Metabolomic and computational studies for antiproliferative potential of Corchorus olitorius methanol root extract and its nanocrystals

Metabolomic and computational studies for antiproliferative potential of Corchorus olitorius methanol root extract and its nanocrystals | 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 Metabolomic and computational studies for antiproliferative potential of Corchorus olitorius methanol root extract and its nanocrystals Marwa Ali, Marwa A. M. Abdel-Razek, Miada F. Abdelwahab, Soad A. Mohamad, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6234677/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted 5 You are reading this latest preprint version Abstract Corchorus olitorius L. Moench (Molokheia) is a common edible plant that is rich in terpenoids and flavonoids. Later, for the first time, this article was planned to study the potential of C. olitorius roots and their nanocrystals against breast cancer (MCF-7), hepatocellular carcinoma (HepG2) and colon cancer (Caco-2) cell lines. Generally, the total methanolic extract of C. olitorius roots (TMECOR) inhibited growth of MCF-7, HepG2 and Caco-2 cells with IC 50 values of 42.68 ± 1.96, 37.14 ± 1.6 and 18.63 ± 1.16 µg/mL, respectively. Whereas, the nanocrystals displayed significantly higher antiproliferative potential especially against HepG-2 and Caco-2 with IC 50 value of 23.288 ± 1.08 and 12.156 ± 0.61 µg/mL, respectively, While MCF-7 showed IC 50 of 62.497 ± 3.63 µg/ mL. To discover which of these compounds is responsible for this activity, metabolomic analysis of TMECOR was studied. It revealed presence of a diversity of metabolites ( 1–15 ) largely dominated by phenolic compounds. In silico network analysis and molecular docking to explore the anticancer efficacy of Corchorus olitorius extract against MCF-7, HepG2, and Caco-2 cancer cell lines. Central hub genes implicated in key oncogenic pathways, such as EGFR and BRAF, were pinpointed and subjected to rigorous docking protocols, using the crystal structures of EGFR (PDB ID: 1M17) and BRAF V600E (PDB ID: 5JRQ). The docking outcomes highlight significant binding affinities for compounds within the extract, notably Chlorogenic acid and Rutin, implying their potential as dual inhibitors for these critical cancer pathways. These findings offer a foundational understanding for subsequent empirical studies and the potential crafting of novel cancer therapies. Corchorus olitorius Cytotoxicity HPLC-HESI-HRMS analysis Molecular Docking Nanocrystals Tiliaceae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Carcer is brought on by abnormal processing of genetic information as a result of genetic alterations (mutations) affecting tumor suppressor genes and oncogenes, or altered epigenetic pathways resulting in chromatin structural abnormalities that are either localized or global (Sarris et al., 2014 ). Changes in the epigenetic landscapes of cancer cells are largely caused by dysregulated expression of different histone methyltransferases. The pathophysiology of hepatocellular carcinoma (HCC), melanoma, breast, esophageal squamous cell carcinoma, and colorectal cancer has been linked to altered expression or activity of G9a, Setdb1, Smyd2, or PR-SET7 methyltransferases. Similarly, the enhancer of zeste homolog 2 (EZH2) methyltransferase promotes cell proliferation, invasion, and angiogenesis in various epithelial cancer cells (Sarris et al., 2016 ). The innovative and developing field of nanobiotechnology includes the study of both biotechnology and nanotechnology. It has numerous uses, particularly in the biomedical industry. Drug nanocrystals are molecular assemblies that can be combined to generate the drug in a crystalline form and are enclosed in a thin stabilizer layer. Nanocrystals technology is a significant alternative to the existing nanocarrier drug delivery technologies in increasing (Goel et al., 2022 ) the bioavailability of medicines that are not readily soluble in water (Rao et al., 2007 ). They typically display remarkable physicochemical and biological activities that differ from those of larger particles because of their tiny size (350–500 nm) (Hanutami and Budiman 2017 ). It's interesting to keep in mind that they have shown significant potential for use in cancer treatments (Joseph and Singhvi 2019 ), orthopedics (Abushammala 2019 ) and dentistry (Samiei et al., 2022 ). Nanocrystal formulation can be used to improve drug delivery, targeting and bioavailability. It is possible to administer the drug orally or intravenously and the limited carrier which mostly consists of a thin layer of surfactant may greatly reduce any toxicity (Jahangir et al., 2020 ). The current model change in the search for alternative applications regarding existing pharmaceuticals is expected to have implications for the current use of herbal medicines and vegetables, particularly in several traditional societies. Focusing additional effort into this field will promote the beneficial use of plant biodiversity, which is already known to be a major source of food, medicine and other resources used by humans who gain influence from the natural world (Elmaidomy et al., 2023 ; Mohammed et al., 2023 ; Ahmed et al., 2022 ; El-Hawwary et al., 2020; Taiwo et al., 2016 ). About 50–60 species make up the genus corchorous of annual herbs, which is a member of the Tiliaceae family. Only two species C. olitorius and C. capsularis are known to produce the best fiber on a commercial basis and are found in warm-temperate and tropical regions of the world (Loumerem and Alercia 2016 ). Additionally, it has been established that the leaves of C. olitorius and C. capsularis have anticancer and antioxidant properties in addition to anti-inflammatory, antiviral, gastroprotective, antinociceptive, analgesic, and antibacterial properties (Abdelrazek et al., 2022 ). The main reason of this diversity in biological activities was attributed to the active metabolites, including terpenes, ionone, flavonoids and steroidal derivatives (Ademiluyi et al., 2015 ). Few studies were reported on C. olitorius neglected roots. This aggravated us to perform the current study including the phytochemical composition of TMECOR via untargeted metabolomic analysis. Additionally, the cytotoxic activity of the TMECOR and its prepared nanocrystals was assessed against three different cell lines. 2. Experimental (Materials and methods) 2.1. Plant material Fresh plant roots (500 g) were collected from a farm from Maghagha city, Minia, Egypt in March 2022. The plant was identified by Prof. Nasser Barakat (Minia University-Faculty of Science-Botany and Microbiology Department). Its voucher number ( Mn-Ph-Cog-060 , Pharmacognosy Department, Faculty of Pharmacy, Minia University). 2.2. Chemicals and reagents Ethanol (Merck, Germany), petroleum ether (Merck, Germany) methanol (98%), DMSO (El-Nasr Company for Pharmaceuticals and Chemicals, Egypt), Insulin (Sigma), acetonitrile and 1% penicillin-streptomycin (Sigma-Aldrich, Germany). 2.3. Extraction The collected Corchorus olitorius roots were dried in shade for two weeks. Afterthat, they were grinded into fine powder resulting in a total amount of 55 g. The resulted fine powder was extracted using 99.8% methanol (1 L, 3×, 2 weeks interval). TMECOR were then evaporated using rotary evaporator (Heidolph®). The yielded viscous pale green TMECOR (5 g) was kept in the refrigerator until further processing. 2.4. Cytotoxic activity The antiproliferative potential was evaluated by the MTT assay of TMECOR and their prepared nanocrystals (Hamed et al., 2022 ; Mosmann 1983 ), using cancer cell lines previously obtained from the American Type Culture Collection (Manassas, VA, USA). The study focused on MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma) and Caco-2 (colon carcinoma) cell lines. The cells were firstly grown in DMEM high glucose (Invitrogen/Life Technologies, USA) with 10% FBS (Hyclone, USA), 1% penicillin-streptomycin and 10 mg/mL of insulin, at 37°C and 5% CO 2 . They were afterwards moved to 96-well plates at a density of 2.2 and 104 cell/cm 2 and then incubated overnight. Subsequently, TMECOR dissolved in DMSO was introduced to the grown cells at varying concentrations (20, 30, 40, 50, and 60 mg/mL). The next day, the MTT test was utilized to evaluate the cell viability as described in (Hamed et al., 2022 ). 2.5. Preparation of Corchorus olitorius nanocrystals Corchorus olitorius nanocrystals were prepared by rotary solvent evaporation and ultrasonication method. Specified amount of TMECOR was dissolved in absolute ethanol (Merck, Germany) and Pet. ether (Merck, Germany) mixture 25:75 ratio. The final amount achieved 50mg/5 mL solution was well sonicated in an ultrasonicate bath at a frequency of 50 kHz (Branson® Ultrasonic Bath,). Tween 80 surfactant (2% by weight) was added. The mixture was then mixed at 1000 rpm for 15 minutes. The resulting solution was placed on a rotary (BUCHI Rotavapor™ R-300 Rotary Evaporator) for solvent evaporation. The resulting powder was collected as Corchorus olitorius nanocrystals and stored at -20°C. 2.6. Particle size (nm) and size distribution The prepared particles were analyzed for their particle size and size distribution in terms of the average volume diameters and polydispersity index by photon correlation spectroscopy using particle size analyzer Dynamic Light Scattering (DLS) (Zetasizer Nano ZN, Malvern Panalytical Ltd, United Kingdom) at fixed angle of 173° at 25°C. Samples were analyzed in triplicate. 2.7. Scanning electron microscopy An SEM instrument (SEM, TESCAN, Warrendale, PA) was utilized for observing the morphology of the prepared nanocrystals. For this, the powder of the nanoparticles was placed on stubs and then coated with a gold layer. 2.8. LC-MS Metabolomic analysis At the Faculty of Pharmacy, Fayoum University, metabolomic profiling of TMECOR was carried out using a 6530 Q-TOF LC/MS (Agilent Technologies, Japan) outfitted with an autosampler (G7129A), a Quat. Pump (G7104C), and a Column Comp (G7116A) for chromatographic separation. The volume of injection was 3 µL. The analytes were separated using an Agilent Technologies Zorbax RP-18 column (150 mm × 3 mm, dp = 2.7 µm). The mass spectra were obtained using ESI in positive and negative ionization modes through a capillary voltage of 4500 V. They were recorded in the range of 50 to 3000 m/z . The drying gas flow and the gas temperature were 8 L/min and 200°C, respectively. The fragmentator and skimmer voltages were established at 130 and 65 V, respectively, while the collision energy was 10 V. A Phenomenex Kinetex 2.6 mm XB-C18 150 × 4.6 mm column, maintained at 30°C and connected to a guard column, was filled with 10 mL of samples (1 mg/mL in methanol). The mobile phase consisted of a mixture of LC-MS grade water (A) and acetonitrile (B), each containing formic acid (0.1%). The flow rate of 500 mL/min was adjusted for the gradient elution, going from 5–20% B in two minutes, 20–98% B in eighteen minutes, 98% B in five minutes, and lastly 98–5% B in two minutes. The MS settings used for the HPLC-HESI-HRMS analysis were as follows: capillary temperature (320°C), spray voltage (+ 3.5 or -2.7 kV), sheath gas (57.50 Pa), sweep gas (3.25 Pa), auxiliary gas (16.25 Pa), probe heater (462.50°C), AGC target (1e6), S-Lens RF (50 cm) resolution (70.000), microscans (1). Mzmine 2.12 was used to obtain a differential analysis of MS data, and ProteoWizard was used to transform the raw data into positive and negative files in the mz/mL format. Finally, metabolite identification was achieved by referring to Dictionary of Natural Products and METLIN databases (DNP, 2020; METLIN, 2020). 2.9. In silico studies The specific action mechanisms of Corchorus olitorius extract and nanocrystals against MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma) and Caco-2 (colon cancer) cell lines were investigated by in silico protein network and docking analyses. These computational strategies were designed to validate the selectivity and targeting efficacy of identified bioactive compounds within Corchorus olitorius , particularly against the mentioned cancer types. By integrating the established biological pathways and simulating their interactions with pivotal molecular targets, our objective was to substantiate the experimental biological relevance. This approach aims to provide a detailed molecular insight into how Corchorus olitorius exerts its therapeutic effects, highlighting the extract and nanocrystals potential for enhanced specificity and effectiveness in treating breast cancer, hepatocellular carcinoma and colon cancer. 2.9.1. Investigating Data to Identify Targets Associated with Cancer Cytotoxicity We utilized data from two pivotal resources to pinpoint the genetic targets implicated in the cytotoxicity against MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma) and Caco-2 (colon cancer) cell lines. The Gene Expression Omnibus (GEO) Database, hosted by the National Center for Biotechnology Information ([NCBI]( https://www.ncbi.nlm.nih.gov/guide/genes-expression/ )), provided exhaustive gene expression profiles. This enabled a comprehensive analysis of the transcriptional behavior of genes involved in cancer cytotoxicity, particularly within the contexts of breast cancer, hepatocellular and colon cancers. Simultaneously, the Pharmacogenomics Knowledgebase ([PharmGKB]( https://www.pharmgkb.org/ )) delivered valuable insights into how genetic variations influence the efficacy and toxicity of drugs. This approach aims to deepen our understanding of the genetic underpinnings that contribute to the therapeutic effectiveness of Corchorus olitorius extract and nanocrystals in targeting these specific cancer types. 2.9.2. Identifying Targets for Cancer Treatment Efficacy via Corchorus olitorius with STITCH We employed the STITCH database ([STITCH: chemical association networks](embl.de)) to decode the interactions between protein targets uncovered through the Gene Expression Omnibus (GEO) Database and Pharmacogenomics Knowledgebase (PharmGKB), alongside the active compounds in Corchorus olitorius . The STITCH database serves as a repository for understanding the complex interactions between chemicals and proteins, amalgamating data from experimental studies, databases and literature to map out how chemicals influence protein activity. This method facilitates the exploration of the relationship between the identified proteins and the bioactive compounds in Corchorus olitorius . Utilizing data from STITCH, which sheds light on the chemical-protein interplay, we aim to reveal the mechanistic pathways by which Corchorus olitorius extract and nanocrystals might deliver their therapeutic benefits in targeting the selected cancer cell lines. This approach highlights the critical role of bioinformatics tools in narrowing down the connection between natural product research and targeted cancer therapy. The process initiated with pinpointing the principal active compounds in Corchorus olitorius . These compounds were then cross-referenced in the STITCH database along with the protein targets identified through the GEO and PharmGKB, generating an intricate network of interactions that potentially underlie the anticancer efficacy of Corchorus olitorius . 