108, TYR161 Brassicasterol -8.5 ± 0.15 PHE63 ,ALA69, GLY104 , TRY 161 Campesterol -8.0 ± 0.15 PHE63 ,TYR67,GLU95, LEU96 , ARG105 Ergosterol -8.5 ± 0.15 PHE63 ,ARG66,TYR67,ASP70, GLY104 ,ARG105 Fucosterol -7.1 ± 0.32 ALA59 , ASP62,GLY104,TYR161 Sitosterol -7.2 ± 0.53 ALA59 ,ASP62, PHE63 ,TYR67, GLY104 , TYR161 Stigmasterol -7.5 ± 0.32 PHE63 ,GLU95,LEU96,TYR97, GLY104 ,ARG105 Amino acids in bold are common to those of the natural ligand at the region of interest of the Bcl-2 protein. Accuracy of the docking analysis was evaluated using the natural ligand standard distance deviation, RMSD; comparing the position of the ligand co-crystallized with Bcl-2 protein to the position predicted in simulation the RMSD the observed value of 3.48 Armstrong (Å) ± 0.15. All sterols interacted with the same active site on the protein that the C 42 H 44 ClN 7 O 6 S 3 (Fig. 1). Only the C 42 H 44 ClN 7 O 6 S 3 exhibit a hydrogen type bond (asparagine, ASN102) on the Bcl-2 protein central hydrophobic groove. The evaluated ligands (sterols) bonded to the Blc-2 protein in the same hydrophobic groove, and almost all of the bonds are of the hydrophobic/electrostatic type. Common residues, PHE63 and GLY104, interacted with almost all the sterols via hydrophobic/electrostatic interactions. The evaluation of each system's root means square fluctuations (RMSF) showed stable behaviour from the beginning of the simulation (Fig. 2). Among evaluated sterols, the integrity of the brassicasterol-target site interaction in the Bcl-2 protein showed the highest structural integrity. Evaluation of pharmacokinetics properties The outcome of ADME analysis, Table 3 , suggested that all evaluated sterols possess interesting pharmacokinetics properties and do not show important toxicity effects. Discussion Several studies have reported that sterols have health-promoting effects on humans, such as anti-cancer activity, anti-inflammatory, antibacterial, and antifungal activity (Salehi et al., 2021 ). Furthermore, long-term studies in animal models and humans have shown no long-term consumption toxic effects of plant sterols (Yuyuan et al., 2022 ). However, the plasma concentration of human sterols is commonly below the minimum therapeutic concentration. This low concentration is due to the low dietary intake of sterols in food, their poor oil solubility, and the unpopularity of nutraceutical formulations (Yuyuan et al., 2022 ). Table 3 ADME – Pharmacokinetic properties and physiological properties. Compound ADME Parameters Drug likeness properties GI-Absorption BBB permeability P-gp CYP1A2 inhibitor CYP2C19 inhibitor CYP2C9 inhibitor CYP2D6 inhibitor CYP3A4 inhibitor Lipinski rule Bio ability score C 42 H 44 ClN 7 O 6 S 3 Low No Yes No No No No Yes No 0.17 Brassicasterol Low No No No No No No No Yes 0.55 Campesterol Low No No No No No No No Yes 0.55 Ergosterol Low No No No No Yes No No Yes 0.55 Fucosterol Low No No No No No No No Yes 0.55 Sitosterol Low No No No No No No No Yes 0.55 Stigmasterol Low No No No No Yes No No Yes 0.55 The evaluation of the cytotoxic effect of secondary metabolites isolated from the Cyathea genus, ethanoic extracts of the Cyathea crinita , Cyathea nilgirensis , and Cyathea gigantea , on MCF-7 cancer cell line was reported. The cytotoxic assay showed a decrease in cell viability and increased growth inhibition in MCF 7 cells. C. crinita showed the maximum percentage cell inhibition, IC 50 400 µg/ml, followed by C. nilgiriensis , IC 50 714.28 µg/ml, and C. gigantea , IC 50 806.45 µg/ml (Janakiraman and Johnson, 2018 ). The evaluation of the aqueous extract of the Cyathea nilgirensis , synthesized using silver nitrate (AgNO 3 ) nanoparticles, showed an inhibition of 56% of Dalton's lymphoma ascites cells at a nanoparticle concentration of 200 µg/ml (Johnson et al., 2018 ). The cyathea extracts show antimicrobial, antioxidant, antimicrobial, antidiabetic, and anti-neurodegenerative activity (Salehi et al., 2021 ). Several studies reported that sterols ( β -sitosterol, stigmasterol, stigmastan-3,5-diene, γ-sitosterol, cholesterol margarate, and 7-dehydrodiosgenin) isolated from plants induced apoptosis and decreased cell viability in MCF-7 cell lines (Ayaz et al., 2019 ; Raju et al. 2021 ; AmeliMojarad et al., 2022 ). Some sterols ( e.g. stigmasterol or campesterol) can reduce the expression of Bcl-2 gene and increase the death of estrogen-induced/related breast cancer cells and other cancer types (Du et al., 2000 ; Awad et al., 2007 ; Raju et al., 2021 ; AmeliMojarad et al., 2022 ). The effect of the sterols has been correlated to the cell pro and anti apoptotic pathways (mitochondrial, death receptor-mediated pathway and up/down-regulation of BCL-2 proteins family). Apoptosis mediated by sterols has been reported by two different pathways: i) sterols activate the BCL-2 initiator proteins (BIM, PUMA, BAD, NOXA, BIK, HRK, BMF, and tBID); at the same time, they inhibit the anti-apoptotic BCL-2-like proteins (Bcl-2, Bcl-xL), allowing the activation of the pro-apoptotic effectors BAX and BAK, which then disrupt the mitochondrial outer membrane release the cytochrome c and promote the activation of caspase-9 on the scaffold protein APAF1 (apoptotic protease-activating factor 1) (Awad et al., 2003 ). ii) the sterols promote the release of specific ligands of the tumor necrosis factor (TNF) family, such as FAS ligand and TN, that bind to their corresponding death receptors (FAS and TNFR1, respectively) on plasma membrane. This interaction activates caspase 8 through FAS-associated death domain protein (FADD) and TNFR-associated death domain protein (TRADD). While TRADD is crucial for apoptosis induction by certain death receptors, such as TNFR, it is not required for others like FAS. Finaly Fadd and TRADD promote the activation of effector caspases, including caspase 3, caspase 7, and caspase 6 (Awad et al., 2003 ; Harlozinska, 2005 ; Riedl and Salvesen, 2007 ; Pluchino et al., 2015 ; Jianjun et al., 2022 ). The anti-apoptotic protein (Bcl-2) targeted in this study has been recognized as a critical component apoptotic signaling pathway of cancer cells. Overexpression of Bcl-2 protein extends the lifespan that are typically short-lived, thus facilitating accumulation of additional oncogenic mutations (Rooswinkel et al., 2014 ). Furthermore, overexpression of Bcl-2, occurrence of oncogene-induced events, and/or loss of micro-RNAs 15a and 16 − 1, have been linked to the progression of an early neoplastic or pre-malignant state (Cimmino et al., 2006 ). Increased expression of Bcl-2 anti apoptotic protein has been reported in both estrogen receptor positive and progesterone receptor positive breast cancer (Qian et al., 2022 ). High expression of Bcl-2 protein is usually observed in breast tumor tissues. If the Bcl-2 proteins can be functionally blocked, the apoptosis of tumor cells can be restored (Qian et al., 2022 ). Different approaches (chemotherapy, targeted therapy, and immunotherapy) are usually used for treat breast cancer. One of the most promising areas of targeted therapy is the evaluation of new molecules that promote the apoptosis process. These molecules can be cataloged as antisense oligonucleotide preparations (Janaghard et al., 2021 ), peptide inhibitors (Reddy and Sankararamakrishnan, 2021 ), and small-molecule inhibitors (Verdine and Walensky, 2007 ). The peptide and small-molecule inhibitor drugs bind specifically to anti-apoptotic proteins (Bcl-2, Bcl-xL, and BAX), inhibiting or inactivating them (Qian et al., 2022 ;). The potential of those molecules in cancer therapy has been demonstrated by the therapeutic effect of the ABT737 and ABT263 (navitoclax) drugs (Roberts et al., 2012 ; Mason et al., 2008 ). The uses of Navitoclax has been focused on treating blood and lung cell cancers. The results are promising, particularly in patients with chronic lymphocytic leukemia (Mason et al., 2008 ). Additionally, recent preclinical studies suggest that combining navitoclax with other anticancer drugs, enhances cancer survival rates (Mason et al., 2008 ; Tan et al., 2011 ). Molecular docking simulations predict the optimal position, orientation, bond energy (binding affinity) and conformation of a small molecule (drug candidate) when bound to a protein. Brassicasterol and ergosterol showed binding energies similar to those of the native ligand (C 42 H 44 ClN 7 O 6 S 3 ), indicating a high affinity between those molecules and the Bcl-2 protein. Gowtham et al. ( 2023 ) reported that the binding energy between the daucosterol and the Bcl-2 protein (PDB id 2W3L) was − 9.0 kcal/mol. Gideon et al. ( 2022 ) reported a binding energy of -9.4 kcal/mol for sitosterol and Bcl-2 protein (PDB id 4lvt). Ayaz et al. ( 2019 ) reported binding energies of -7.6 to -7.7 kcal/mol between tyrosine kinase and β-sitosterol and stigmasterol. Raju et al. ( 2021 ) reported binding