Growth inhibitory action of Red Trapa natans shell methanolic extract (RTNSME) in Ehrlich ascites carcinoma (EAC) cells in Swiss Albino Mice by inducing apoptosis through inactivation of NFkB

Growth inhibitory action of Red Trapa natans shell methanolic extract (RTNSME) in Ehrlich ascites carcinoma (EAC) cells in Swiss Albino Mice by inducing apoptosis through inactivation of NFkB | 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 Growth inhibitory action of Red Trapa natans shell methanolic extract (RTNSME) in Ehrlich ascites carcinoma (EAC) cells in Swiss Albino Mice by inducing apoptosis through inactivation of NFkB Saharia Yeasmin Asha, Mst. Ayesha Siddika, Mahmud Ismail, Arifur Rahman, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6950088/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Trapa natans L., commonly known as water chestnut, is a small aquatic herb belonging to the family Trapaceae . Its fruit is valued for both nutritional and pharmaceutical properties. This study investigates the anticancer potential of the methanolic extract of red Trapa natans shell (RTNSME) against Ehrlich ascites carcinoma (EAC) cells. RTNSME demonstrated significant anticancer activity, inhibiting EAC cell growth by 76.05% compared to the untreated control. Fluorescence microscopy of DAPI-stained cells revealed classic apoptotic features such as DNA fragmentation, nuclear condensation, cell shrinkage, and membrane alterations in RTNSME-treated cells. These findings were supported by RT-PCR analysis of apoptosis-related genes. RTNSME upregulated the expression of p53 and Bax , while downregulating the anti-apoptotic gene Bcl-2 . Additionally, RTNSME suppressed the activity of the nuclear factor kappa B (NF-κB), further indicating its role in promoting apoptosis. GC-MS analysis of RTNSME identified 35 phytochemical constituents, including known bioactive and anticancer compounds such as canthaxanthin, dipyridamole, and colchiceinamide. Molecular docking studies suggested that canthaxanthin exhibits the strongest binding affinity and is likely the most potent compound against EAC cells. Overall, this study demonstrates that RTNSME exerts significant anticancer effects on EAC cells, primarily through NF-κB inactivation and induction of apoptosis. Trapa natans L apoptosis NF-kB GC/MS molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cancer remains one of the deadliest diseases worldwide, claiming the lives of over six million people annually ( 1 ). While both synthetic drugs and natural compounds with anticancer properties have shown promise in cancer treatment ( 2 , 3 ), current chemotherapeutic agents are often expensive and associated with severe side effects. Therefore, the development of cost-effective and safer therapeutic alternatives is a critical priority in cancer research ( 4 ). Due to their lower toxicity, wide availability, and bioactivity, natural compounds are increasingly being explored for their anticancer potential ( 5 ). Trapa natans L., commonly known as water chestnut or water caltrop, is an annual aquatic floating herb belonging to the family Trapaceae ( 6 ). It typically grows in lakes, ponds, ditches, and other stagnant water bodies, and is widely cultivated in tropical, subtropical, and temperate regions of the world ( 7 ). Two primary varieties of Trapa natans are recognized. Various extracts of its fruit have been reported to exhibit a broad spectrum of pharmacological properties, including antimicrobial ( 8 ), analgesic ( 9 ), anti-inflammatory ( 10 ), antidiabetic ( 11 ), antioxidant, antiulcer, and antifungal activities ( 12 ). Previous studies have also demonstrated the anticancer potential of Trapa natans in several human cancer cell lines, such as human colon cancer (Colo-205), human ductal breast carcinoma (T47D), and human breast adenocarcinoma (MCF-7) cells ( 13 ). However, its anticancer activity against Ehrlich ascites carcinoma (EAC) cells has not yet been investigated. Our earlier study revealed that the methanolic extract of red Trapa natans shell (RTNSME) contains a high concentration of phytochemicals and exhibits strong antioxidant activity ( 14 ). Building on these findings, the present study aims to evaluate the anti-proliferative effects of RTNSME on EAC cells and to elucidate its underlying molecular mechanisms, with a focus on apoptosis and NF-κB signaling. Materials and Methods Preparation of Red Trapa natans Shell Methanolic Extract (RTNSME) Mature Trapa natans fruits were procured from a local market in Rajshahi, Bangladesh. The species was taxonomically authenticated by a specialist in the Department of Botany, University of Rajshahi. The red-colored fruits were thoroughly washed with distilled water to remove surface impurities. The shells were manually separated, shade-dried, and then ground into a fine powder using a mechanical grinder. The powdered shell material was stored in an airtight container at room temperature until extraction. The extraction process followed the method described by Luqman A. Olayaki et al. (2014) ( 