2.9.3. Construction of a Protein-Protein Interaction (PPI) Network for Cancer-Related Targets Following the identification of genes implicated in cancer cytotoxicity effects on MCF-7, HepG2 and Caco-2 cell lines from our preliminary analyses, these genes were introduced into the STRING database (Von Mering et al., 2005 ) to develop a Protein-Protein Interaction (PPI) network. This step utilized STRING database to accurately map the interactions between a broad array of proteins, applying a minimum combined score threshold of 0.4 to ensure the relevance of functional interactions highlighted. The network was then visualized and meticulously analyzed using Cytoscape (Shannon et al., 2003 ), focusing particularly on identifying hub genes within the network. These hub genes, pinpointed through the use of CytoHubba plugin in Cytoscape, stand out due to their numerous connections within the network, suggesting their pivotal role in mechanisms related to the cytotoxic effects of Corchorus olitorius on these specific cancer cell lines. This method underscores the significant value of integrating bioinformatic tools to elucidate the intricate molecular interactions that underpin the anticancer efficacy of Corchorus olitorius extract and nanocrystals. 2.9.4. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment for Anticancer Targets To delineate the molecular functions (MF), biological processes (BP), cellular components (CC) and critical signaling pathways associated with the genes implicated in the cytotoxic effects on selected cell lines, we conducted Gene Ontology (GO) and pathway enrichment analyses. These analyses were instrumental in understanding the specific roles genes play within biological contexts: BP for their functions in biological activities, CC for their localization within cellular structures, and MF for their precise molecular actions. We employed Shiny GO ([ http://bioinformatics.sdstate.edu/go/](http://bioinformatics.sdstate.edu/go/) ) for the enrichment analysis, setting a stringent False Discovery Rate (FDR) threshold to ensure accuracy and used Srplot ([ https://www.bioinformatics.com.cn/en](https://www.bioinformatics.com.cn/en) ) for the visualization of results. This methodology offered an in-depth view of the genes in molecular interactions and biological pathways relevant to the anticancer efficacy of Corchorus olitorius extract and nanocrystals, significantly enriching our comprehension of their functional importance in combating breast cancer, hepatocellular carcinoma and colon cancer. 2.9.5. Molecular Docking Analysis Within the scope of our research on the cytotoxic effects of Corchorus olitorius extract and nanocrystals against the selected cancer cell lines, the veracity of our network pharmacology predictions was substantiated through molecular docking analysis. This technique verified the interactions between crucial protein targets and active compounds found in Corchorus olitorius , underscoring potential synergistic effects for cancer treatment. Utilizing Discovery Studio Client version 16.1.0.15350 (Studio 2008) and the RCSB Protein Data Bank ([ http://www.rcsb.org/](http://www.rcsb.org/) ), the process entailed detailed preparation of proteins and ligands for the docking procedure. This essential validation phase bolsters our investigation into viable anticancer agents, in line with our objective to leverage Corchorus olitorius bioactive compounds for enhanced therapeutic outcomes in targeting breast cancer, hepatocellular carcinoma and colon cancer. 3. Results 3.1. Cytotoxic activity The cytotoxic activity of TMECOR root extract was investigated using the MTT viability assay in comparison to the anticancer drug Staurosporine® as a positive control. The assay was carried out against a number of cancer cell lines (MCF-7, HepG2 and Caco-2). The results exposed that the crude extract presented a high inhibitory activity against (MCF-7, HepG2 and Caco-2) cells, with IC 50 values of 42.68 ± 1.96, 37.14 ± 1.6 and 18.63 ± 1.16 µg/mL, respectively. While, the nanocrystals showed higher cytotoxic activity especially against HepG2 and Caco-2 with IC 50 value of 23.288 ± 1.08 and 12.156 ± 0.61 µg/mL, respectively, showing an enhanced cytotoxic activity for molokheia roots. While MCF-7 showed IC 50 of 62.497 ± 3.63 µg/mL as described in Table 1 . Table 1 Cytotoxic activities of TMECOR and its nanocrystals. Sample IC 50 values (mean ± S.E.M; µg/mL) MCF-7 HepG2 Caco-2 TMECOR 42.68 ± 1.96 37.14 ± 1.6 18.63 ± 1.16 TMECOR nanocrystals 62.497 ± 3.63 23.288 ± 1.08 12.156 ± 0.61 Staurosporine® 8.1454 ± 0.47 5.88 ± 0.37 3.47 ± 0.15 3.2. Synthesis and characterization of nanocrystals The nanocrystals showed a mean particle size of 370 ± 25 nm and a poly dispersity index value of 0.2–0.5 indicating a narrow size distribution. The data of particle size distribution relative to their PI were presented in Fig. 1 that enhance the bioavailability, in addition to zeta potential − 22 ± 4.9 mV indicating high stability (Kakran et al. 2012 ; Yadi et al. 2018 ). SEM results Fig. 2 approved different particle size distribution and presented that the nanocrystals had grouped quasispheroidal within the sample (Bohlouli et al. 2021 ). 3.3. Metabolomics study The phenolic compounds were predominating among the secondary metabolites that were identified through metabolic profiling of C. olitorius utilizing the untargeted HPLC-HESI-HRMS metabolomics technique (Table S1 ; Figure S1 ). From the (DNP, 2020; METLIN, 2020) databases and from Figures S2 and S3, the mass ion peak at m/z 137.0613 [M + H] + for the anticipated molecular formula C 10 H 16 was dereplicated as limonene ( 1 ); a monoterpene derivative previously identified among the volatile compounds of the crushed C. olitorius leaves and flowers (Driss et al., 2016 ). Also, the mass ion peak at m/z 165.9943 [M + H] + was described as coumaric acid ( 2 ) a phenolic acid that was earlier identified in molokheia leaves (Driss et al., 2016 ). Compound ( 3 ) was identified as vanillic acid in arrangement with the mass ion peak at m/z 167.9792 [M-H]ˉ and the molecular formula C 8 H 8 O 4 , which was also previously reported as a phenolic acid metabolite in C. capsularis flowers (Ademiluyi et al., 2015 ). Moreover, the mass ion peak at m/z 181.1225 [M + H] + for the suggested molecular formula C 9 H 8 O 4 was dereplicated as caffeic acid ( 4 ); a phenolic acid in C. capsularis flowers (Ademiluyi et al., 2015 ). Compound ( 5 ) was dereplicated as quinic acid, also a phenolic acid derivative corresponding to the molecular formula C 7 H 12 O 6 and the mass ion peak at m/z 191.0668 [M-H]ˉ. It was previously isolated from C. capsularis flowers (Ademiluyi et al., 2015 ). Another phenolic acid called ferulic acid ( 6 ), was characterized in consonance with the molecular formula C 10 H 10 O 4 and mass ion peak at m/z 195.1034 [M + H] + . It was formerly identified from C. capsularis (Ademiluyi et al., 2015 ). Compound ( 7 ) was dereplicated as the flavonoid derivative apigenin with a molecular formula of C 15 H 10 O 5 and mass ion peak at m/z 269.2327 [M-H]ˉ,which was previously isolated from C. olitorius (Yakoub et al., 2018 ). A flavonol derivative ( 8 ) was also dereplicated as kaempferol corresponding to the molecular formula C 15 H 10 O 6 and the mass ion peak at m/z 287.1865 [M + H] + (Yakoub et al., 2018 ). Phytol a diterpene derivative ( 9 ) was reported in both C. olitorius and C. capsularis with the molecular formula C 20 H 40 O and the mass ion peaks at m/z 295.2218 [M-H]ˉ(Mekhael et al., 2019). Another flavone derivative was dereplicated as Cirsiliol ( 10 ) in alignment with the molecular formula C 17 H 14 O 7 and the mass ion peak at m/z 329.2333 [M-H] − (Yakoub et al., 2018 ). The mass ion peaks at m/z 353.2129 [M-H]ˉand 361.2477 [M + H] + for the predicted molecular formulas C 16 H 18 O 9 and C 18 H 16 O 8 were identified as chlorogenic ( 11 ) (Azuma et al., 1999 ) and rosmarinic ( 12 ) (Yakoub et al., 2018 ) acids, respectively. These phenolic acids were formerly isolated from C. olitorius . Whereas the molecular ion peak at m/z 385.1975 [M-H]ˉ corresponding to the molecular formula C 19 H 30 O 8 was characterized as corchoiononside C ( 13 ), which was earlier isolated from C. olitorius leaves as an ionone derivative (Yoshikawa et al., 1997 ). Additionally, two flavonol derivatives with mass ion peaks at m/z 417.2971 and 609.5092 [M-H]ˉwere dereplicated as kaempferol 3 O - α -L-arabinopyranoside (Kohda et al., 1994 ) and rutin (Ademiluyi et al., 2015 ) respectively. These flavones were reported previously from C. olitorius leaves. 3. Studies 3. In silico Studies 3.1. Therapeutic Targets for Cancer Treatment Based on our experimental findings, a dataset of 51 target proteins associated with the cytotoxic effects on breast cancer, hepatocellular carcinoma and colon cancer was meticulously compiled from the NCBI-GEO and PharmGKB databases. These proteins, presented in Table S2 , encompass critical targets and biomarkers pivotal to our insights into the anticancer effects of Corchorus olitorius . This collection not only highlights the importance of these proteins in the realm of cancer remedy but also emphasizes their potential as therapeutic targets, underpinned by the experimental evidence gathered through our research activities. 3.2. STITCH Database Analysis of Cancer Treatment Targets and Corchorus Olitorius Extract In this detailed investigation, we utilize the STITCH database to meticulously examine the interactions between the principal active components in Corchorus olitorius extract namely, Chlorogenic acid, Rosmarinic acid and Rutin and the protein targets identified through GEO and PharmGKB analyses Fig. 3 . This analysis is pivotal in furthering our understanding of the anticancer potential of Corchorus olitorius extract, particularly against MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma) and Caco-2 (colon cancer) cell lines. The study highlights the synergistic action of Corchorus olitorius extract, comparing its comprehensive benefits against those of its individual compounds. The identified synergy suggests complex interactions between the combined bioactive molecules in the extract and proteins associated with cancer, potentially augmenting the extract therapeutic efficacy in cancer treatment. This mechanism of action signifies a promising avenue for enhancing the protective effects against cancer through the bioactive components of Corchorus olitorius . 3.3. Construction of the Protein-Protein Interaction (PPI) Network In our investigation into the anticancer efficacy of Corchorus olitorius , proteins delineated from the research were amalgamated into the STRING database, version 12.0 ( https://string-db.org/cgi/input?sessionId=barlI0uOHF46 ), to establish initial PPI networks that reveal their direct and functional relationships. This integration enabled the depiction of the PPI network through Cytoscape software, (version 3.10.1). By employing the Analyzer feature in Cytoscape, we constructed a detailed protein interaction network, incorporating 98 nodes and 1679 interaction linkages, yielding an average node connectivity of 34.3. The complexities of this network are displayed in Fig. 4 , underlining the extensive analysis of protein interactions crucial to our research on enhancing the anticancer potential of Corchorus olitorius against MCF-7, HepG2 and Caco-2 cell lines. 3.4. Analysis of Overrepresented Gene Ontology Terms in Corchorus Olitorius Extract The Gene Ontology (GO) enrichment analysis for our study on the anticancer properties of Corchorus olitorius extract was performed using Shiny GO v0.80. Our analysis revealed significant participation of proteins in Biological Processes (BP) such as "Regulation of apoptotic signaling pathway", "Regulation of angiogenesis", "Response to estradiol", "Regulation of Wnt signaling" and "Regulation of cell cycle," pivotal in mediating the cytotoxic effects on cancer cells. In the Cellular Component (CC) category, terms like "Transcription repressor complex", "Bcl-2 protein complex" and "Nucleoplasm" were prominent, indicating the subcellular locales impacted by Corchorus olitorius . Molecular Function (MF) insights, with terms such as "DNA-binding transcription factor binding", "Estrogen receptor binding" and "Tyrosine-kinase activity," suggest interactions within signaling pathways that may be crucial for the anticancer activity of the extract. These observations are in harmony with our objective to unravel the molecular interactions through which Corchorus olitorius extract exerts its effects on MCF-7, HepG2 and Caco-2 cell lines. Table S3 provides a detailed account of the statistically significant GO terms and their relevance. The visualizations in Fig. 5 succinctly illustrate the enriched Gene Ontology (GO) terms across the domains of Biological Process (BP), Cellular Component (CC) and Molecular Function (MF), related to the cytotoxic activities of Corchorus olitorius extract. 3.5. Analysis of Dominant KEGG Pathways in Corchorus Olitorius Extract Research The KEGG pathway analysis is a cornerstone of our study, connecting the molecular activity of Corchorus olitorius extracts and nanocrystals to specific biological pathways impacted in the treatment of MCF-7, HepG2 and Caco-2 cancer cell lines. Our findings, detailed in Table S4, are illustrated in a bubble plot Fig. 6 and reveal significant enrichment in pathways such as "Colorectal cancer pathway", "Hepatocellular carcinoma pathway", "Estrogen signaling pathway", "ErbB (Epidermal Growth Factor Receptor) signalling pathway", "Wnt signaling pathway", "PI3K-Akt signaling pathway" and "Apoptosis". These pathways are essential for deciphering how our treatment strategy may modulate gene expression and protein interactions to exert cytotoxic effects on cancer cells, shedding light on potential mechanisms of action and identifying therapeutic targets for intervention in cancer treatment. Anticancer Activity. In our research, the KEGG pathway analysis of "Pathways in Cancer" underscores the potential molecular targets affected by Corchorus olitorius extract in exerting cytotoxic effects on MCF-7, HepG2 and Caco-2 cell lines. Key pathways such as the PI3K-Akt signaling pathway, Estrogen signaling pathway and apoptosis pathways have been highlighted Fig. 7 . These pathways are critical in regulating cell proliferation, survival and metabolism, which are processes that can be dysregulated in cancer. The identification of these pathways provides insights into the anticancer mechanisms of Corchorus olitorius extract, suggesting that it may exert its therapeutic effects by modulating pathways commonly involved in the cellular response to cancerous growth. The Colorectal Cancer Pathway depicted here reflects the molecular interactions that Corchorus olitorius extracts and nanocrystals may target to exert cytotoxic effects on colorectal cancer. This pathway, central to the progression from normal epithelium through dysplasia to carcinoma, presents potential targets that could be influenced by our anticancer agents to disrupt the cancer cell cycle and promote apoptosis Fig. 8 . The involvement of key elements such as Wnt signaling, ErbB signalling and PI3K-Akt pathways indicates that the mechanism of action may include inhibiting pathways crucial for tumor growth and survival. Deciphering these interactions is important for the development of targeted therapies to treat colorectal cancer effectively. 