energies of -6.2 to -10.0 kcal/mol between several phytosterols (γ-tocopherol, stigmastan-3,5-diene, α-sitosterol, cholesterol margarate, stigmastan-3,5-diene) and the estrogen receptor (ERα), progesterone receptor, and epidermal growth factor receptor correlated with human breast cancer. Marisa et al. ( 2021 ) evaluated the potential of phytosterols ( α -tocopherol, β -sitosterol, campesterol, stigmasterol, fucosterol) as breast cancer drugs by docking approach. They report high binding energies 6.8 to -8.9 kcal/mol, suggests a possible inactivation of the estrogen receptor (ERα). Lower the ∆G°, means a higher stability of the binding between the ligands and the interest protein; but for a more accurate evaluation, it is important to evaluate the solvation/desolvation energy, the specific interactions ligand (drug) –receptor (target protein), and energy contribution protein-ligand due molecular movements (rotational, translational, vibrational). Czabotar et al. ( 2014 ) indicate that physical interactions on anti-apoptotic proteins of the BCL-2 family occur at the amphipathic helix groove, primarily through insertion of four hydrophobic residues along one face into hydrophobic pockets within groove. Additionally, a salt bridge is formed between a conserved Asp residue and a conserved Arg residue in α-1 helix (Sattler et al., 1997 ; Czabotar et al., 2014 ). However, mutational studies suggest that the ligand and the BCL-2 protein bonds occur across the entire amphipathic helix-helix-groove interface (Chen et al., 2013 ). Ayaz et al. ( 2019 ) reported multiple electrostatic bonds, and only one hydrogen bond was reported between the β -sitosterol and stigmasterol and the tyrosine kinase. Marisa et al. ( 2021 ) reported multiple binding between the ER α and α -tocopherol, β -sitosterol, campesterol, stigmasterol, and fucosterol. The main binding interaction was electrostatic, even in the case of the evaluated native ligand (raloxifene). Raju et al. ( 2021 ) reported multiple binding residues (no hydrogen bonding was reported) with the ASP351, methionine 759 (MET759), and ASP800 as the key residues for the evaluated protein. Hydrogen bonds stabilizing the structures ligand-proteins due to their participation in secondary structures- The absence of hydrogen bonding in almost all evaluated ligands may indicate that these bonds are unstable (Bitencourt-Ferreiraet al., 2019 ). However, the information from the simulation indicates a good binding energy (values near the exhibited for the evaluated natural ligands) and that the bonds are located in the same active region as the natural ligands (the amphipathic helix groove region). The pharmacological and pharmacokinetic properties of the sterol molecules were evaluated, and although they exhibit good properties as drug candidates (all evaluated sterols did not violate any of the Lipinski rules), their low predicted gastrointestinal absorption limits their potential as druggable molecules. Assessment of in silico ADME is a credible technique to approve the potential of a drug candidate. Drugs/bioactive compounds may exhibit inhibiting effects on various cytochromes (specially the cytochrome P450 (CYP) 3A4, and its genetic variants, is one of the major factors to consider in the interindividual drug metabolism differences (Yamaguchi et al., 2021 ). The most significant predicted effect of the evaluated sterols was the inhibition of cytochrome CYP2C9. The in silico evaluation of the ADME properties of the lupeol, the stigmasterol, and the swertiamarin isolated from rhizomes and leaves of Cyathea nilgiriensis, Cyathea gigantea, and Cyathea crinita (Janakiraman et al., 2023 ) didn´t show significant differences compared to those reported for the sterol fraction isolated from the rhizome of Cyathea bicrenata Janakiraman et al. ( 2023 ) evaluated the in silico binding energy of some sterols from Cyathea nilgiriensis, Cyathea gigantea , and Cyathea crinita against proteins correlated with P450 cytochrome oxidation and proteins associated with cell death pathways and concluded that the evaluated molecules can be considered selective and nontoxic compounds with anticancer properties. Finally, some authors suggest that the anti-carcinogenic effect of the sterols on the cancer cell survival/proliferation is correlated to other mechanisms such as: i) Reduction of estrogen synthesis, reduction of the LDL-cholesterol synthesis (Janakiraman et al., 2023 ). ii) The anti-inflammatory activity, modulation of the TNF-α, IL-6, NF-kB (Valerio and Awad, 2001; Rideout et al., 2015 ). iii) Effect on the cell 0membranes, membrane structure and fluidity (Teixeira et al., 2021 ). iv) Stimulation the immune system function (Bouic et al., 1996 ; Awad et al., 2000 ). Conclusion The sterol fraction isolated from Cyathea bicrenata rhizome demonstrates promising cytotoxic activity against human breast cancer cells while exhibiting low toxicity to normal epithelial cells. These findings suggest that the sterols have potential as alternative therapeutic agents in breast cancer treatment, possibly through interaction with anti-apoptotic proteins like Bcl-2. The overall results of the current research evidence that the Cyathea bicrenata can be further explored for its pharmacology activities for the discovery of plant-based anti-breast cancer drug candidates. Nonetheless, the following observations should be considered: i) It is necessary to carry out experiments where the influence of other analytes of similar polarity to those obtained in the evaluated phase (sterol fraction), synergies between these unidentified analytes and the identified sterols; as well as the exclusive effect of the identified sterols (and the possible effects of the concentration of each of them). ii) Stability of the ligands with the site of interest (RMSD dynamics) must be simulated with other solvents that emulate the biological conditions closest to reality. iii) Effect of the cell membrane on the transport of the evaluated ligands must be evaluate. iv) Additional studies are still required to elucidate how the identified sterols inactivate the Bcl-2. Abbreviations g Gram µg microgram (1x10 -6 gram) h Hour min Minute l Liter µl microliter (1x10 -6 liter) mm millimeter (1x10 -3 meter) µm micrometer(1x10 -6 meter) Å Armstrong V Volt kV kilovolt (1x10 3 volt) kPa kilopascal (1x10 3 Pascal) kcal kilocalorie(1x10 3 calorie) µmol micromole(1x10 -6 mol) ΔG° free Gibbs energy MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte trazolium bromide CO 2 carbon dioxide ASP aspartame MET methionine TYR tyrosine ALA alanine GLY glycine PHE phenylalanine GLU glutamine VAL valine ARG arginine ASN asparagine MCF-7 Human cancer cell line - acronym of Michigan Cancer Foundation-7 HMEC human dermal micro-vascular endothelial cell line NF-kB nuclear enhancer actor of kappa light chains of activated B cells TNF-α tumor necrosis factor alpha IL-6 interleukin − 6 ERα estrogen receptor alpha CYP cytochrome LDL-cholesterol Low-density lipoprotein cholesterol IC 50 Half maximal inhibitory concentration RMSF root mean square deviations SPE Solid phase extraction UV-Vis ultraviolet – visible radiation spectrum ADMET absorption, distribution, metabolism, and excretion of a drug molecule PDB Protein Data Bank Declarations Ethics approval and consent to participate. Not applicable Consent for publication. The authors declare that they agree to the publication of the present work. Availability of data and material . The corresponding author can provide simulation files and information on in vitro evaluation upon request. Competing interests. The authors have no relevant financial or non-financial interests to disclose. Funding. This research received no external funding for the in vitro assay and/or in silico evaluation. Thanks to the Consejo Duranguense de Ciencia y Tecnología (COCyTED) and the Comisión de Operación y Fomento de Actividades Académicas (COFAA) del Instituto Politécnico Nacional for the grants to cover the publication fees. Authors' contributions. Mass spectrometry experimental B.B., Ch. H. M.L.; methodology and writing, B.B., M.L.; in silico simulation B.B., V.F.; in vitro experimental Ch. H. All authors have read and approved the published version of the manuscript. Acknowledgements. 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According to the 2020 global cancer statistics, there were approximately 2.26\u0026nbsp;million new cases (Ferlay et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHowever, earlier diagnosis and advancements in treatment have the potential to reduce breast cancer-related deaths. The gap between developed and underdeveloped countries is a significant problem. Women from low and middle-income countries show high mortality rates while those in high-income countries show low breast cancer mortality rates (Francies et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBreast cancer has been correlated with numerous endogenous and exogenous risk factors, such as age, family history, race/ethnicity, breast tissue density, pregnancy and breastfeeding, aberrant DNA mutations, prolonged estrogen exposure, and endometrial or ovarian cancer (Admoun and Mayrovitz, 2022).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSignificant advances have been made in tools for detecting early stages of breast cancer, as mammography, digital breast tomosynthesis, magnetic resonance imaging, and biopsies (UN-WHO, 2023; Mousumi et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Despite prevention programs and improved access to therapeutic programs (pharmacological, surgical and irradiation), breast cancer remains the most common cause of death in women, with an increasing incidence in men. Therefore, recent investigations have centered on developing new treatments that are more personalized based on the cancer stage. These include endocrine therapy, chemotherapy, or small-molecule inhibitor therapy combined with chemotherapy (Kaur et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, not all patients can benefit from personalized treatment. To deal with this problem, research providers must go beyond conventional treatments and employ other approaches.\u003c/p\u003e\u003cp\u003eChemotherapeutic drugs play a pivotal role in cancer treatment because they have toxic effects on cancer cells but adverse reactions to healthy cells, resulting in undesirable side effects (UN-WHO, 2023). Therefore, plants' secondary metabolites have been of great interest in recent years due to their cytotoxic activity against cancer cells and low toxicity to non-cancerous cells (Kaur et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePlant sterols are a group of metabolites with effects on cell homeostasis. Plant cells synthesize a complex array of sterol mixtures, the most abundant are \u003cem\u003eβ\u003c/em\u003e-sitosterol, campesterol, and stigmasterol (Du et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies have shown that sterols possess anticancer potential by inhibiting cancer cell viability and proliferation, regulating cell cycle, and promoting cell apoptosis (Evtyugin et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., 2023). Furthermore, they have little influence on the proliferation of normal cells. For this reason, sterols have been shown to possess interesting properties and are considered potential drug targets for various types of cancer.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eApoptosis is essential for holding a fine and delicate balance in healthy cells. However, alteration of apoptosis may be a responsible factor not only for cancer development and progression but also for cancer resistance to treatments (Qian et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). BCL-2 protein family includes pro-apoptotic and anti-apoptotic proteins that influence the intracellular signaling chains related to the apoptosis pharmacoloical therapy (Singh et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The anti-apoptotic protein Bcl-2 plays a key role in cancer cell survival by inhibiting apoptosis (Singh et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Czabotar and Garc\u0026iacute;a-Saez, 2023). Therefore, its inactivation by small molecules, triggers the apoptotic mechanism of cancer cells (Zhou et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn human cells, six BCL-2 proteins (Bcl-xL, Bcl-2, MCL1, A1, and Bcl-B) have been reported with anti-apoptotic effects (Oltersdorf et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The human Bcl-2 protein shares four BH domains and adopts similar globular structures; the helical bundle surrounding a central hydrophobic core helix (helix α5) forming a critical interface/groove for interactions between the pro-apoptotic/anti-apoptotic ligand and the protein receptor (Oltersdorf et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIts have been reported the effect of some molecules against human cancer cell (inhibition the Bcl-2 anti apoptotic behavior). ABT-737, ABT263\u0026mdash;navitoclax, ABT-199- venetoclax, S55746, APG-2575. Those molecules bind strongly to BH3 domain in the Bcl-2 protein and inactivate it (Zhou et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSeveral species of the \u003cem\u003eCyathea\u003c/em\u003e genus are traditionally used for therapeutic purposes in many countries, included the breast cancer; although the biological effects of its secondary metabolites have not been studied. Chaparro-Hern\u0026aacute;ndez et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported a review of several bioactive compounds for the \u003cem\u003eCyathea\u003c/em\u003e genus like triterpenes, sterols, saponins, and flavonoids.\u003c/p\u003e\u003cp\u003eThis study aimed to assess the biological effect of sterols present in the rhizome of \u003cem\u003eCyathea bicrenata\u003c/em\u003e on breast cancer cells (MCF-7).Additionally, molecular docking approaches were used to evaluate the binding energy and stability of sterol trajectories on the active site of Bcl-2 protein.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eReagents and equipment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll the reagents for isolate the sterol fractions, for cellular evaluation and mass spectrometry were acquired from Sigma Aldrich M\u0026eacute;xico and JT Baker M\u0026eacute;xico.\u003c/p\u003e\u003cp\u003eThe C8/SAX SPE cartridges were acquired from Finetech Ltd.\u003c/p\u003e\u003cp\u003eThe MCF-7 (human breast carcinoma) and HMEC (human mammary epithelial) cell lines were sourced from the Faculty of Medicine at the Universidad Aut\u0026oacute;noma Benito Ju\u0026aacute;rez de Oaxaca, located in Oaxaca, Mexico.\"\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlant material\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCyathea bicrenata\u003c/em\u003e was gathered in Oaxaca, Mexico (17.523913, -96.509311; 25670 above sea level). The species was identified and documented at the herbarium of the Centro Interdisciplinario de Investigaci\u0026oacute;n para el Desarrollo Integral Regional, Unidad Oaxaca\u0026mdash;Instituto Polit\u0026eacute;cnico Nacional (OAX-FLO-129-0402).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlant extract procedure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe rhizome of \u003cem\u003eCyathea bicrenata\u003c/em\u003e was cleaned with 1% (volume-volume; v/v) of sodium hypochlorite solution, then thoroughly rinsed with distilled water.\u003c/p\u003e\u003cp\u003eThe rhizome of \u003cem\u003eCyathea bicrenata\u003c/em\u003e was dried to a constant weight (relative humidity\u0026thinsp;\u0026lt;\u0026thinsp;10%). Afterward, it is ground and stored at 25 Celsius degree (\u0026deg;C).\u003c/p\u003e\u003cp\u003eFive grams of dry rhizome were macerated in 25 milliliters (ml) of hexane for 24 hours (h) at 25\u0026deg;C and sonicated for 30 minutes (min). The resulting mixture was centrifuged at 3000 revolutions per minute (rpm) for 2 min. The supernatant was collected and filtered through Whatman #4 paper to eliminate residual particulate matter.\u003c/p\u003e\u003cp\u003eThe fern extract was concentrated using a nitrogen stream and subsequently stored at 25\u0026deg;C in darkness.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePurification of sterols\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe purification of the fern extract was carried out according to the modified methodology of Jenner and Brown (Jenner and Brown, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The C8/SAX SPE cartridge was pre-conditioned by eluting 2 ml methanol, followed by 2 ml of formic acid solution (40 millimolar, mM\u0026mdash;pH 4.5).\u003c/p\u003e\u003cp\u003eThe fern extract was re-suspended in 2 ml hexane and eluted through the SPE cartridge. Finally, cartridge was washed with 2 ml of a 40% (v/v) methanol\u0026mdash;formic acid solution (40 mM\u0026mdash;pH 4.5). The SPE cartridge was dried in nitrogen flow for 1\u0026ndash;2 min. The pre-conditioning and washing eluates were discarded.\u003c/p\u003e\u003cp\u003eThe sterol fraction was eluted by passing 2 ml hexane through the loaded-dried cartridge. The solution, called the sterol fraction, was concentrated using a nitrogen stream and subsequently stored at 25\u0026deg;C in darkness.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eCharacterization of compounds by mass spectrometry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chromatography was carried out according to the modified methodology of Korne et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The sterol fraction was re-suspended in 1 ml of hexane and eluted by polyvinylidene difluoride (PDVF) filter (0.25 \u0026micro;m) before being introduced to the chromatograph. The liquid chromatographic was performed using a gradient method with two mobile phases: phase A, consisting of a water:methanol (85:15, v/v) mixture, and phase B, composed of methanol:acetone:hexane (2:2:1, v/v/v), at a flow rate of 0.4 milliliter per minute mL/min.