3.6. Identification of Crucial Hub Genes The CytoHubba plugin identified crucial hub genes within the PPI network pertinent to the cytotoxic effects of Corchorus olitorius extracts and nanocrystals on MCF-7, HepG2 and Caco-2 cancer cell lines. In breast cancer, Corchorus olitorius extract could theoretically affect key pathways mediated by hub genes such as ESR1. ESR1 (Tamoxifen therapy) is crucial for hormone-driven cancer proliferation. BCL2, another hub gene has a significant role in cell death pathways and is being examined in trials with drugs like Venetoclax. For hepatocellular carcinoma (HCC), the extract might impact genes like MYC, which regulates cell cycle and apoptosis but lacks direct inhibitors. It could also influence the AKT1/MTOR pathway, a common oncogenic pathway in HCC that is targeted by Everolimus. Additionally, TP53, frequently mutated in HCC, represents a therapeutic target, with strategies focusing on restoring its function or targeting pathways that are altered due to TP53 mutations. In colorectal cancer (CRC), KRAS, a gene commonly mutated and difficult to target, along with CTNNB1, integral to the Wnt signaling pathway, could be modulated by constituents of the extract. Similarly, the therapeutic approaches could influence TP53 mutations, common in CRC, to reactivate its tumor suppressor functions. Also, ERBB1 (EGFR) Which is targeted by drugs like Erlotinib, is essential for cell growth and survival. Notably, BRAF mutations in CRC are being targeted through combination therapies, such as the FDA-approved encorafenib and cetuximab, pointing to the potential of combination treatments in enhancing efficacy. The analysis of these hub genes Fig. 9 highlights a potential pathway-modulating effect of Corchorus olitorius extract, suggesting that it could complement FDA-approved drugs by targeting these genes and their associated pathways, potentially inhibiting cancer progression and promoting cell death. 3.7. Molecular Docking In our study, molecular docking serves as a crucial tool to examine the roles of pivotal hubs within the cancer network like EGFR and BRAF as potential targets in the treatment of cancer. Both EGFR and BRAF are well-established in oncogenic signaling pathways, EGFR is a primary receptor tyrosine kinase implicated in tumor growth and spread, while BRAF mutations activate the MAPK/ERK signaling pathway, leading to cancer cell survival and proliferation. Modeling the combined inhibition of EGFR and BRAF may offer a synergistic therapeutic approach, aiming to disrupt concurrent pathways and enhance the efficacy of treatment. The benefits of this dual inhibition strategy include a potential reduction in drug resistance, as cancer cells often activate alternative survival pathways to evade therapy. By simultaneously blocking both EGFR and BRAF pathways, this approach could result in more comprehensive pathway inhibition, reduced cell viability and impeded cancer progression. 5.7.1. Molecular Modeling with EGFR The redocking of EGFR ligand (PDB ID: 1M17) within its active site demonstrated an RMSD of 1.03 Å, confirming the accuracy of our docking methods and a binding energy indicative of a significant interaction potential. For the series of 15 compounds examined against EGFR, detailed in Table S5, specific bioactive compounds from Corchorus olitorius such as chlorogenic acid 11 and rutin 15 demonstrated superior binding affinities, surpassing the native ligand with docking scores of -7.276 and − 8.498 kcal/mol, respectively. Their interactions with key residues-LYS 721, ASP 831 and others-suggest a promising inhibitory action on EGFR, essential for the management of various cancers. 5.7.2. Molecular Modeling with BRAF V600E Similarly, the BRAF V600E crystal structure was sourced from the RCSB Protein Data Bank (PDB ID: 5JRQ). The redocking of the native ligand validated our simulation with an RMSD of 1.623 Å and a favorable binding energy. The assessment of 15 compounds against BRAF V600E , as outlined in Table S6, highlighted compounds like caffeic acid 4 and corchoiononside C 13 from Corchorus olitorius . These compounds showed enhanced docking scores of -4.794 and − 7.471 kcal/mol, respectively, indicating strong binding capabilities. The significant interactions with crucial amino acids such as CYS 532 and LYS 483 support their potential role as dual inhibitors, targeting both EGFR and BRAF pathways. The combined dual inhibition of EGFR and BRAF by compounds found in Corchorus olitorius could offer several benefits, including a broader inhibition of cancer cell signaling pathways, potentially leading to reduced tumor growth and resistance to single-target therapies. Figure 10 would visually illustrate these promising interactions, showcasing the potential of these compounds as dual inhibitors that may enhance the efficacy of existing cancer therapies. 4. Discussion The work was studied for the first time on C. olitorius roots. It uncovered the marked in vitro cytotoxic inhibitory aptitude of TMECOR, against breast, liver and colon cell lines. The previous studies are in the same line with my results, which the gold nanoparticle molokheia leaves extract tested against breast, liver and colon cancer cell lines also. The results ensured the cytotoxic effect of it with IC 50 12.2, 10.3 and 11.2 µg/mL, respectively (Ismail et al., 2023). Furthermore, the prepared nanocrystals notably enhanced the cytotoxic activity of TMECOR, particularly against HepG2 (37%) and Caco-2 (34%). According to the US NCI guidelines, these data remarkably reflect the strong (IC 50 < 20 mg/mL) to moderate (IC 50 = 21–50 mg/mL) cytotoxic potential of TMECOR towards the aforementioned cell lines a result that indicates the cytotoxic properties of such ignored plant part, which is commonly considered as a vegetable waste product. To determine which of active constituents are responsible for this activity, LC-HRMS-based metabolomics analysis was studied. They belong to various chemical classes, including flavonoids, terpenes, ionones and phenolic acids, illustrated in Table S2 and Figure S6. This metabolic profile distinctly explicates the cytotoxic potential of TMECOR. For instance, phytol ( 9 ) (Shariare et al., 2021 ), apigenin ( 7 ) (Imran et al., 2020 ), rutin ( 15 ) (Caparica et al., 2020 ), Cirsiliol ( 10 ) (Jia et al., 2021 ) and kaempferol ( 8 ) (Imran et al., 2019 ) have all been previously reported to exert potent cytotoxic effects. 5. Conclusion The current study focused on in vitro cytotoxic activites against three different cell lines (MCF-7, HepG2 and Caco-2) of TMECOR as a natural source. Interestingly, the nanocrystals gave considerable cytotoxic activity in liver and colon cell lines in comparison with Staurosporine®. Consequently, molokheia roots may be used to develop anticancer herbal drug in the future. This study introduces a comprehensive in silico methodology that intertwines network pharmacology with molecular docking to delineate the anticancer mechanisms of compounds derived from Corchorus olitorius against MCF-7, HepG2, and Caco-2 cancer cell lines. Through the construction of a protein-protein interaction (PPI) network, pivotal hub genes such as EGFR, BRAF, MYC, and TP53 were highlighted, playing a critical role in oncogenic signaling. Molecular docking simulations predicted the binding efficacy of Corchorus olitorius compounds at these proteins' active sites. Utilizing crystal structures of EGFR (PDB ID: 1M17) and BRAF V600E (PDB ID: 5JRQ), our docking approach was substantiated, achieving RMSDs that ensure reliable predictions. The results showcased compounds, notably chlorogenic acid and rutin, with binding energies exceeding those of native ligands, pointing to a substantial therapeutic potential. These computational insights open a pathway for the experimental exploration and crafting of novel anticancer agents aimed at targeting key elements in cancer pathways. Declarations Disclosure statement: The authors declare no conflict of interest. Consent for publication : Not applicable . Availability of data and materials: All data generated or analyzed during this study are included in this article and its supplementary information files . Ethical approval: Not applicable. Competing interests: The authors declare no competing interests. Author contributions Marwa A.M. Abdelrazek and Miada F. Abdelwahab : writing original draft, visualization, validation, investigation, formal analysis, data curation. Soad A. Mohamed : methodology, conceptualization. Hesham A. Abo Zeid : validation, software, methodology, conceptualization. Usama R. Abdelmohsen and Ashraf N.E Hamed : reviewing, supervision, methodology, investigation, data curation, conceptualization. Acknowlegment: The authors acknowledge EKB (Egyptian Knowledge Bank) Minia University. Funding : Not applicable. References Ademiluyi AO, Oboh G, Aragbaiye FP, Oyeleye SI, Ogunsuyi OB (2015) Antioxidant properties and in vitro α-amylase and α-glucosidase inhibitory properties of phenolics constituents from different varieties of Corchorus spp. J Taibah Univ Med Sci 10(3):278–287 Abushammala H (2019) On the para/ortho reactivity of isocyanate groups during the carbamation of cellulose nanocrystals using 2, 4-toluene diisocyanate. Polymers 11:1164 Ahmed SS, Fahim JR, Youssif KA, AboulMagd AM, Amin MN, Abdelmohsen UR, Hamed ANE (2022) Metabolomics of the secondary metabolites of Ammi visnaga L. roots (family Apiaceae) and evaluation of their biological potential. South Afr J Bot 149:860–869 Azuma K, Nakayama M, Koshioka M, Ippoushi K, Yamaguchi Y, Kohata K, Yamauchi Y, Ito H, Higashio H (1999) Phenolic antioxidants from the leaves of Corchorus olitorius L. J Agric Food Chem 47:3963–3966 Abdelrazek MAM, Abdelwahab MF, Abdelmohsen UR, Hamed ANE (2022) Pharmacological and phytochemical biodiversity of Corchorus olitorius . RSC Ad 12:35103–35114 Bohlouli S, Jafarmadar Gharehbagh F, Dalir Abdolahinia E, Kouhsoltani M, Ebrahimi G, Roshangar L, Imani A, Sharifi S, Maleki Dizaj S (2021) Preparation, Characterization, and Evaluation of Rutin Nanocrystals as an Anticancer Agent against Head and Neck Squamous Cell Carcinoma Cell Line. J. Nanomater. 2021:9980451 Caparica R, Júlio A, Araújo MEM, Baby AR, Fonte P, Costa JG, de Santos T (2020) Anticancer activity of rutin and its combination with ionic liquids on renal cells. Biomolecules 10(2):233 Driss D, Kaoubaa M, Mansour RB, Kallel F, eddine Abdelmalek B, and Chaabouni S. E (2016) Antioxidant, antimutagenic and cytotoxic properties of essential oil from Corchorus olitorius L. flowers and leaf. Free Radic Antioxid 6(1):34–43 El-Hawwary SS, Saber FR, Almaksoud A, Elimam HM, Sayed H, Abdelmohsen AM, U. R (2022) Cytotoxic potential of three Sabal species grown in Egypt: a metabolomic and docking-based study. Nat Prod Res 36(4):1109–1114 Elmaidomy AH, Zawily E, Salem A, Altemani AK, Algehainy FH, Altemani NA, Rateb AH, Abdelmohsen ME, Shady UR, N. H (2023) New cytotoxic dammarane type saponins from Ziziphus spina-christi. Sci Rep 13(1):20612 Goel H, Siddiqui L, Mahtab A, Talegaonkar S (2022) Fabrication design, process technologies, and convolutions in the scale-up of nanotherapeutic delivery systems in Nanoparticle Therapeutics. Elsevier . 2022:47–131 Hanutami NPB, Budiman A (2017) Review Artikel: Penggunaan Teknologi Nano Pada Formulasi Obat Herbal. Farmaka 15(2):30–42 Hamed ANE, Abdelaty NA, Attia EZ, Amin MN, Ali TFS, Afifi AH, Abdelmohsen UR, Desoukey SY (2022) Antiproliferative potential of moluccella laevis L. aerial parts family lamiaceae (labiatae), supported by phytochemical investigation and molecular docking study. Nat Prod Res 36:1391–1395 Ismail EH, Saqer AMA, Assirey E, Naqvi A, Okasha RM (2018) Successful Green Synthesis of Gold Nanoparticles using a Corchorus olitorius Extract and Their Antiproliferative Effect in Cancer Cells. Mol Sci 19(9):2612 Imran M, Gondal TA, Atif M, Shahbaz M, Qaisarani TB, Mughal MH, Salehi B, Martorell M, Sharifi-Rad J (2020) Apigenin as an anticancer agent. Phytother Res 34(8):1812–1828 Imran M, Salehi B, Sharifi-Rad J, Aslam Gondal T, Saeed F, Imran A, Shahbaz M, Fokou PVT, Arshad MU, Khan H, Guerreiro SG, Martins N, Estevinho LM (2019) Kaempferol: A key emphasis to its anticancer potential. Molecules 24(12):2277 Joseph E, Singhvi G (2019) Multifunctional nanocrystals for cancer therapy: a potential nanocarrier. Nanomater. Drug Deliv.Ther . 2019:91–116 Jahangir MA, Imam SS, Muheem A, Chettupalli A, Al-Abbasi F, Nadeem MS, Kazmi I, Afzal M, Alshehri S (2020) Nanocrystals: characterization overview, applications in drug delivery, and their toxicity concerns. J. Pharm. Innov. 2020:1–12 Jia X, Huang C, Hu Y, Wu Q, Liu F, Nie W, Chen X, Li X, Dong Z, Liu K (2021) Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth in vitro and in vivo . J Exp Clin Cancer Res 40(1):1–15 Kakran M, Shegokar R, Sahoo NG, Gohla S, Li L, Müller RH (2012) Long-term stability of quercetin nanocrystals prepared by different methods. J Pharm Pharmacol 64:1394–1402 Kohda H, Tanaka S, Yamaoka Y, Morinaga S, Ohhara Y (1994) Constituents of Corchorus olitorius L. Nat Med 48(3):213–214 Loumerem M, Alercia A (2016) Descriptors for jute ( Corchorus olitorius L). Genet Resour Crop Evol 63(7):1103–1111 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63 Mohammed MHH, Hamed ANE, Elhabal SF, Mokhtar FA, Abdelmohsen UR, Fouad MA, Kamel MS (2023) Metabolic profiling and cytotoxic activities of ethanol extract of Dypsis leptocheilos aerial parts and its green synthesized silver nanoparticles supported by network pharmacology analysis. South Afr J Bot 161:648–665 Mekhael Maged KG, Hassan A, Hanna A, Simon A, Tóth G, Duddeck H (2019) Phytochemical investigation of Corchorus olitorius and Corchorus capsularis (family Tiliaceae ) that grow in Egypt. Pharm J 18:123–134 Rao CNR, Thomas PJ, Kulkarni GU (2007) Synthesis of nanocrystals. Nanocrystals: Synthesis, Properties and Applications. 