\u003c/p\u003e\u003cp\u003eThe chromatographic gradient methodology was as follows: 0\u0026ndash;0.1 min, 100% A; 0.1\u0026ndash;0.3 min, 65% A; 0.3\u0026ndash;7.3 min, 65% A; 7.3\u0026ndash;7.4 min, 45% A; 7.1\u0026ndash;17.4 min, 45% A; 17.4\u0026ndash;17.5 min, 0% A; 17.5\u0026ndash;32.5 min, 0% A. Separation was performed using a C18 column (50mm x 4.6mm x 2.6\u0026micro;m).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNegative mode electro spray ionization was employed, with a mass range of 50 to 3000 mass/charge relation (m/z). Mass spectrometry was conducted under the following conditions:sheath gas temperature was set at 250\u0026deg;C with a flow rate of 11 liter per minute (l/min), nitrogen gas temperature at 300\u0026deg;C with a flow rate of 5 l/min, and a nebulizer gas pressure was maintained at 310.2 kilo Pascal (kPa).The capillary voltage was adjusted to 3.5 kilo volt (kV), while the nozzle voltage was set to 500 volt (V).. A 50 micro liter (\u0026micro;L) aliquot of sterol fraction was introduced into mass spectrometer. Compound identification was achievedby comparing the mass fragmentation spectra pattern and the mass of the molecular ion with the spectral libraries of the NIST20 software (NIST 2020). Diagnostic ions were used to confirm compound identification.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eCell culture conditions\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor the growth of the MCF-7 tumor line, Roswell Park culture medium was used, to which the following was added: glutamine (2 mM), sodium pyruvate (1 mM), no essential amino acids (0.1 mM), sodium bicarbonate (1.5 gram per liter, g/l), 10% (v/v) fetal calf serum, 50 micro gram per milliliter (\u0026micro;g/ml) of gentamycin and 50 \u0026micro;g/ml amphotericin-B.\u003c/p\u003e\u003cp\u003eHuman mammary epithelial cells (HMEC) were cultured in serum-free mammary epithelium growth medium (MEGM) containing sodium pyruvate (0.11grm per liter, g/l), L-glutamine (1.461 g/l), dextrose (1.0 g/l), 10% (v/v) fetal calf serum, 50 \u0026micro;g/ml of gentamycin and 50 \u0026micro;g/m amphotericin-B.\u003c/p\u003e\u003cp\u003eBoth celular lines were cultured at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, and conserved in a logarithmic growth phase. Cell growth (by Neubauer chamber), division, and morphology (inverse microscopy) were monitored periodically.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003ein vitro cytotoxic activity assay\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe citotoxic assay were carried out acording the modified method of Razak et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMCF-7 and HMEC cells, at a concentration of 5x10\u003csup\u003e6\u003c/sup\u003e cells per milliliter (cells/ml), were cultivated in 0.2 ml of HepG2 growth medium in a humidified atmosphere at 37\u0026deg;C for 24 h.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCells were exposed to escalating concentrations of the sterol fraction ranging from 0 to 200 \u0026micro;g/mL, and viability was assessed at 24 and 48 hours by measuring absorbance at 570 nm. A 0.75% dimethyl sulfoxide (DMSO) solution served as the negative control.\u003c/p\u003e\u003cp\u003e\u003cb\u003ein silico simulation. Docking evaluation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess interactions with the sterol profile, the crystallographic structure of Bcl-2 (PDB ID: 4IEH) was retrieved from the Protein Data Bank repository maintained by the Research Collaboratory for Structural Bioinformatics (Protein Data Bank, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) sterols and C\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eClN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e (Blc-2 inhibitor) molecule structures were acquire from Chemical Information database (Pubchem, 2023)\u003c/p\u003e\u003cp\u003eThe Avogadro ver 1.2.0 software (Avogadro, 2022) was used to pretreat the evaluated ligands (adjust physical structure). USCF Chimera ver. 1.17.3 software (Sanner et al, 2011) was used to pretreat the Bcl-2 protein.\u003c/p\u003e\u003cp\u003eAutoDockTools ver. 1.5.7 software (NBCR, 2011) was employed to do \u003cem\u003ein silico\u003c/em\u003e evaluation.\u003c/p\u003e\u003cp\u003eDocking outcomes from the in silico analysis were reported based on the lowest (most negative) scoring function values, representing Gibbs free energy.\u003c/p\u003e\u003cp\u003eThe molecular dynamics trajectory of different ligands was evaluated using program HEX software (HEX, 2023). The docked protein target and ligand complexes underwent a molecular dynamic simulation (RMSF). Each docked structure was subjected to 50000 minimization steps, 600 picosecond (ps) of heating (using the Langevin thermostat), and 1.75 nanosecond (ns) of density equilibration (constant pressure at 101 kPa). The water model used was TIP3P, and the protein force fields were constructed using antechamber and parmchk.\u003c/p\u003e\u003cp\u003e\u003cb\u003ein silico simulation. Pharmacokinetics properties simulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSwissADME software (SwissADME, 2017) was used to evaluate the drug interactions profile of the sterol fraction.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis and experimental design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll \u003cem\u003ein vitro\u003c/em\u003e experiments involving the sterol fraction were carried out in triplicate to validate consistency of the results. The significance of concentration and time of evaluation on cytotoxicity were evaluated by ANOVA (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.05\u003c/em\u003e). Statistica software 7.0 (Statsoft, 2004) was used to treat data.\u003c/p\u003e\u003cp\u003eThe concentration of the mean lethal dose (IC\u003csub\u003e50\u003c/sub\u003e) was calculated using a linear regression model.\u003c/p\u003e\u003cp\u003eThe molecular docking (calculation of free Gibbs energy, ΔG\u0026deg;; and coupling region on the protein) was performed in triplicate, and mean energy and its standard deviation are reported.\u003c/p\u003e\u003cp\u003eFor compound identification by mass spectrometry, the process was performed in duplicate.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eSterol profile\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe sterol profile of the \u003cem\u003eCyathea bicrenata\u003c/em\u003e rhizome was composed of six molecules (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSome identified sterols, such as ergosterol, \u003cem\u003eβ\u003c/em\u003e-sitosterol, campesterol, lupeol, and daucosterol, have already been reported in \u003cem\u003eCyathea nilgirensis, Cyathea gigantea, Cyathea crinita, Cyathea latebrosa\u003c/em\u003e, and \u003cem\u003eCyathea spinulosa\u003c/em\u003e (Talukdar et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Janakiraman et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As far as the authors are aware, brassicasterol, fucosterol, stigmasterol, and ergosterol have not been previously identified in any Cyathea species.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eCytotoxicity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe sterol fraction significantly inhibited the proliferation of both MCF-7 and HMEC cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eFor the MCF-7 cells, the linear model (R\u003csup\u003e2\u003c/sup\u003e\u003csub\u003eadj\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.86 p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) indicated an IC\u003csub\u003e50\u003c/sub\u003e of 157.79\u0026thinsp;\u0026plusmn;\u0026thinsp;8.33 \u0026micro;g/ml at 24 h and 120.23\u0026thinsp;\u0026plusmn;\u0026thinsp;6.37 \u0026micro;g/ml at 48 h. For the HMEC cells, the linear model (R\u003csup\u003e2\u003c/sup\u003e\u003csub\u003eadj\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.90 p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) indicated an IC\u003csub\u003e50\u003c/sub\u003e of \u003cem\u003e3239.84\u0026thinsp;\u0026plusmn;\u0026thinsp;8.40\u003c/em\u003e \u0026micro;g/ml \u003cem\u003eat 24 h and 706.06\u0026thinsp;\u0026plusmn;\u0026thinsp;10.93\u003c/em\u003e \u0026micro;g/ml \u003cem\u003eat 48 h.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"451\" height=\"494\"\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eDocking evaluation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe native ligand for Blc-2 (C\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eClN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e /ABT-737) showed the higher binding energy, -8.9 kilocalories per mol, kcal/mol (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among the sterols tested, brassicasterol showed the strongest binding affinity, the highest with binding energy of -8.6 kcal/mol.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003ein sillico\u003c/em\u003e interaction parameters of ligands and Bcl-2 protein.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLigand\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEnergy value\u003c/p\u003e\u003cp\u003e(kcal/mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAmino acid residue interactions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eClN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e\u003csub\u003e(natural ligand)\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eALA59,PHE63,PHE71, MET74,LEU96,ASN102,GLY104,VAL107,ALA108, TYR161\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBrassicasterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003ePHE63\u003c/b\u003e,ALA69, \u003cb\u003eGLY104\u003c/b\u003e, TRY 161\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCampesterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003ePHE63\u003c/b\u003e,TYR67,GLU95, \u003cb\u003eLEU96\u003c/b\u003e, ARG105\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eErgosterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003ePHE63\u003c/b\u003e,ARG66,TYR67,ASP70,\u003cb\u003eGLY104\u003c/b\u003e,ARG105\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFucosterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eALA59\u003c/b\u003e, ASP62,GLY104,TYR161\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSitosterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eALA59\u003c/b\u003e,ASP62,\u003cb\u003ePHE63\u003c/b\u003e,TYR67,\u003cb\u003eGLY104\u003c/b\u003e,\u003cb\u003eTYR161\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStigmasterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e-7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003ePHE63\u003c/b\u003e,GLU95,LEU96,TYR97,\u003cb\u003eGLY104\u003c/b\u003e,ARG105\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAmino acids in bold are common to those of the natural ligand at the region of interest of the Bcl-2 protein.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAccuracy of the docking analysis was evaluated using the natural ligand standard distance deviation, RMSD; comparing the position of the ligand co-crystallized with Bcl-2 protein to the position predicted in simulation the RMSD the observed value of 3.48 Armstrong (\u0026Aring;)\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15.\u003c/p\u003e\u003cp\u003eAll sterols interacted with the same active site on the protein that the C\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eClN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e (Fig.\u0026nbsp;1). Only the C\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eClN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e exhibit a hydrogen type bond (asparagine, ASN102) on the Bcl-2 protein central hydrophobic groove.\u003c/p\u003e\u003cp\u003eThe evaluated ligands (sterols) bonded to the Blc-2 protein in the same hydrophobic groove, and almost all of the bonds are of the hydrophobic/electrostatic type. Common residues, PHE63 and GLY104, interacted with almost all the sterols via hydrophobic/electrostatic interactions.\u003c/p\u003e\u003cp\u003eThe evaluation of each system's root means square fluctuations (RMSF) showed stable behaviour from the beginning of the simulation (Fig.\u0026nbsp;2). Among evaluated sterols, the integrity of the brassicasterol-target site interaction in the Bcl-2 protein showed the highest structural integrity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of pharmacokinetics properties\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe outcome of ADME analysis, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, suggested that all evaluated sterols possess interesting pharmacokinetics properties and do not show important toxicity effects.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSeveral studies have reported that sterols have health-promoting effects on humans, such as anti-cancer activity, anti-inflammatory, antibacterial, and antifungal activity (Salehi et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, long-term studies in animal models and humans have shown no long-term consumption toxic effects of plant sterols (Yuyuan et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, the plasma concentration of human sterols is commonly below the minimum therapeutic concentration. This low concentration is due to the low dietary intake of sterols in food, their poor oil solubility, and the unpopularity of nutraceutical formulations (Yuyuan et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eADME \u0026ndash; Pharmacokinetic properties and physiological properties.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u003cp\u003eADME\u003c/p\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003eDrug likeness properties\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGI-Absorption\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBBB permeability\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP-gp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCYP1A2 inhibitor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCYP2C19 inhibitor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCYP2C9 inhibitor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCYP2D6 inhibitor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eCYP3A4 inhibitor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eLipinski rule\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eBio ability score\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eClN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBrassicasterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCampesterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eErgosterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFucosterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSitosterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStigmasterol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe evaluation of the cytotoxic effect of secondary metabolites isolated from the \u003cem\u003eCyathea\u003c/em\u003e genus, ethanoic extracts of the \u003cem\u003eCyathea crinita\u003c/em\u003e, \u003cem\u003eCyathea nilgirensis\u003c/em\u003e, and \u003cem\u003eCyathea gigantea\u003c/em\u003e, on MCF-7 cancer cell line was reported. The cytotoxic assay showed a decrease in cell viability and increased growth inhibition in MCF 7 cells. \u003cem\u003eC. crinita\u003c/em\u003e showed the maximum percentage cell inhibition, IC\u003csub\u003e50\u003c/sub\u003e 400 \u0026micro;g/ml, followed by \u003cem\u003eC. nilgiriensis\u003c/em\u003e, IC\u003csub\u003e50\u003c/sub\u003e 714.28 \u0026micro;g/ml, and \u003cem\u003eC. gigantea\u003c/em\u003e, IC\u003csub\u003e50\u003c/sub\u003e 806.45 \u0026micro;g/ml (Janakiraman and Johnson, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe evaluation of the aqueous extract of the \u003cem\u003eCyathea nilgirensis\u003c/em\u003e, synthesized using silver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e) nanoparticles, showed an inhibition of 56% of Dalton's lymphoma ascites cells at a nanoparticle concentration of 200 \u0026micro;g/ml (Johnson et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe cyathea extracts show antimicrobial, antioxidant, antimicrobial, antidiabetic, and anti-neurodegenerative activity (Salehi et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral studies reported that sterols (\u003cem\u003eβ\u003c/em\u003e-sitosterol, stigmasterol, stigmastan-3,5-diene, γ-sitosterol, cholesterol margarate, and 7-dehydrodiosgenin) isolated from plants induced apoptosis and decreased cell viability in MCF-7 cell lines (Ayaz et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Raju et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; AmeliMojarad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSome sterols (\u003cem\u003ee.g.\u003c/em\u003e stigmasterol or campesterol) can reduce the expression of Bcl-2 gene and increase the death of estrogen-induced/related breast cancer cells and other cancer types (Du et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Awad et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Raju et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; AmeliMojarad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe effect of the sterols has been correlated to the cell pro and anti apoptotic pathways (mitochondrial, death receptor-mediated pathway and up/down-regulation of BCL-2 proteins family).