25–68 Sarris M, Nikolaou K, Talianidis I (2014) Context-specific regulation of cancer epigenomes by histone and transcription factor methylation. Oncogene 33(10):1207–1217 Sarris ME, Moulos P, Haroniti A, Giakountis A, Talianidis I (2016) Smyd3 is a transcriptional potentiator of multiple cancer-promoting genes and required for liver and colon cancer development. Cancer Cell 29(3):354–366 Shariare MH, Noor HB, Khan JH, Uddin J, Ahamad SR, Altamimi MA, Alanazi FK, Kazi M (2021) Liposomal drug delivery of Corchorus olitorius leaf extract containing phytol using design of experiment (DoE): In-vitro anticancer and in-vivo anti-inflammatory studies. Colloids Surf B 199:111543 Samiei M, Arablouye Moghaddam F, Dalir Abdolahinia E, Ahmadian E, Sharifi S, Maleki Dizaj S (2022) Influence of Curcumin Nanocrystals on the Early Osteogenic Differentiation and Proliferation of Dental Pulp Stem Cells. J. Nanomater. 2022, 1–8 Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13(11):2498–2504 Taiwo BJ, Taiwo GO, Olubiyi OO, Fatokun AA (2016) Polyphenolic compounds with anti-tumour potential from Corchorus olitorius (L.) Tiliaceae, a Nigerian leaf vegetable. Bioorg Med Chem Lett 3:1–7 Von Mering C, Jensen LJ, Snel B, Hooper SD, Krupp M, Foglierini M, Jouffre N, Huynen MA, Bork P (2005) STRING: known and predicted protein–protein associations, integrated and transferred across organisms. Nucleic Acids Res 33(suppl1):D433–D437 Yadi M, Mostafavi E, Saleh B, Davaran S, Aliyeva I, Khalilov R, Nikzamir M, Nikzamir N, Akbarzadeh A, Panahi Y (2018) Current developments in green synthesis of metallic nanoparticles using plant extracts: a review. Artif Cells Nanomed Biotechnol 46:S336–S343 Yakoub ARB, Abdehedi O, Jridi M, Elfalleh W, Nasri M, Ferchichi A (2018) Flavonoids, phenols, antioxidant, and antimicrobial activities in various extracts from Tossa jute leave ( Corchorus olitorus L). Ind Crops Prod 118:206–213 Yoshikawa M, Shimada H, Saka M, Yoshizumi S, Yamahara J, Matsuda H (1997) Medicinal foodstuffs. V. Moroheiya. Absolute stereostructures of corchoionosides A, B, and C, histamine release inhibitors from the leaves of Vietnamese Corchorus olitorius L. ( Tiliaceae ). Chem. Pharm. Bull . 45:464–469 Supplementary Files GraphicalAbstract.pdf Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted Reviewers agreed at journal 21 Mar, 2025 Reviewers invited by journal 21 Mar, 2025 Editor invited by journal 20 Mar, 2025 Editor assigned by journal 19 Mar, 2025 First submitted to journal 17 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6234677","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431997672,"identity":"38b1bb18-250a-4cb3-a92d-febde79f890e","order_by":0,"name":"Marwa Ali","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYDCCA1Caj5n98IMPQAYbO7Fa2Jh50gxngBlEa2FgMJDmAbEIaeG7fTrxc2WbnTzQPQnGNr+2yfMxMzB++JiDW4vkudzNkmfbkg3bmBkPPM7tuw1kMDBLztyGW4vBGd4Nko3bmBmBKhOMc3tugxhszLz4tWz+2bit3h6o0kDasue2PTFatgFtOZwI1sLw43YiQS2SQC2Wjf+OJ7eBArm34TaQwdiM1y98QIfdbDhTbdvPf/zwgx9/btvOb28++OEjHi2ogLENTDYQqx4E/pCieBSMglEwCkYKAABVgE7edZDdkAAAAABJRU5ErkJggg==","orcid":"","institution":"Minia University Faculty of Pharmacy","correspondingAuthor":true,"prefix":"","firstName":"Marwa","middleName":"","lastName":"Ali","suffix":""},{"id":431997673,"identity":"7bcf690e-e998-4460-beae-3ca229943682","order_by":1,"name":"Marwa A. M. Abdel-Razek","email":"","orcid":"","institution":"Minia University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Marwa","middleName":"A. M.","lastName":"Abdel-Razek","suffix":""},{"id":431997674,"identity":"5f68ab93-48f5-46bf-88ee-606b6f455306","order_by":2,"name":"Miada F. Abdelwahab","email":"","orcid":"","institution":"Minia University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Miada","middleName":"F.","lastName":"Abdelwahab","suffix":""},{"id":431997675,"identity":"b6443436-93cd-4f66-a498-fc1633d73f19","order_by":3,"name":"Soad A. Mohamad","email":"","orcid":"","institution":"Deraya University","correspondingAuthor":false,"prefix":"","firstName":"Soad","middleName":"A.","lastName":"Mohamad","suffix":""},{"id":431997676,"identity":"70eb9a88-d7f5-49aa-9875-ba87a559ba5e","order_by":4,"name":"Hesham A. Abou-Zied","email":"","orcid":"","institution":"Deraya University","correspondingAuthor":false,"prefix":"","firstName":"Hesham","middleName":"A.","lastName":"Abou-Zied","suffix":""},{"id":431997677,"identity":"5a30511a-f2bd-46a9-a0d0-7b28d4017230","order_by":5,"name":"Usama Ramadan Abdelmohsen","email":"","orcid":"","institution":"Deraya University","correspondingAuthor":false,"prefix":"","firstName":"Usama","middleName":"Ramadan","lastName":"Abdelmohsen","suffix":""},{"id":431997678,"identity":"3e91d92e-4268-4e72-af12-1f9ed6320428","order_by":6,"name":"Ashraf N. E. Hamed","email":"","orcid":"","institution":"Minia University Faculty of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Ashraf","middleName":"N. E.","lastName":"Hamed","suffix":""}],"badges":[],"createdAt":"2025-03-15 20:42:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6234677/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6234677/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43450-025-00665-5","type":"published","date":"2025-07-01T15:57:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79632942,"identity":"d6249c15-b68c-4fa7-99ec-2be92687ba60","added_by":"auto","created_at":"2025-04-01 03:39:19","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95943,"visible":true,"origin":"","legend":"\u003cp\u003eMean particle size (left) and zeta potential of nanocrystals suspensions (right).\u003c/p\u003e","description":"","filename":"1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/fa63d6fb13e0179c03818cf7.jpeg"},{"id":79632932,"identity":"fc5cac09-d107-4958-bfd1-59ae52810c65","added_by":"auto","created_at":"2025-04-01 03:39:19","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93462,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of molokheia NCs with power of magnification (15KV×3.500).\u003c/p\u003e","description":"","filename":"2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/1092c58c8910ecc2cd826a8f.jpeg"},{"id":79633542,"identity":"8f1b284a-a251-4b15-8946-570d41fedc23","added_by":"auto","created_at":"2025-04-01 03:47:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":157419,"visible":true,"origin":"","legend":"\u003cp\u003eSTITCH Analysis of Cancer Treatment Targets and Key Active Compounds in \u003cem\u003eCorchorus Olitorius\u003c/em\u003e Extract.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/795e8ba6a1e23b16bd93829a.png"},{"id":79633892,"identity":"37636167-b9ac-4530-ba6c-fcf5bcf464ba","added_by":"auto","created_at":"2025-04-01 03:55:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1609601,"visible":true,"origin":"","legend":"\u003cp\u003ePPI Network Analysis of \u003cem\u003eCorchorus Olitorius\u003c/em\u003eExtract Against MCF-7, HepG2 and Caco-2 Cancer Cell Lines.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/6998a1ea25a94a2047c8e643.png"},{"id":79633545,"identity":"b0ccd46c-391f-4ce9-8ec3-25f254c30afe","added_by":"auto","created_at":"2025-04-01 03:47:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154791,"visible":true,"origin":"","legend":"\u003cp\u003eBubble chart representing Gene Ontology categorizations for \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract: Biological Process, Cellular Component, and Molecular Function, related to anticancer activity.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/db298da2d6c1db20dd944a49.png"},{"id":79632945,"identity":"feb7af0d-63bc-48f7-887b-01fc58aa3c64","added_by":"auto","created_at":"2025-04-01 03:39:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":60869,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG Pathway Analysis Bubble Chart for Target Genes Related to \u003cem\u003eCorchorus olitorius\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/7bbf629e5de6193d20e9f600.png"},{"id":79633541,"identity":"a9eb57d2-b42b-4796-847d-afd009f9cf97","added_by":"auto","created_at":"2025-04-01 03:47:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":290597,"visible":true,"origin":"","legend":"\u003cp\u003ePathways in Cancer Targeted by \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/c8857edc869e56891422596d.png"},{"id":79633550,"identity":"7395bd76-fa3b-4c19-a313-9537db0806b9","added_by":"auto","created_at":"2025-04-01 03:47:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":166237,"visible":true,"origin":"","legend":"\u003cp\u003eThe Colorectal Cancer Pathway illustrating molecular targets and interactions potentially modulated by Corchorus olitorius.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/fb5f5acf830b5f90b1269357.png"},{"id":79632966,"identity":"049f5eef-44be-45be-92d6-adec26e538f9","added_by":"auto","created_at":"2025-04-01 03:39:20","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":423964,"visible":true,"origin":"","legend":"\u003cp\u003eKey hub genes associated with the anticancer activity of \u003cem\u003eCorchorus olitorius\u003c/em\u003eextract in breast cancer, hepatocellular carcinoma and colorectal cancer\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/5dd41e49375947a6a0848dba.png"},{"id":79633546,"identity":"05c361e2-46bd-421e-9fa6-0afe5a7c0384","added_by":"auto","created_at":"2025-04-01 03:47:19","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":5756749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(\u003c/strong\u003eA) Two-dimensional and three-dimensional docking of compound \u003cstrong\u003e11\u003c/strong\u003e within the binding pocket of EGFR (PDB ID: 1M17). (B) Visualization of compound \u003cstrong\u003e15\u003c/strong\u003e orientation in the EGFR binding site (PDB ID: 1M17). (C) Compound \u003cstrong\u003e4\u003c/strong\u003e binding in the active site of BRAF \u003csup\u003eV600E\u003c/sup\u003e (PDB ID: 5JRQ). (D) Compound \u003cstrong\u003e13\u003c/strong\u003e docking in the active site of BRAF \u003csup\u003eV600E\u003c/sup\u003e (PDB ID: 5JRQ).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/f3763a0d634bdbbbd3959d58.png"},{"id":86179821,"identity":"2482fcc8-9394-4fbc-90f6-8ec53e9e97f1","added_by":"auto","created_at":"2025-07-07 16:19:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11518802,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/65babc22-a517-4f00-b25b-cb3bcd663223.pdf"},{"id":79632940,"identity":"03d550c2-cd7a-425f-b23e-a995df865968","added_by":"auto","created_at":"2025-04-01 03:39:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":247679,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/8e80b6500b06941ddc24f935.pdf"},{"id":79633544,"identity":"da96c821-b8a9-4c95-803c-ad48ed1c99f8","added_by":"auto","created_at":"2025-04-01 03:47:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":466942,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6234677/v1/bc88bd9983b9d0668857aa11.docx"}],"financialInterests":"","formattedTitle":"Metabolomic and computational studies for antiproliferative potential of Corchorus olitorius methanol root extract and its nanocrystals","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCarcer is brought on by abnormal processing of genetic information as a result of genetic alterations (mutations) affecting tumor suppressor genes and oncogenes, or altered epigenetic pathways resulting in chromatin structural abnormalities that are either localized or global (Sarris et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Changes in the epigenetic landscapes of cancer cells are largely caused by dysregulated expression of different histone methyltransferases. The pathophysiology of hepatocellular carcinoma (HCC), melanoma, breast, esophageal squamous cell carcinoma, and colorectal cancer has been linked to altered expression or activity of G9a, Setdb1, Smyd2, or PR-SET7 methyltransferases. Similarly, the enhancer of zeste homolog 2 (EZH2) methyltransferase promotes cell proliferation, invasion, and angiogenesis in various epithelial cancer cells (Sarris et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe innovative and developing field of nanobiotechnology includes the study of both biotechnology and nanotechnology. It has numerous uses, particularly in the biomedical industry. Drug nanocrystals are molecular assemblies that can be combined to generate the drug in a crystalline form and are enclosed in a thin stabilizer layer. Nanocrystals technology is a significant alternative to the existing nanocarrier drug delivery technologies in increasing (Goel et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) the bioavailability of medicines that are not readily soluble in water (Rao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). They typically display remarkable physicochemical and biological activities that differ from those of larger particles because of their tiny size (350\u0026ndash;500 nm) (Hanutami and Budiman \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It's interesting to keep in mind that they have shown significant potential for use in cancer treatments (Joseph and Singhvi \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), orthopedics (Abushammala \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and dentistry (Samiei et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nanocrystal formulation can be used to improve drug delivery, targeting and bioavailability. It is possible to administer the drug orally or intravenously and the limited carrier which mostly consists of a thin layer of surfactant may greatly reduce any toxicity (Jahangir et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe current model change in the search for alternative applications regarding existing pharmaceuticals is expected to have implications for the current use of herbal medicines and vegetables, particularly in several traditional societies. Focusing additional effort into this field will promote the beneficial use of plant biodiversity, which is already known to be a major source of food, medicine and other resources used by humans who gain influence from the natural world (Elmaidomy et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mohammed et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ahmed et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; El-Hawwary et al., 2020; Taiwo et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAbout 50\u0026ndash;60 species make up the genus \u003cem\u003ecorchorous\u003c/em\u003e of annual herbs, which is a member of the Tiliaceae family. Only two species \u003cem\u003eC. olitorius\u003c/em\u003e and \u003cem\u003eC. capsularis\u003c/em\u003e are known to produce the best fiber on a commercial basis and are found in warm-temperate and tropical regions of the world (Loumerem and Alercia \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, it has been established that the leaves of \u003cem\u003eC. olitorius\u003c/em\u003e and \u003cem\u003eC. capsularis\u003c/em\u003e have anticancer and antioxidant properties in addition to anti-inflammatory, antiviral, gastroprotective, antinociceptive, analgesic, and antibacterial properties (Abdelrazek et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The main reason of this diversity in biological activities was attributed to the active metabolites, including terpenes, ionone, flavonoids and steroidal derivatives (Ademiluyi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFew studies were reported on \u003cem\u003eC. olitorius\u003c/em\u003e neglected roots. This aggravated us to perform the current study including the phytochemical composition of TMECOR \u003cem\u003evia\u003c/em\u003e untargeted metabolomic analysis. Additionally, the cytotoxic activity of the TMECOR and its prepared nanocrystals was assessed against three different cell lines.