\u003c/p\u003e\u003cp\u003eApoptosis mediated by sterols has been reported by two different pathways: i) sterols activate the BCL-2 initiator proteins (BIM, PUMA, BAD, NOXA, BIK, HRK, BMF, and tBID); at the same time, they inhibit the anti-apoptotic BCL-2-like proteins (Bcl-2, Bcl-xL), allowing the activation of the pro-apoptotic effectors BAX and BAK, which then disrupt the mitochondrial outer membrane release the cytochrome c and promote the activation of caspase-9 on the scaffold protein APAF1 (apoptotic protease-activating factor 1) (Awad et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). ii) the sterols promote the release of specific ligands of the tumor necrosis factor (TNF) family, such as FAS ligand and TN, that bind to their corresponding death receptors (FAS and TNFR1, respectively) on plasma membrane. This interaction activates caspase 8 through FAS-associated death domain protein (FADD) and TNFR-associated death domain protein (TRADD). While TRADD is crucial for apoptosis induction by certain death receptors, such as TNFR, it is not required for others like FAS. Finaly Fadd and TRADD promote the activation of effector caspases, including caspase 3, caspase 7, and caspase 6 (Awad et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Harlozinska, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Riedl and Salvesen, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Pluchino et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Jianjun et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe anti-apoptotic protein (Bcl-2) targeted in this study has been recognized as a critical component apoptotic signaling pathway of cancer cells. Overexpression of Bcl-2 protein extends the lifespan that are typically short-lived, thus facilitating accumulation of additional oncogenic mutations (Rooswinkel et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, overexpression of Bcl-2, occurrence of oncogene-induced events, and/or loss of micro-RNAs 15a and 16\u0026thinsp;\u0026minus;\u0026thinsp;1, have been linked to the progression of an early neoplastic or pre-malignant state (Cimmino et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIncreased expression of Bcl-2 anti apoptotic protein has been reported in both estrogen receptor positive and progesterone receptor positive breast cancer (Qian et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). High expression of Bcl-2 protein is usually observed in breast tumor tissues. If the Bcl-2 proteins can be functionally blocked, the apoptosis of tumor cells can be restored (Qian et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDifferent approaches (chemotherapy, targeted therapy, and immunotherapy) are usually used for treat breast cancer. One of the most promising areas of targeted therapy is the evaluation of new molecules that promote the apoptosis process. These molecules can be cataloged as antisense oligonucleotide preparations (Janaghard et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), peptide inhibitors (Reddy and Sankararamakrishnan, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and small-molecule inhibitors (Verdine and Walensky, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe peptide and small-molecule inhibitor drugs bind specifically to anti-apoptotic proteins (Bcl-2, Bcl-xL, and BAX), inhibiting or inactivating them (Qian et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;). The potential of those molecules in cancer therapy has been demonstrated by the therapeutic effect of the ABT737 and ABT263 (navitoclax) drugs (Roberts et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mason et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The uses of Navitoclax has been focused on treating blood and lung cell cancers. The results are promising, particularly in patients with chronic lymphocytic leukemia (Mason et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Additionally, recent preclinical studies suggest that combining navitoclax with other anticancer drugs, enhances cancer survival rates (Mason et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tan et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMolecular docking simulations predict the optimal position, orientation, bond energy (binding affinity) and conformation of a small molecule (drug candidate) when bound to a protein.\u003c/p\u003e\u003cp\u003eBrassicasterol and ergosterol showed binding energies similar to those of the native ligand (C\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eClN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e), indicating a high affinity between those molecules and the Bcl-2 protein.\u003c/p\u003e\u003cp\u003eGowtham et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that the binding energy between the daucosterol and the Bcl-2 protein (PDB id 2W3L) was \u0026minus;\u0026thinsp;9.0 kcal/mol. Gideon et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported a binding energy of -9.4 kcal/mol for sitosterol and Bcl-2 protein (PDB id 4lvt).\u003c/p\u003e\u003cp\u003eAyaz et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported binding energies of -7.6 to -7.7 kcal/mol between tyrosine kinase and β-sitosterol and stigmasterol.\u003c/p\u003e\u003cp\u003eRaju et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported binding energies of -6.2 to -10.0 kcal/mol between several phytosterols (γ-tocopherol, stigmastan-3,5-diene, α-sitosterol, cholesterol margarate, stigmastan-3,5-diene) and the estrogen receptor (ERα), progesterone receptor, and epidermal growth factor receptor correlated with human breast cancer.\u003c/p\u003e\u003cp\u003eMarisa et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) evaluated the potential of phytosterols (\u003cem\u003eα\u003c/em\u003e-tocopherol, \u003cem\u003eβ\u003c/em\u003e-sitosterol, campesterol, stigmasterol, fucosterol) as breast cancer drugs by docking approach. They report high binding energies 6.8 to -8.9 kcal/mol, suggests a possible inactivation of the estrogen receptor (ERα).\u003c/p\u003e\u003cp\u003eLower the ∆G\u0026deg;, means a higher stability of the binding between the ligands and the interest protein; but for a more accurate evaluation, it is important to evaluate the solvation/desolvation energy, the specific interactions ligand (drug) \u0026ndash;receptor (target protein), and energy contribution protein-ligand due molecular movements (rotational, translational, vibrational).\u003c/p\u003e\u003cp\u003eCzabotar et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) indicate that physical interactions on anti-apoptotic proteins of the BCL-2 family occur at the amphipathic helix groove, primarily through insertion of four hydrophobic residues along one face into hydrophobic pockets within groove. Additionally, a salt bridge is formed between a conserved Asp residue and a conserved Arg residue in α-1 helix (Sattler et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Czabotar et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, mutational studies suggest that the ligand and the BCL-2 protein bonds occur across the entire amphipathic helix-helix-groove interface (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAyaz et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported multiple electrostatic bonds, and only one hydrogen bond was reported between the \u003cem\u003eβ\u003c/em\u003e-sitosterol and stigmasterol and the tyrosine kinase.\u003c/p\u003e\u003cp\u003eMarisa et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported multiple binding between the ER\u003cem\u003eα\u003c/em\u003e and \u003cem\u003eα\u003c/em\u003e-tocopherol, \u003cem\u003eβ\u003c/em\u003e-sitosterol, campesterol, stigmasterol, and fucosterol. The main binding interaction was electrostatic, even in the case of the evaluated native ligand (raloxifene).\u003c/p\u003e\u003cp\u003eRaju et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported multiple binding residues (no hydrogen bonding was reported) with the ASP351, methionine 759 (MET759), and ASP800 as the key residues for the evaluated protein.\u003c/p\u003e\u003cp\u003eHydrogen bonds stabilizing the structures ligand-proteins due to their participation in secondary structures- The absence of hydrogen bonding in almost all evaluated ligands may indicate that these bonds are unstable (Bitencourt-Ferreiraet al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the information from the simulation indicates a good binding energy (values near the exhibited for the evaluated natural ligands) and that the bonds are located in the same active region as the natural ligands (the amphipathic helix groove region).\u003c/p\u003e\u003cp\u003eThe pharmacological and pharmacokinetic properties of the sterol molecules were evaluated, and although they exhibit good properties as drug candidates (all evaluated sterols did not violate any of the Lipinski rules), their low predicted gastrointestinal absorption limits their potential as druggable molecules.\u003c/p\u003e\u003cp\u003eAssessment of in silico ADME is a credible technique to approve the potential of a drug candidate. Drugs/bioactive compounds may exhibit inhibiting effects on various cytochromes (specially the cytochrome P450 (CYP) 3A4, and its genetic variants, is one of the major factors to consider in the interindividual drug metabolism differences (Yamaguchi et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe most significant predicted effect of the evaluated sterols was the inhibition of cytochrome CYP2C9.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein silico\u003c/em\u003e evaluation of the ADME properties of the lupeol, the stigmasterol, and the swertiamarin isolated from rhizomes and leaves of \u003cem\u003eCyathea nilgiriensis, Cyathea gigantea, and Cyathea crinita\u003c/em\u003e (Janakiraman et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) didn\u0026acute;t show significant differences compared to those reported for the sterol fraction isolated from the rhizome of \u003cem\u003eCyathea bicrenata\u003c/em\u003e\u003c/p\u003e\u003cp\u003eJanakiraman et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) evaluated the \u003cem\u003ein silico\u003c/em\u003e binding energy of some sterols from \u003cem\u003eCyathea nilgiriensis, Cyathea gigantea\u003c/em\u003e, and Cyathea \u003cem\u003ecrinita\u003c/em\u003e against proteins correlated with P450 cytochrome oxidation and proteins associated with cell death pathways and concluded that the evaluated molecules can be considered selective and nontoxic compounds with anticancer properties.\u003c/p\u003e\u003cp\u003eFinally, some authors suggest that the anti-carcinogenic effect of the sterols on the cancer cell survival/proliferation is correlated to other mechanisms such as: i) Reduction of estrogen synthesis, reduction of the LDL-cholesterol synthesis (Janakiraman et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). ii) The anti-inflammatory activity, modulation of the TNF-α, IL-6, NF-kB (Valerio and Awad, 2001; Rideout et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). iii) Effect on the cell 0membranes, membrane structure and fluidity (Teixeira et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). iv) Stimulation the immune system function (Bouic et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Awad et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe sterol fraction isolated from \u003cem\u003eCyathea bicrenata\u003c/em\u003e rhizome demonstrates promising cytotoxic activity against human breast cancer cells while exhibiting low toxicity to normal epithelial cells. These findings suggest that the sterols have potential as alternative therapeutic agents in breast cancer treatment, possibly through interaction with anti-apoptotic proteins like Bcl-2. The overall results of the current research evidence that the \u003cem\u003eCyathea bicrenata\u003c/em\u003e can be further explored for its pharmacology activities for the discovery of plant-based anti-breast cancer drug candidates. Nonetheless, the following observations should be considered: i) It is necessary to carry out experiments where the influence of other analytes of similar polarity to those obtained in the evaluated phase (sterol fraction), synergies between these unidentified analytes and the identified sterols; as well as the exclusive effect of the identified sterols (and the possible effects of the concentration of each of them). ii) Stability of the ligands with the site of interest (RMSD dynamics) must be simulated with other solvents that emulate the biological conditions closest to reality. iii) Effect of the cell membrane on the transport of the evaluated ligands must be evaluate. iv) Additional studies are still required to elucidate how the identified sterols inactivate the Bcl-2.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eg\u003c/div\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eGram\u003c/div\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u0026micro;g\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003emicrogram (1x10\u003csup\u003e-6\u003c/sup\u003e gram)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eh\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eHour\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003emin\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eMinute\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003el\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eLiter\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u0026micro;l\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003emicroliter (1x10\u003csup\u003e-6\u003c/sup\u003e liter)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003emm\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003emillimeter (1x10\u003csup\u003e-3\u003c/sup\u003e meter)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u0026micro;m\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003emicrometer(1x10\u003csup\u003e-6\u003c/sup\u003e meter)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u0026Aring;\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eArmstrong\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eV\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eVolt\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003ekV\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003ekilovolt (1x10\u003csup\u003e3\u003c/sup\u003e volt)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003ekPa\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003ekilopascal (1x10\u003csup\u003e3\u003c/sup\u003e Pascal)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003ekcal\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003ekilocalorie(1x10\u003csup\u003e3\u003c/sup\u003e calorie)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u0026micro;mol\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003emicromole(1x10\u003csup\u003e-6\u003c/sup\u003e mol)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eΔG\u0026deg;\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003efree Gibbs energy\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eMTT\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003e3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003etrazolium bromide\u003c/span\u003e\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eCO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003ecarbon dioxide\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eASP\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003easpartame\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eMET\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003emethionine\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eTYR\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003etyrosine\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eALA\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003ealanine\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eGLY\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eglycine\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003ePHE\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003ephenylalanine\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eGLU\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eglutamine\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eVAL\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003evaline\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eARG\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003earginine\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eASN\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003easparagine\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eMCF-7\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eHuman cancer cell line - acronym of Michigan Cancer Foundation-7\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eHMEC\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003ehuman dermal micro-vascular endothelial cell line\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eNF-kB\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003enuclear enhancer actor of kappa light chains of activated B cells\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eTNF-α\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003etumor necrosis factor alpha\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eIL-6\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003einterleukin \u0026minus;\u0026thinsp;6\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eERα\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eestrogen receptor alpha\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eCYP\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003ecytochrome\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eLDL-cholesterol\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eLow-density