\u003c/p\u003e"},{"header":"2. Experimental (Materials and methods)","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant material\u003c/h2\u003e \u003cp\u003eFresh plant roots (500 g) were collected from a farm from Maghagha city, Minia, Egypt in March 2022. The plant was identified by Prof. Nasser Barakat (Minia University-Faculty of Science-Botany and Microbiology Department). Its voucher number (\u003cb\u003eMn-Ph-Cog-060\u003c/b\u003e, Pharmacognosy Department, Faculty of Pharmacy, Minia University).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Chemicals and reagents\u003c/h2\u003e \u003cp\u003eEthanol (Merck, Germany), petroleum ether (Merck, Germany) methanol (98%), DMSO (El-Nasr Company for Pharmaceuticals and Chemicals, Egypt), Insulin (Sigma), acetonitrile and 1% penicillin-streptomycin (Sigma-Aldrich, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Extraction\u003c/h2\u003e \u003cp\u003eThe collected \u003cem\u003eCorchorus olitorius\u003c/em\u003e roots were dried in shade for two weeks. Afterthat, they were grinded into fine powder resulting in a total amount of 55 g. The resulted fine powder was extracted using 99.8% methanol (1 L, 3\u0026times;, 2 weeks interval). TMECOR were then evaporated using rotary evaporator (Heidolph\u0026reg;). The yielded viscous pale green TMECOR (5 g) was kept in the refrigerator until further processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cytotoxic activity\u003c/h2\u003e \u003cp\u003eThe antiproliferative potential was evaluated by the MTT assay of TMECOR and their prepared nanocrystals (Hamed et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mosmann \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), using cancer cell lines previously obtained from the American Type Culture Collection (Manassas, VA, USA). The study focused on MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma) and Caco-2 (colon carcinoma) cell lines. The cells were firstly grown in DMEM high glucose (Invitrogen/Life Technologies, USA) with 10% FBS (Hyclone, USA), 1% penicillin-streptomycin and 10 mg/mL of insulin, at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. They were afterwards moved to 96-well plates at a density of 2.2 and 104 cell/cm\u003csup\u003e2\u003c/sup\u003e and then incubated overnight.\u003c/p\u003e \u003cp\u003eSubsequently, TMECOR dissolved in DMSO was introduced to the grown cells at varying concentrations (20, 30, 40, 50, and 60 mg/mL). The next day, the MTT test was utilized to evaluate the cell viability as described in (Hamed et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Preparation of \u003cem\u003eCorchorus olitorius\u003c/em\u003e nanocrystals\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCorchorus olitorius\u003c/em\u003e nanocrystals were prepared by rotary solvent evaporation and ultrasonication method. Specified amount of TMECOR was dissolved in absolute ethanol (Merck, Germany) and Pet. ether (Merck, Germany) mixture 25:75 ratio. The final amount achieved 50mg/5 mL solution was well sonicated in an ultrasonicate bath at a frequency of 50 kHz (Branson\u0026reg; Ultrasonic Bath,). Tween 80 surfactant (2% by weight) was added. The mixture was then mixed at 1000 rpm for 15 minutes. The resulting solution was placed on a rotary (BUCHI Rotavapor\u0026trade; R-300 Rotary Evaporator) for solvent evaporation. The resulting powder was collected as \u003cem\u003eCorchorus olitorius\u003c/em\u003e nanocrystals and stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Particle size (nm) and size distribution\u003c/h2\u003e \u003cp\u003eThe prepared particles were analyzed for their particle size and size distribution in terms of the average volume diameters and polydispersity index by photon correlation spectroscopy using particle size analyzer Dynamic Light Scattering (DLS) (Zetasizer Nano ZN, Malvern Panalytical Ltd, United Kingdom) at fixed angle of 173\u0026deg; at 25\u0026deg;C. Samples were analyzed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Scanning electron microscopy\u003c/h2\u003e \u003cp\u003eAn SEM instrument (SEM, TESCAN, Warrendale, PA) was utilized for observing the morphology of the prepared nanocrystals. For this, the powder of the nanoparticles was placed on stubs and then coated with a gold layer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. LC-MS Metabolomic analysis\u003c/h2\u003e \u003cp\u003eAt the Faculty of Pharmacy, Fayoum University, metabolomic profiling of TMECOR was carried out using a 6530 Q-TOF LC/MS (Agilent Technologies, Japan) outfitted with an autosampler (G7129A), a Quat. Pump (G7104C), and a Column Comp (G7116A) for chromatographic separation. The volume of injection was 3 \u0026micro;L. The analytes were separated using an Agilent Technologies Zorbax RP-18 column (150 mm \u0026times; 3 mm, dp\u0026thinsp;=\u0026thinsp;2.7 \u0026micro;m). The mass spectra were obtained using ESI in positive and negative ionization modes through a capillary voltage of 4500 V. They were recorded in the range of 50 to 3000 \u003cem\u003em/z\u003c/em\u003e. The drying gas flow and the gas temperature were 8 L/min and 200\u0026deg;C, respectively. The fragmentator and skimmer voltages were established at 130 and 65 V, respectively, while the collision energy was 10 V. A Phenomenex Kinetex 2.6 mm XB-C18 150 \u0026times; 4.6 mm column, maintained at 30\u0026deg;C and connected to a guard column, was filled with 10 mL of samples (1 mg/mL in methanol). The mobile phase consisted of a mixture of LC-MS grade water (A) and acetonitrile (B), each containing formic acid (0.1%). The flow rate of 500 mL/min was adjusted for the gradient elution, going from 5\u0026ndash;20% B in two minutes, 20\u0026ndash;98% B in eighteen minutes, 98% B in five minutes, and lastly 98\u0026ndash;5% B in two minutes. The MS settings used for the HPLC-HESI-HRMS analysis were as follows: capillary temperature (320\u0026deg;C), spray voltage (+\u0026thinsp;3.5 or -2.7 kV), sheath gas (57.50 Pa), sweep gas (3.25 Pa), auxiliary gas (16.25 Pa), probe heater (462.50\u0026deg;C), AGC target (1e6), S-Lens RF (50 cm) resolution (70.000), microscans (1). Mzmine 2.12 was used to obtain a differential analysis of MS data, and ProteoWizard was used to transform the raw data into positive and negative files in the mz/mL format. Finally, metabolite identification was achieved by referring to Dictionary of Natural Products and METLIN databases (DNP, 2020; METLIN, 2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. \u003cem\u003eIn silico\u003c/em\u003e studies\u003c/h2\u003e \u003cp\u003eThe specific action mechanisms of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract and nanocrystals against MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma) and Caco-2 (colon cancer) cell lines were investigated by \u003cem\u003ein silico\u003c/em\u003e protein network and docking analyses. These computational strategies were designed to validate the selectivity and targeting efficacy of identified bioactive compounds within \u003cem\u003eCorchorus olitorius\u003c/em\u003e, particularly against the mentioned cancer types. By integrating the established biological pathways and simulating their interactions with pivotal molecular targets, our objective was to substantiate the experimental biological relevance. This approach aims to provide a detailed molecular insight into how \u003cem\u003eCorchorus olitorius\u003c/em\u003e exerts its therapeutic effects, highlighting the extract and nanocrystals potential for enhanced specificity and effectiveness in treating breast cancer, hepatocellular carcinoma and colon cancer.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1. Investigating Data to Identify Targets Associated with Cancer Cytotoxicity\u003c/h2\u003e \u003cp\u003eWe utilized data from two pivotal resources to pinpoint the genetic targets implicated in the cytotoxicity against MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma) and Caco-2 (colon cancer) cell lines. The Gene Expression Omnibus (GEO) Database, hosted by the National Center for Biotechnology Information ([NCBI](\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/guide/genes-expression/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/guide/genes-expression/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)), provided exhaustive gene expression profiles. This enabled a comprehensive analysis of the transcriptional behavior of genes involved in cancer cytotoxicity, particularly within the contexts of breast cancer, hepatocellular and colon cancers. Simultaneously, the Pharmacogenomics Knowledgebase ([PharmGKB](\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pharmgkb.org/\u003c/span\u003e\u003cspan address=\"https://www.pharmgkb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)) delivered valuable insights into how genetic variations influence the efficacy and toxicity of drugs. This approach aims to deepen our understanding of the genetic underpinnings that contribute to the therapeutic effectiveness of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract and nanocrystals in targeting these specific cancer types.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.9.2. Identifying Targets for Cancer Treatment Efficacy via \u003cem\u003eCorchorus olitorius\u003c/em\u003e with STITCH\u003c/h2\u003e \u003cp\u003eWe employed the STITCH database ([STITCH: chemical association networks](embl.de)) to decode the interactions between protein targets uncovered through the Gene Expression Omnibus (GEO) Database and Pharmacogenomics Knowledgebase (PharmGKB), alongside the active compounds in \u003cem\u003eCorchorus olitorius\u003c/em\u003e. The STITCH database serves as a repository for understanding the complex interactions between chemicals and proteins, amalgamating data from experimental studies, databases and literature to map out how chemicals influence protein activity. This method facilitates the exploration of the relationship between the identified proteins and the bioactive compounds in \u003cem\u003eCorchorus olitorius\u003c/em\u003e. Utilizing data from STITCH, which sheds light on the chemical-protein interplay, we aim to reveal the mechanistic pathways by which \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract and nanocrystals might deliver their therapeutic benefits in targeting the selected cancer cell lines. This approach highlights the critical role of bioinformatics tools in narrowing down the connection between natural product research and targeted cancer therapy. The process initiated with pinpointing the principal active compounds in \u003cem\u003eCorchorus olitorius\u003c/em\u003e. These compounds were then cross-referenced in the STITCH database along with the protein targets identified through the GEO and PharmGKB, generating an intricate network of interactions that potentially underlie the anticancer efficacy of \u003cem\u003eCorchorus olitorius\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.9.3. Construction of a Protein-Protein Interaction (PPI) Network for Cancer-Related Targets\u003c/h2\u003e \u003cp\u003eFollowing the identification of genes implicated in cancer cytotoxicity effects on MCF-7, HepG2 and Caco-2 cell lines from our preliminary analyses, these genes were introduced into the STRING database (Von Mering et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) to develop a Protein-Protein Interaction (PPI) network. This step utilized STRING database to accurately map the interactions between a broad array of proteins, applying a minimum combined score threshold of 0.4 to ensure the relevance of functional interactions highlighted. The network was then visualized and meticulously analyzed using Cytoscape (Shannon et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), focusing particularly on identifying hub genes within the network. These hub genes, pinpointed through the use of CytoHubba plugin in Cytoscape, stand out due to their numerous connections within the network, suggesting their pivotal role in mechanisms related to the cytotoxic effects of \u003cem\u003eCorchorus olitorius\u003c/em\u003e on these specific cancer cell lines. This method underscores the significant value of integrating bioinformatic tools to elucidate the intricate molecular interactions that underpin the anticancer efficacy of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract and nanocrystals.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.9.4. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment for Anticancer Targets\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo delineate the molecular functions (MF), biological processes (BP), cellular components (CC) and critical signaling pathways associated with the genes implicated in the cytotoxic effects on selected cell lines, we conducted Gene Ontology (GO) and pathway enrichment analyses. These analyses were instrumental in understanding the specific roles genes play within biological contexts: BP for their functions in biological activities, CC for their localization within cellular structures, and MF for their precise molecular actions. We employed Shiny GO ([\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go/](http://bioinformatics.sdstate.edu/go/)\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go/](http://bioinformatics.sdstate.edu/go/)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the enrichment analysis, setting a stringent False Discovery Rate (FDR) threshold to ensure accuracy and used Srplot ([\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/en](https://www.bioinformatics.com.cn/en)\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/en](https://www.bioinformatics.com.cn/en)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the visualization of results. This methodology offered an in-depth view of the genes in molecular interactions and biological pathways relevant to the anticancer efficacy of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract and nanocrystals, significantly enriching our comprehension of their functional importance in combating breast cancer, hepatocellular carcinoma and colon cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.9.5. Molecular Docking Analysis\u003c/h2\u003e \u003cp\u003eWithin the scope of our research on the cytotoxic effects of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract and nanocrystals against the selected cancer cell lines, the veracity of our network pharmacology predictions was substantiated through molecular docking analysis. This technique verified the interactions between crucial protein targets and active compounds found in \u003cem\u003eCorchorus olitorius\u003c/em\u003e, underscoring potential synergistic effects for cancer treatment. Utilizing Discovery Studio Client version 16.1.0.15350 (Studio 2008) and the RCSB Protein Data Bank ([\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org/](http://www.rcsb.org/)\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org/](http://www.rcsb.org/)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the process entailed detailed preparation of proteins and ligands for the docking procedure. This essential validation phase bolsters our investigation into viable anticancer agents, in line with our objective to leverage \u003cem\u003eCorchorus olitorius\u003c/em\u003e bioactive compounds for enhanced therapeutic outcomes in targeting breast cancer, hepatocellular carcinoma and colon cancer.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Cytotoxic activity\u003c/h2\u003e \u003cp\u003eThe cytotoxic activity of TMECOR root extract was investigated using the MTT viability assay in comparison to the anticancer drug Staurosporine\u0026reg; as a positive control. The assay was carried out against a number of cancer cell lines (MCF-7, HepG2 and Caco-2). The results exposed that the crude extract presented a high inhibitory activity against (MCF-7, HepG2 and Caco-2) cells, with IC\u003csub\u003e50\u003c/sub\u003e values of 42.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96, 37.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 and 18.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 \u0026micro;g/mL, respectively. While, the nanocrystals showed higher cytotoxic activity especially against HepG2 and Caco-2 with IC\u003csub\u003e50\u003c/sub\u003e value of 23.288\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 and 12.156\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 \u0026micro;g/mL, respectively, showing an enhanced cytotoxic activity for molokheia roots. While MCF-7 showed IC\u003csub\u003e50\u003c/sub\u003e of 62.497\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63 \u0026micro;g/mL as described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCytotoxic activities of TMECOR and its nanocrystals.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e values (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M; \u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMCF-7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHepG2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCaco-2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTMECOR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e42.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e37.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTMECOR nanocrystals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e62.497\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.288\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e12.156\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStaurosporine\u0026reg;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.1454\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Synthesis and characterization of nanocrystals\u003c/h2\u003e \u003cp\u003eThe nanocrystals showed a mean particle size of 370\u0026thinsp;\u0026plusmn;\u0026thinsp;25 nm and a poly dispersity index value of 0.2\u0026ndash;0.5 indicating a narrow size distribution. The data of particle size distribution relative to their PI were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e that enhance the bioavailability, in addition to zeta potential \u0026minus;\u0026thinsp;22\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 mV indicating high stability (Kakran et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yadi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). SEM results Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e approved different particle size distribution and presented that the nanocrystals had grouped quasispheroidal within the sample (Bohlouli et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Metabolomics study\u003c/h2\u003e \u003cp\u003eThe phenolic compounds were predominating among the secondary metabolites that were identified through metabolic profiling of \u003cem\u003eC. olitorius\u003c/em\u003e utilizing the untargeted HPLC-HESI-HRMS metabolomics technique (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). From the (DNP, 2020; METLIN, 2020) databases and from Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3, the mass ion peak at \u003cem\u003em/z\u003c/em\u003e 137.0613 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003efor the anticipated molecular formula C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003e was dereplicated as limonene (\u003cb\u003e1\u003c/b\u003e); a monoterpene derivative previously identified among the volatile compounds of the crushed \u003cem\u003eC. olitorius\u003c/em\u003e leaves and flowers (Driss et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Also, the mass ion peak at \u003cem\u003em/z\u003c/em\u003e 165.9943 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e was described as coumaric acid (\u003cb\u003e2\u003c/b\u003e) a phenolic acid that was earlier identified in molokheia leaves (Driss et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Compound (\u003cb\u003e3\u003c/b\u003e) was identified as vanillic acid in arrangement with the mass ion peak at \u003cem\u003em/z\u003c/em\u003e 167.9792 [M-H]ˉ and the molecular formula C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, which was also previously reported as a phenolic acid metabolite in \u003cem\u003eC. capsularis\u003c/em\u003e flowers (Ademiluyi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, the mass ion peak at \u003cem\u003em/z\u003c/em\u003e 181.1225 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e for the suggested molecular formula C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e was dereplicated as caffeic acid (\u003cb\u003e4\u003c/b\u003e); a phenolic acid in \u003cem\u003eC. capsularis\u003c/em\u003e flowers (Ademiluyi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Compound (\u003cb\u003e5\u003c/b\u003e) was dereplicated as quinic acid, also a phenolic acid derivative corresponding to the molecular formula C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e and the mass ion peak at \u003cem\u003em/z\u003c/em\u003e 191.0668 [M-H]ˉ. It was previously isolated from \u003cem\u003eC. capsularis\u003c/em\u003e flowers (Ademiluyi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Another phenolic acid called ferulic acid (\u003cb\u003e6\u003c/b\u003e), was characterized in consonance with the molecular formula C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and mass ion peak at \u003cem\u003em/z\u003c/em\u003e 195.1034 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e. It was formerly identified from \u003cem\u003eC. capsularis\u003c/em\u003e (Ademiluyi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Compound (\u003cb\u003e7\u003c/b\u003e) was dereplicated as the flavonoid derivative apigenin with a molecular formula of C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e and mass ion peak at \u003cem\u003em/z\u003c/em\u003e 269.2327 [M-H]ˉ,which was previously isolated from \u003cem\u003eC. olitorius\u003c/em\u003e (Yakoub et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A flavonol derivative (\u003cb\u003e8\u003c/b\u003e) was also dereplicated as kaempferol corresponding to the molecular formula C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e and the mass ion peak at \u003cem\u003em/z\u003c/em\u003e 287.1865 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e (Yakoub et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Phytol a diterpene derivative (\u003cb\u003e9\u003c/b\u003e) was reported in both \u003cem\u003eC. olitorius\u003c/em\u003e and \u003cem\u003eC. capsularis\u003c/em\u003e with the molecular formula C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e40\u003c/sub\u003eO and the mass ion peaks at \u003cem\u003em/z\u003c/em\u003e 295.2218 [M-H]ˉ(Mekhael et al., 2019). Another flavone derivative was dereplicated as Cirsiliol (\u003cb\u003e10\u003c/b\u003e) in alignment with the molecular formula C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e and the mass ion peak at \u003cem\u003em/z\u003c/em\u003e 329.2333 [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e (Yakoub et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The mass ion peaks at \u003cem\u003em/z\u003c/em\u003e 353.2129 [M-H]ˉand 361.2477 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e for the predicted molecular formulas C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e and C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e were identified as chlorogenic (\u003cb\u003e11\u003c/b\u003e) (Azuma et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and rosmarinic (\u003cb\u003e12\u003c/b\u003e) (Yakoub et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) acids, respectively. These phenolic acids were formerly isolated from \u003cem\u003eC. olitorius\u003c/em\u003e. Whereas the molecular ion peak at \u003cem\u003em/z\u003c/em\u003e 385.1975 [M-H]ˉ corresponding to the molecular formula C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e was characterized as corchoiononside C (\u003cb\u003e13\u003c/b\u003e), which was earlier isolated from \u003cem\u003eC. olitorius\u003c/em\u003e leaves as an ionone derivative (Yoshikawa et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Additionally, two flavonol derivatives with mass ion peaks at \u003cem\u003em/z\u003c/em\u003e 417.2971 and 609.5092 [M-H]ˉwere dereplicated as kaempferol 3\u003cem\u003eO\u003c/em\u003e-\u003cem\u003eα\u003c/em\u003e-L-arabinopyranoside (Kohda et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) and rutin (Ademiluyi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) respectively. These flavones were reported previously from \u003cem\u003eC. olitorius\u003c/em\u003e leaves.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3. Studies\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e3. \u003cem\u003eIn silico\u003c/em\u003e Studies\u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Therapeutic Targets for Cancer Treatment\u003c/h2\u003e \u003cp\u003eBased on our experimental findings, a dataset of 51 target proteins associated with the cytotoxic effects on breast cancer, hepatocellular carcinoma and colon cancer was meticulously compiled from the NCBI-GEO and PharmGKB databases. These proteins, presented in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, encompass critical targets and biomarkers pivotal to our insights into the anticancer effects of \u003cem\u003eCorchorus olitorius\u003c/em\u003e. This collection not only highlights the importance of these proteins in the realm of cancer remedy but also emphasizes their potential as therapeutic targets, underpinned by the experimental evidence gathered through our research activities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2. STITCH Database Analysis of Cancer Treatment Targets and \u003cem\u003eCorchorus Olitorius\u003c/em\u003e Extract\u003c/h2\u003e \u003cp\u003eIn this detailed investigation, we utilize the STITCH database to meticulously examine the interactions between the principal active components in \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract namely, Chlorogenic acid, Rosmarinic acid and Rutin and the protein targets identified through GEO and PharmGKB analyses Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. This analysis is pivotal in furthering our understanding of the anticancer potential of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract, particularly against MCF-7 (breast cancer), HepG2 (hepatocellular carcinoma) and Caco-2 (colon cancer) cell lines. The study highlights the synergistic action of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract, comparing its comprehensive benefits against those of its individual compounds. The identified synergy suggests complex interactions between the combined bioactive molecules in the extract and proteins associated with cancer, potentially augmenting the extract therapeutic efficacy in cancer treatment. This mechanism of action signifies a promising avenue for enhancing the protective effects against cancer through the bioactive components of \u003cem\u003eCorchorus olitorius\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Construction of the Protein-Protein Interaction (PPI) Network\u003c/h2\u003e \u003cp\u003eIn our investigation into the anticancer efficacy of \u003cem\u003eCorchorus olitorius\u003c/em\u003e, proteins delineated from the research were amalgamated into the STRING database, version 12.