lipoprotein cholesterol\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eHalf maximal inhibitory concentration\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eRMSF\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eroot mean square deviations\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eSPE\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eSolid phase extraction\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eUV-Vis\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eultraviolet \u0026ndash; visible radiation spectrum\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003eADMET\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eabsorption, distribution, metabolism, and excretion of a drug molecule\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003ePDB\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eProtein Data Bank\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003cbr/\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u003c/strong\u003e Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u003c/strong\u003e The authors declare that they agree to the publication of the present work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e. The corresponding author can provide simulation files and information on \u003cem\u003ein vitro\u003c/em\u003e evaluation upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u003c/strong\u003e \u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u003c/strong\u003e This research received no external funding for the \u003cem\u003ein vitro\u003c/em\u003e assay and/or \u003cem\u003ein silico\u0026nbsp;\u003c/em\u003eevaluation. Thanks to the Consejo Duranguense de Ciencia y Tecnolog\u0026iacute;a (COCyTED) and the Comisi\u0026oacute;n de Operaci\u0026oacute;n y Fomento de Actividades Acad\u0026eacute;micas (COFAA) del Instituto Polit\u0026eacute;cnico Nacional for the grants to cover the publication fees.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions.\u003c/strong\u003e Mass spectrometry experimental B.B., Ch. H. M.L.; methodology and writing, B.B., M.L.; \u003cem\u003ein silico\u003c/em\u003e simulation B.B., V.F.; \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eexperimental Ch. H. All authors have read and approved the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements.\u0026nbsp;\u003c/strong\u003eThe authors would like to give special thanks to Ph. D. Socorro Canseco for using the facilities at her charge for the\u003cem\u003e\u0026nbsp;in vitro\u0026nbsp;\u003c/em\u003eassays.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdmoun C, Mayrovitz H (2022) The Etiology of Breast Cancer. In: Mayrovitz N (ed) Breast Cancer. Exon Publications, Brisbane.\u003c/li\u003e\n\u003cli\u003eAmeliMojarad M, AmeliMojarad M, Pourmahdian A (2022) The inhibitory role of stigmasterol on tumor growth by inducing apoptosis in Balb/c mouse with spontaneous breast tumor (SMMT). BMC Pharmacol Toxicol 23:35-42.\u003c/li\u003e\n\u003cli\u003eAvogadro. Available from: https://avogadro.cc/ Accessed 10 Nov2023.\u003c/li\u003e\n\u003cli\u003eAwad A, Chinnam M, Fink C, Bradford P (2007) Beta-Sitosterol activates Fas signaling in human breast cancer cells. Phytomedicine 14:747-54. \u003c/li\u003e\n\u003cli\u003eAwad A, Downie D, Fink C (2000) Inhibition of growth and stimulation of apoptosis by \u003cem\u003e\u0026beta;\u003c/em\u003e-sitosterol treatment of human breast cancer MDA-MB-231 cells in culture. Int J. Mol Med 5:541-545.\u003c/li\u003e\n\u003cli\u003eAwad A, Roy R, Fink C (2003) Beta-sitosterol, a plant sterol, induces apoptosis and activates key caspases in MDA-MB-231 human breast cancer cells. Oncol Rep 10:497-500.\u003c/li\u003e\n\u003cli\u003eAyaz M, Sadiq A, Wadood A, Junaid M, Ullah F, Zaman N (2019) Cytotoxicity and molecular docking studies on phytosterols isolated from \u003cem\u003ePolygonum hydropiper\u003c/em\u003e L. Steroids 141:30-35.\u003c/li\u003e\n\u003cli\u003eBitencourt-Ferreira G, Veit-Acosta M, de Azevedo W (2019) Methods in Molecular Biology: Hydrogen bonds in protein - ligand complexes. In: de Azevedo W (ed) Docking Screens for Drug Discovery. 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Acta Pharmacol Sin 44:475-485. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bulletin-of-the-national-research-centre","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnrc","sideBox":"Learn more about [Bulletin of the National Research Centre](https://BNRC.springeropen.com)","snPcode":"42269","submissionUrl":"https://submission.springernature.com/new-submission/42269/3","title":"Bulletin of the National Research Centre","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cyathea bicrenata, cytotoxicity evaluation, MCF-7/HMEC cell line, docking simulation, Bcl-2 protein","lastPublishedDoi":"10.21203/rs.3.rs-6813685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6813685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHerbal infusions are considered as a kind of therapeutic approach that aids, and in many cases replaces, conventional pharmacological treatments. However, the identification of its compounds and their activities in human health continue to be the subject of multidisciplinary research. This research aimed to evaluate the cytotoxic effect of the sterols fraction isolated from \u003cem\u003eCyathea bicrenata\u003c/em\u003e Liebm (\u003cem\u003eCyatheae\u003c/em\u003e) rhizome in MCF-7 carcinoma cell line and simulate their blocking effect in the anti-apoptotic Bcl-2 protein. The sterols fraction was isolated by solid phase extraction (SPE). Mass spectrometry (LC-MS/MS) was employed to analyze the fraction containing a high concentration of sterols.). Cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay method. Binding energy and the stability of sterols trajectories on pro apoptotic Bcl-2 protein active site was carried on by \u003cem\u003ein silico\u003c/em\u003e simulation. Six sterols were identified. The sterol-rich fraction showed a cytotoxicity (IC\u003csub\u003e50-24h)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;157.79 micrograms per milliliter (\u0026micro;g/ml) and IC\u003csub\u003e50-48h\u003c/sub\u003e=120.23 \u0026micro;g/ml for the MCF-7 line. The sterol-rich fraction generated an apoptotic response in the MCF-7 cell line; while the control cell line (normal human epithelial cell, HMEC) exhibited a lower response. The sterols from \u003cem\u003eC. bicrenata\u003c/em\u003e rhizome exhibited properties as an alternative pharmacological treatment of human breast cancer.\u003c/p\u003e","manuscriptTitle":"Sterols from Cyathea Bicrenata Liebm Rhizome and their Breast Cancer Activity in vitro and in silico Evaluation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 12:46:03","doi":"10.21203/rs.3.rs-6813685/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T11:51:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-19T22:58:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T20:21:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248826487782821370669712956919836034662","date":"2025-07-09T16:14:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246619085138590110352155328720300175177","date":"2025-07-07T12:46:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-07T12:36:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-07T07:51:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-06T22:50:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bulletin of the National Research Centre","date":"2025-06-30T18:11:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bulletin-of-the-national-research-centre","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnrc","sideBox":"Learn more about [Bulletin of the National Research Centre](https://BNRC.springeropen.com)","snPcode":"42269","submissionUrl":"https://submission.springernature.com/new-submission/42269/3","title":"Bulletin of the National Research Centre","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"26f35e94-81a2-4b49-9684-dd6d3e0d99bf","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T16:05:26+00:00","versionOfRecord":{"articleIdentity":"rs-6813685","link":"https://doi.org/10.1186/s42269-025-01353-z","journal":{"identity":"bulletin-of-the-national-research-centre","isVorOnly":false,"title":"Bulletin of the National Research Centre"},"publishedOn":"2025-09-02 15:57:11","publishedOnDateReadable":"September 2nd, 2025"},"versionCreatedAt":"2025-07-10 12:46:03","video":"","vorDoi":"10.1186/s42269-025-01353-z","vorDoiUrl":"https://doi.org/10.1186/s42269-025-01353-z","workflowStages":[]},"version":"v1","identity":"rs-6813685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6813685","identity":"rs-6813685","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}