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/cgi/input?sessionId=barlI0uOHF46\u003c/span\u003e\u003cspan address=\"https://string-db.org/cgi/input?sessionId=barlI0uOHF46\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), to establish initial PPI networks that reveal their direct and functional relationships. This integration enabled the depiction of the PPI network through Cytoscape software, (version 3.10.1). By employing the Analyzer feature in Cytoscape, we constructed a detailed protein interaction network, incorporating 98 nodes and 1679 interaction linkages, yielding an average node connectivity of 34.3. The complexities of this network are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, underlining the extensive analysis of protein interactions crucial to our research on enhancing the anticancer potential of \u003cem\u003eCorchorus olitorius\u003c/em\u003e against MCF-7, HepG2 and Caco-2 cell lines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Analysis of Overrepresented Gene Ontology Terms in \u003cem\u003eCorchorus Olitorius\u003c/em\u003e Extract\u003c/h2\u003e \u003cp\u003eThe Gene Ontology (GO) enrichment analysis for our study on the anticancer properties of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract was performed using Shiny GO v0.80. Our analysis revealed significant participation of proteins in Biological Processes (BP) such as \"Regulation of apoptotic signaling pathway\", \"Regulation of angiogenesis\", \"Response to estradiol\", \"Regulation of Wnt signaling\" and \"Regulation of cell cycle,\" pivotal in mediating the cytotoxic effects on cancer cells. In the Cellular Component (CC) category, terms like \"Transcription repressor complex\", \"Bcl-2 protein complex\" and \"Nucleoplasm\" were prominent, indicating the subcellular locales impacted by \u003cem\u003eCorchorus olitorius\u003c/em\u003e. Molecular Function (MF) insights, with terms such as \"DNA-binding transcription factor binding\", \"Estrogen receptor binding\" and \"Tyrosine-kinase activity,\" suggest interactions within signaling pathways that may be crucial for the anticancer activity of the extract. These observations are in harmony with our objective to unravel the molecular interactions through which \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract exerts its effects on MCF-7, HepG2 and Caco-2 cell lines. Table S3 provides a detailed account of the statistically significant GO terms and their relevance. The visualizations in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e succinctly illustrate the enriched Gene Ontology (GO) terms across the domains of Biological Process (BP), Cellular Component (CC) and Molecular Function (MF), related to the cytotoxic activities of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Analysis of Dominant KEGG Pathways in Corchorus Olitorius Extract Research\u003c/h2\u003e \u003cp\u003eThe KEGG pathway analysis is a cornerstone of our study, connecting the molecular activity of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extracts and nanocrystals to specific biological pathways impacted in the treatment of MCF-7, HepG2 and Caco-2 cancer cell lines. Our findings, detailed in Table S4, are illustrated in a bubble plot Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and reveal significant enrichment in pathways such as \"Colorectal cancer pathway\", \"Hepatocellular carcinoma pathway\", \"Estrogen signaling pathway\", \"ErbB (Epidermal Growth Factor Receptor) signalling pathway\", \"Wnt signaling pathway\", \"PI3K-Akt signaling pathway\" and \"Apoptosis\". These pathways are essential for deciphering how our treatment strategy may modulate gene expression and protein interactions to exert cytotoxic effects on cancer cells, shedding light on potential mechanisms of action and identifying therapeutic targets for intervention in cancer treatment.\u003c/p\u003e \u003cp\u003eAnticancer Activity.\u003c/p\u003e \u003cp\u003eIn our research, the KEGG pathway analysis of \"Pathways in Cancer\" underscores the potential molecular targets affected by \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract in exerting cytotoxic effects on MCF-7, HepG2 and Caco-2 cell lines. Key pathways such as the PI3K-Akt signaling pathway, Estrogen signaling pathway and apoptosis pathways have been highlighted Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. These pathways are critical in regulating cell proliferation, survival and metabolism, which are processes that can be dysregulated in cancer. The identification of these pathways provides insights into the anticancer mechanisms of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract, suggesting that it may exert its therapeutic effects by modulating pathways commonly involved in the cellular response to cancerous growth.\u003c/p\u003e \u003cp\u003eThe Colorectal Cancer Pathway depicted here reflects the molecular interactions that \u003cem\u003eCorchorus olitorius\u003c/em\u003e extracts and nanocrystals may target to exert cytotoxic effects on colorectal cancer. This pathway, central to the progression from normal epithelium through dysplasia to carcinoma, presents potential targets that could be influenced by our anticancer agents to disrupt the cancer cell cycle and promote apoptosis Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The involvement of key elements such as Wnt signaling, ErbB signalling and PI3K-Akt pathways indicates that the mechanism of action may include inhibiting pathways crucial for tumor growth and survival. Deciphering these interactions is important for the development of targeted therapies to treat colorectal cancer effectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Identification of Crucial Hub Genes\u003c/h2\u003e \u003cp\u003eThe CytoHubba plugin identified crucial hub genes within the PPI network pertinent to the cytotoxic effects of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extracts and nanocrystals on MCF-7, HepG2 and Caco-2 cancer cell lines. In breast cancer, \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract could theoretically affect key pathways mediated by hub genes such as ESR1. ESR1 (Tamoxifen therapy) is crucial for hormone-driven cancer proliferation. BCL2, another hub gene has a significant role in cell death pathways and is being examined in trials with drugs like Venetoclax. For hepatocellular carcinoma (HCC), the extract might impact genes like MYC, which regulates cell cycle and apoptosis but lacks direct inhibitors. It could also influence the AKT1/MTOR pathway, a common oncogenic pathway in HCC that is targeted by Everolimus. Additionally, TP53, frequently mutated in HCC, represents a therapeutic target, with strategies focusing on restoring its function or targeting pathways that are altered due to TP53 mutations. In colorectal cancer (CRC), KRAS, a gene commonly mutated and difficult to target, along with CTNNB1, integral to the Wnt signaling pathway, could be modulated by constituents of the extract. Similarly, the therapeutic approaches could influence TP53 mutations, common in CRC, to reactivate its tumor suppressor functions. Also, ERBB1 (EGFR) Which is targeted by drugs like Erlotinib, is essential for cell growth and survival. Notably, BRAF mutations in CRC are being targeted through combination therapies, such as the FDA-approved encorafenib and cetuximab, pointing to the potential of combination treatments in enhancing efficacy. The analysis of these hub genes Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e highlights a potential pathway-modulating effect of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract, suggesting that it could complement FDA-approved drugs by targeting these genes and their associated pathways, potentially inhibiting cancer progression and promoting cell death.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Molecular Docking\u003c/h2\u003e \u003cp\u003eIn our study, molecular docking serves as a crucial tool to examine the roles of pivotal hubs within the cancer network like EGFR and BRAF as potential targets in the treatment of cancer. Both EGFR and BRAF are well-established in oncogenic signaling pathways, EGFR is a primary receptor tyrosine kinase implicated in tumor growth and spread, while BRAF mutations activate the MAPK/ERK signaling pathway, leading to cancer cell survival and proliferation. Modeling the combined inhibition of EGFR and BRAF may offer a synergistic therapeutic approach, aiming to disrupt concurrent pathways and enhance the efficacy of treatment. The benefits of this dual inhibition strategy include a potential reduction in drug resistance, as cancer cells often activate alternative survival pathways to evade therapy. By simultaneously blocking both EGFR and BRAF pathways, this approach could result in more comprehensive pathway inhibition, reduced cell viability and impeded cancer progression.\u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e5.7.1. Molecular Modeling with EGFR\u003c/h2\u003e \u003cp\u003eThe redocking of EGFR ligand (PDB ID: 1M17) within its active site demonstrated an RMSD of 1.03 \u0026Aring;, confirming the accuracy of our docking methods and a binding energy indicative of a significant interaction potential. For the series of 15 compounds examined against EGFR, detailed in Table S5, specific bioactive compounds from \u003cem\u003eCorchorus olitorius\u003c/em\u003e such as chlorogenic acid 11 and rutin 15 demonstrated superior binding affinities, surpassing the native ligand with docking scores of -7.276 and \u0026minus;\u0026thinsp;8.498 kcal/mol, respectively. Their interactions with key residues-LYS 721, ASP 831 and others-suggest a promising inhibitory action on EGFR, essential for the management of various cancers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e5.7.2. Molecular Modeling with BRAF \u003csup\u003eV600E\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eSimilarly, the BRAF \u003csup\u003eV600E\u003c/sup\u003e crystal structure was sourced from the RCSB Protein Data Bank (PDB ID: 5JRQ). The redocking of the native ligand validated our simulation with an RMSD of 1.623 \u0026Aring; and a favorable binding energy. The assessment of 15 compounds against BRAF \u003csup\u003eV600E\u003c/sup\u003e, as outlined in Table S6, highlighted compounds like caffeic acid 4 and corchoiononside C 13 from \u003cem\u003eCorchorus olitorius\u003c/em\u003e. These compounds showed enhanced docking scores of -4.794 and \u0026minus;\u0026thinsp;7.471 kcal/mol, respectively, indicating strong binding capabilities. The significant interactions with crucial amino acids such as CYS 532 and LYS 483 support their potential role as dual inhibitors, targeting both EGFR and BRAF pathways. The combined dual inhibition of EGFR and BRAF by compounds found in \u003cem\u003eCorchorus olitorius\u003c/em\u003e could offer several benefits, including a broader inhibition of cancer cell signaling pathways, potentially leading to reduced tumor growth and resistance to single-target therapies. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e would visually illustrate these promising interactions, showcasing the potential of these compounds as dual inhibitors that may enhance the efficacy of existing cancer therapies.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":" \u003cp\u003eThe work was studied for the first time on \u003cem\u003eC. olitorius\u003c/em\u003e roots. It uncovered the marked \u003cem\u003ein vitro\u003c/em\u003e cytotoxic inhibitory aptitude of TMECOR, against breast, liver and colon cell lines. The previous studies are in the same line with my results, which the gold nanoparticle molokheia leaves extract tested against breast, liver and colon cancer cell lines also. The results ensured the cytotoxic effect of it with IC\u003csub\u003e50\u003c/sub\u003e 12.2, 10.3 and 11.2 \u0026micro;g/mL, respectively (Ismail et al., 2023).\u003c/p\u003e \u003cp\u003eFurthermore, the prepared nanocrystals notably enhanced the cytotoxic activity of TMECOR, particularly against HepG2 (37%) and Caco-2 (34%). According to the US NCI guidelines, these data remarkably reflect the strong (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;20 mg/mL) to moderate (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;21\u0026ndash;50 mg/mL) cytotoxic potential of TMECOR towards the aforementioned cell lines a result that indicates the cytotoxic properties of such ignored plant part, which is commonly considered as a vegetable waste product.\u003c/p\u003e \u003cp\u003eTo determine which of active constituents are responsible for this activity, LC-HRMS-based metabolomics analysis was studied. They belong to various chemical classes, including flavonoids, terpenes, ionones and phenolic acids, illustrated in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Figure S6. This metabolic profile distinctly explicates the cytotoxic potential of TMECOR. For instance, phytol (\u003cb\u003e9\u003c/b\u003e) (Shariare et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), apigenin (\u003cb\u003e7\u003c/b\u003e) (Imran et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), rutin (\u003cb\u003e15\u003c/b\u003e) (Caparica et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Cirsiliol (\u003cb\u003e10\u003c/b\u003e) (Jia et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and kaempferol (\u003cb\u003e8\u003c/b\u003e) (Imran et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) have all been previously reported to exert potent cytotoxic effects.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe current study focused on \u003cem\u003ein vitro\u003c/em\u003e cytotoxic activites against three different cell lines (MCF-7, HepG2 and Caco-2) of TMECOR as a natural source. Interestingly, the nanocrystals gave considerable cytotoxic activity in liver and colon cell lines in comparison with Staurosporine\u0026reg;. Consequently, molokheia roots may be used to develop anticancer herbal drug in the future. This study introduces a comprehensive in silico methodology that intertwines network pharmacology with molecular docking to delineate the anticancer mechanisms of compounds derived from \u003cem\u003eCorchorus olitorius\u003c/em\u003e against MCF-7, HepG2, and Caco-2 cancer cell lines. Through the construction of a protein-protein interaction (PPI) network, pivotal hub genes such as EGFR, BRAF, MYC, and TP53 were highlighted, playing a critical role in oncogenic signaling. Molecular docking simulations predicted the binding efficacy of \u003cem\u003eCorchorus olitorius\u003c/em\u003e compounds at these proteins' active sites. Utilizing crystal structures of EGFR (PDB ID: 1M17) and BRAF V600E (PDB ID: 5JRQ), our docking approach was substantiated, achieving RMSDs that ensure reliable predictions. The results showcased compounds, notably chlorogenic acid and rutin, with binding energies exceeding those of native ligands, pointing to a substantial therapeutic potential. These computational insights open a pathway for the experimental exploration and crafting of novel anticancer agents aimed at targeting key elements in cancer pathways.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure statement:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e:\u0026nbsp;Not applicable\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e All data generated or analyzed during this study are included in this article and its supplementary information files\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eEthical approval: \u003c/span\u003e\u003c/strong\u003e\u003cspan dir=\"LTR\"\u003eNot applicable.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eCompeting interests: \u003c/span\u003e\u003c/strong\u003e\u003cspan dir=\"LTR\"\u003eThe authors declare no competing interests.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"LTR\"\u003eMarwa A.M. Abdelrazek \u003c/span\u003e\u003c/strong\u003e\u003cspan dir=\"LTR\"\u003eand\u003cstrong\u003e\u0026nbsp;Miada F. Abdelwahab\u003c/strong\u003e: writing original draft, visualization, validation, investigation, formal analysis, data curation. \u003cstrong\u003eSoad A. Mohamed\u003c/strong\u003e: methodology, conceptualization. \u003cstrong\u003eHesham A. Abo Zeid\u003c/strong\u003e: validation, software, methodology, conceptualization. \u003cstrong\u003eUsama R. Abdelmohsen\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;Ashraf N.E Hamed\u003c/strong\u003e: reviewing, supervision, methodology, investigation, data curation, conceptualization.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowlegment:\u003c/strong\u003e The authors acknowledge EKB (Egyptian Knowledge Bank) Minia University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdemiluyi AO, Oboh G, Aragbaiye FP, Oyeleye SI, Ogunsuyi OB (2015) Antioxidant properties and in vitro α-amylase and α-glucosidase inhibitory properties of phenolics constituents from different varieties of Corchorus spp. J Taibah Univ Med Sci 10(3):278\u0026ndash;287\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbushammala H (2019) On the para/ortho reactivity of isocyanate groups during the carbamation of cellulose nanocrystals using 2, 4-toluene diisocyanate. Polymers 11:1164\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed SS, Fahim JR, Youssif KA, AboulMagd AM, Amin MN, Abdelmohsen UR, Hamed ANE (2022) Metabolomics of the secondary metabolites of Ammi visnaga L. roots (family Apiaceae) and evaluation of their biological potential. South Afr J Bot 149:860\u0026ndash;869\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzuma K, Nakayama M, Koshioka M, Ippoushi K, Yamaguchi Y, Kohata K, Yamauchi Y, Ito H, Higashio H (1999) Phenolic antioxidants from the leaves of \u003cem\u003eCorchorus olitorius\u003c/em\u003e L. J Agric Food Chem 47:3963\u0026ndash;3966\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdelrazek MAM, Abdelwahab MF, Abdelmohsen UR, Hamed ANE (2022) Pharmacological and phytochemical biodiversity of \u003cem\u003eCorchorus olitorius\u003c/em\u003e. RSC Ad 12:35103\u0026ndash;35114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBohlouli S, Jafarmadar Gharehbagh F, Dalir Abdolahinia E, Kouhsoltani M, Ebrahimi G, Roshangar L, Imani A, Sharifi S, Maleki Dizaj S (2021) Preparation, Characterization, and Evaluation of Rutin Nanocrystals as an Anticancer Agent against Head and Neck Squamous Cell Carcinoma Cell Line. \u003cem\u003eJ. Nanomater.\u003c/em\u003e 2021:9980451\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaparica R, J\u0026uacute;lio A, Ara\u0026uacute;jo MEM, Baby AR, Fonte P, Costa JG, de Santos T (2020) Anticancer activity of rutin and its combination with ionic liquids on renal cells. Biomolecules 10(2):233\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDriss D, Kaoubaa M, Mansour RB, Kallel F, eddine Abdelmalek B, and Chaabouni S. E (2016) Antioxidant, antimutagenic and cytotoxic properties of essential oil from \u003cem\u003eCorchorus olitorius\u003c/em\u003e L. flowers and leaf. Free Radic Antioxid 6(1):34\u0026ndash;43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Hawwary SS, Saber FR, Almaksoud A, Elimam HM, Sayed H, Abdelmohsen AM, U. R (2022) Cytotoxic potential of three Sabal species grown in Egypt: a metabolomic and docking-based study. Nat Prod Res 36(4):1109\u0026ndash;1114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElmaidomy AH, Zawily E, Salem A, Altemani AK, Algehainy FH, Altemani NA, Rateb AH, Abdelmohsen ME, Shady UR, N. H (2023) New cytotoxic dammarane type saponins from Ziziphus spina-christi. Sci Rep 13(1):20612\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoel H, Siddiqui L, Mahtab A, Talegaonkar S (2022) Fabrication design, process technologies, and convolutions in the scale-up of nanotherapeutic delivery systems in Nanoparticle Therapeutics. \u003cem\u003eElsevier\u003c/em\u003e. 2022:47\u0026ndash;131\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanutami NPB, Budiman A (2017) Review Artikel: Penggunaan Teknologi Nano Pada Formulasi Obat Herbal. Farmaka 15(2):30\u0026ndash;42\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamed ANE, Abdelaty NA, Attia EZ, Amin MN, Ali TFS, Afifi AH, Abdelmohsen UR, Desoukey SY (2022) Antiproliferative potential of \u003cem\u003emoluccella laevis\u003c/em\u003e L. aerial parts family lamiaceae (labiatae), supported by phytochemical investigation and molecular docking study. Nat Prod Res 36:1391\u0026ndash;1395\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsmail EH, Saqer AMA, Assirey E, Naqvi A, Okasha RM (2018) Successful Green Synthesis of Gold Nanoparticles using a \u003cem\u003eCorchorus olitorius\u003c/em\u003e Extract and Their Antiproliferative Effect in Cancer Cells. Mol Sci 19(9):2612\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImran M, Gondal TA, Atif M, Shahbaz M, Qaisarani TB, Mughal MH, Salehi B, Martorell M, Sharifi-Rad J (2020) Apigenin as an anticancer agent. Phytother Res 34(8):1812\u0026ndash;1828\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImran M, Salehi B, Sharifi-Rad J, Aslam Gondal T, Saeed F, Imran A, Shahbaz M, Fokou PVT, Arshad MU, Khan H, Guerreiro SG, Martins N, Estevinho LM (2019) Kaempferol: A key emphasis to its anticancer potential. Molecules 24(12):2277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoseph E, Singhvi G (2019) Multifunctional nanocrystals for cancer therapy: a potential nanocarrier. Nanomater. \u003cem\u003eDrug Deliv.Ther\u003c/em\u003e. 2019:91\u0026ndash;116\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJahangir MA, Imam SS, Muheem A, Chettupalli A, Al-Abbasi F, Nadeem MS, Kazmi I, Afzal M, Alshehri S (2020) Nanocrystals: characterization overview, applications in drug delivery, and their toxicity concerns. \u003cem\u003eJ. Pharm. Innov.\u003c/em\u003e 2020:1\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia X, Huang C, Hu Y, Wu Q, Liu F, Nie W, Chen X, Li X, Dong Z, Liu K (2021) Cirsiliol targets tyrosine kinase 2 to inhibit esophageal squamous cell carcinoma growth \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. J Exp Clin Cancer Res 40(1):1\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKakran M, Shegokar R, Sahoo NG, Gohla S, Li L, M\u0026uuml;ller RH (2012) Long-term stability of quercetin nanocrystals prepared by different methods. J Pharm Pharmacol 64:1394\u0026ndash;1402\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohda H, Tanaka S, Yamaoka Y, Morinaga S, Ohhara Y (1994) Constituents of Corchorus olitorius L. Nat Med 48(3):213\u0026ndash;214\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoumerem M, Alercia A (2016) Descriptors for jute (\u003cem\u003eCorchorus olitorius\u003c/em\u003e L). Genet Resour Crop Evol 63(7):1103\u0026ndash;1111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55\u0026ndash;63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammed MHH, Hamed ANE, Elhabal SF, Mokhtar FA, Abdelmohsen UR, Fouad MA, Kamel MS (2023) Metabolic profiling and cytotoxic activities of ethanol extract of Dypsis leptocheilos aerial parts and its green synthesized silver nanoparticles supported by network pharmacology analysis. South Afr J Bot 161:648\u0026ndash;665\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMekhael Maged KG, Hassan A, Hanna A, Simon A, T\u0026oacute;th G, Duddeck H (2019) Phytochemical investigation of \u003cem\u003eCorchorus olitorius\u003c/em\u003e and \u003cem\u003eCorchorus capsularis\u003c/em\u003e (family \u003cem\u003eTiliaceae\u003c/em\u003e) that grow in Egypt. Pharm J 18:123\u0026ndash;134\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao CNR, Thomas PJ, Kulkarni GU (2007) Synthesis of nanocrystals. Nanocrystals: Synthesis, Properties and Applications. 25\u0026ndash;68\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarris M, Nikolaou K, Talianidis I (2014) Context-specific regulation of cancer epigenomes by histone and transcription factor methylation. Oncogene 33(10):1207\u0026ndash;1217\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarris ME, Moulos P, Haroniti A, Giakountis A, Talianidis I (2016) Smyd3 is a transcriptional potentiator of multiple cancer-promoting genes and required for liver and colon cancer development. Cancer Cell 29(3):354\u0026ndash;366\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShariare MH, Noor HB, Khan JH, Uddin J, Ahamad SR, Altamimi MA, Alanazi FK, Kazi M (2021) Liposomal drug delivery of Corchorus olitorius leaf extract containing phytol using design of experiment (DoE): In-vitro anticancer and in-vivo anti-inflammatory studies. Colloids Surf B 199:111543\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamiei M, Arablouye Moghaddam F, Dalir Abdolahinia E, Ahmadian E, Sharifi S, Maleki Dizaj S (2022) Influence of Curcumin Nanocrystals on the Early Osteogenic Differentiation and Proliferation of Dental Pulp Stem Cells. \u003cem\u003eJ. Nanomater.\u003c/em\u003e 2022, 1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13(11):2498\u0026ndash;2504\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaiwo BJ, Taiwo GO, Olubiyi OO, Fatokun AA (2016) Polyphenolic compounds with anti-tumour potential from Corchorus olitorius (L.) Tiliaceae, a Nigerian leaf vegetable. Bioorg Med Chem Lett 3:1\u0026ndash;7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVon Mering C, Jensen LJ, Snel B, Hooper SD, Krupp M, Foglierini M, Jouffre N, Huynen MA, Bork P (2005) STRING: known and predicted protein\u0026ndash;protein associations, integrated and transferred across organisms. Nucleic Acids Res 33(suppl1):D433\u0026ndash;D437\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadi M, Mostafavi E, Saleh B, Davaran S, Aliyeva I, Khalilov R, Nikzamir M, Nikzamir N, Akbarzadeh A, Panahi Y (2018) Current developments in green synthesis of metallic nanoparticles using plant extracts: a review. Artif Cells Nanomed Biotechnol 46:S336\u0026ndash;S343\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYakoub ARB, Abdehedi O, Jridi M, Elfalleh W, Nasri M, Ferchichi A (2018) Flavonoids, phenols, antioxidant, and antimicrobial activities in various extracts from Tossa jute leave (\u003cem\u003eCorchorus olitorus\u003c/em\u003e L). Ind Crops Prod 118:206\u0026ndash;213\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshikawa M, Shimada H, Saka M, Yoshizumi S, Yamahara J, Matsuda H (1997) Medicinal foodstuffs. V. Moroheiya. Absolute stereostructures of corchoionosides A, B, and C, histamine release inhibitors from the leaves of Vietnamese \u003cem\u003eCorchorus olitorius\u003c/em\u003e L. (\u003cem\u003eTiliaceae\u003c/em\u003e). \u003cem\u003eChem. Pharm. Bull\u003c/em\u003e. 45:464\u0026ndash;469\u003c/span\u003e\u003c/li\u003e\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":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Corchorus olitorius, Cytotoxicity, HPLC-HESI-HRMS analysis, Molecular Docking, Nanocrystals, Tiliaceae","lastPublishedDoi":"10.21203/rs.3.rs-6234677/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6234677/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eCorchorus olitorius\u003c/em\u003e L. Moench (Molokheia) is a common edible plant that is rich in terpenoids and flavonoids. Later, for the first time, this article was planned to study the potential of \u003cem\u003eC. olitorius\u003c/em\u003e roots and their nanocrystals against breast cancer (MCF-7), hepatocellular carcinoma (HepG2) and colon cancer (Caco-2) cell lines. Generally, the total methanolic extract of \u003cem\u003eC. olitorius\u003c/em\u003e roots (TMECOR) inhibited growth of MCF-7, HepG2 and Caco-2 cells with IC\u003csub\u003e50\u003c/sub\u003e values of 42.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96, 37.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 and 18.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 \u0026micro;g/mL, respectively. Whereas, the nanocrystals displayed significantly higher antiproliferative potential especially against HepG-2 and Caco-2 with IC\u003csub\u003e50\u003c/sub\u003e value of 23.288\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 and 12.156\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 \u0026micro;g/mL, respectively, While MCF-7 showed IC\u003csub\u003e50\u003c/sub\u003e of 62.497\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63 \u0026micro;g/ mL. To discover which of these compounds is responsible for this activity, metabolomic analysis of TMECOR was studied. It revealed presence of a diversity of metabolites (\u003cb\u003e1\u0026ndash;15\u003c/b\u003e) largely dominated by phenolic compounds. \u003cem\u003eIn silico\u003c/em\u003e network analysis and molecular docking to explore the anticancer efficacy of \u003cem\u003eCorchorus olitorius\u003c/em\u003e extract against MCF-7, HepG2, and Caco-2 cancer cell lines. Central hub genes implicated in key oncogenic pathways, such as EGFR and BRAF, were pinpointed and subjected to rigorous docking protocols, using the crystal structures of EGFR (PDB ID: 1M17) and BRAF \u003csup\u003eV600E\u003c/sup\u003e (PDB ID: 5JRQ). The docking outcomes highlight significant binding affinities for compounds within the extract, notably Chlorogenic acid and Rutin, implying their potential as dual inhibitors for these critical cancer pathways. These findings offer a foundational understanding for subsequent empirical studies and the potential crafting of novel cancer therapies.\u003c/p\u003e","manuscriptTitle":"Metabolomic and computational studies for antiproliferative potential of Corchorus olitorius methanol root extract and its nanocrystals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 03:39:14","doi":"10.21203/rs.3.rs-6234677/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-21T09:27:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-21T09:20:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2025-03-20T18:42:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-19T10:27:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2025-03-17T22:54:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"adc76b44-1024-4d65-b2bc-8340ade2b6f1","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:11:24+00:00","versionOfRecord":{"articleIdentity":"rs-6234677","link":"https://doi.org/10.1007/s43450-025-00665-5","journal":{"identity":"revista-brasileira-de-farmacognosia","isVorOnly":false,"title":"Revista Brasileira de Farmacognosia"},"publishedOn":"2025-07-01 15:57:55","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-01 03:39:14","video":"","vorDoi":"10.1007/s43450-025-00665-5","vorDoiUrl":"https://doi.org/10.1007/s43450-025-00665-5","workflowStages":[]},"version":"v1","identity":"rs-6234677","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6234677","identity":"rs-6234677","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}