15 ), with slight modifications. Approximately 150 grams of the powdered shell were soaked in 500 mL of methanol and kept under continuous stirring for 24 hours at room temperature. After initial extraction, the mixture was filtered through Whatman No. 1 filter paper, and an additional 100 mL of methanol was added to the residue for re-extraction. The combined filtrates were concentrated using a rotary evaporator at 37°C to remove the solvent. The resulting dried extract (RTNSME) was stored at 4°C in a refrigerator for further use in experiments. Animals Swiss albino mice (6 to 8 weeks old, weighing 25 ± 4 g) were obtained from the Department of Pharmacy, Jahangirnagar University, Bangladesh. The animals were housed in standard laboratory conditions with a 12-hour light/dark cycle and maintained at room temperature with proper ventilation. Six mice were kept per cage. All animals were provided with standard laboratory chow and drinking water ad libitum throughout the experimental period. Ethical Clearance All experimental procedures involving animals were conducted in accordance with ethical guidelines and approved by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) for experimentation on Animals, Humans, Microbes, and Living Natural Sources, Institute of Biological Sciences, University of Rajshahi, Bangladesh (Approval No. 117/320 (47)/IAMEBBC/IBSc). Cell Line Ehrlich ascites carcinoma (EAC) cells were kindly provided by the Department of Pharmacy, Jahangirnagar University, Bangladesh. All procedures involving biosafety and animal handling were conducted in compliance with the guidelines of the Institute of Biological Sciences, University of Rajshahi, Bangladesh. Estimation of Tumor Weight Tumor weight was estimated following the method described by ( 16 ). Experimental mice were divided into groups, each consisting of five mice. On day 0, each mouse was intraperitoneally inoculated with 1.0 × 10⁶ EAC cells. After 24 hours, the mice were treated with RTNSME at doses of 25, 50, and 100 mg/kg body weight, respectively, for 20 consecutive days. Tumor weight was measured every 48 hours throughout the treatment period. Cell Growth Inhibition Determination Cell growth inhibition was assessed as previously described ( 17 ). On day 0, mice were intraperitoneally inoculated with 1 × 10⁶ EAC cells. After 24 hours, RTNSME was administered at doses of 25, 50, and 100 mg/kg body weight per mouse per day to the respective treatment groups for five consecutive days. A control group received only normal saline. On day 6, all animals were sacrificed, and the ascitic fluid was collected. EAC cells were harvested and counted using a hemocytometer to evaluate the extent of cell growth inhibition. DAPI Staining for Morphological Appearance and Nuclear Damage of EAC Cells The induction of apoptosis in EAC cells was assessed as previously described ( 18 ). Both treated and untreated control EAC cells were analyzed using a fluorescence inverted microscope (Olympus IX71, Japan). Initially, the cells were collected and stained with 25 µL of DAPI solution (1 mg/mL) at 37°C for 20 minutes. After staining, the cells were washed three times with PBS in the dark to remove excess dye. Morphological changes, including nuclear condensation and fragmentation, were observed under the fluorescence microscope to assess nuclear damage and apoptotic characteristics. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Total RNA was extracted from cells using an RNA extraction kit according to the manufacturer’s instructions. cDNA was synthesized via reverse transcription PCR following the protocol provided with the kit. The primers used for the experiment are listed in Table 1 . The PCR products were subjected to electrophoresis on a 1.0% agarose gel, which was then stained with ethidium bromide. The gel was visualized under a UV transilluminator to assess the amplification of target genes. Table 1 The sequence of primers used for PCR amplification. Gene name Primer sequence references GAPDH Forward: (5′-GTGGAAGGACTCATGACCACAG-3′) Reverse: (5′-CTGGTGCTCAGTGTAGCCCAG-3′) p53 Forward: (5′-CACAAAAACAGGTTAAACCCAG-3′) Reverse: (5′-AGCACATAGGAGGCAGAGAC-3′) Bcl-2 Forward: (5′-GTGGAGGAGCTCTTCAGGGA-3′) Reverse: (5′-AGGCACCCAGGGTGATGCAA-3′) Bax Forward: (5'-GGCCCACCAGCTCTGAGCAGA-3') Reverse: (3'-GCCACGTGGGCGTCCCAAAGT-5') NF-kB Forward: (5'-AACAAAATGCCCCACGGTTA-3') Reverse: (3'-GGGACGATGCAATGGACTGT-5') Gas Chromatography-Mass Spectrometry (GC-MS) Analysis GC-MS analysis of RTNSME was performed using a Varian GC spectrophotometer (Model CP-3800, USA) coupled with a Varian Saturn-2200 mass spectrometer. The system was equipped with a flame ionization detector and a VF-5 ms capillary column (30 m × 0.25 mm, 0.25 µm). The instrument was operated in electron impact mode at 70 eV ionization voltage, with the injector temperature set at 250°C and the detector temperature set at 280°C. A 1 µL sample was injected with helium as the carrier gas at a flow rate of 1 mL/min. The column temperature was initially set to 40°C for 1 minute, then ramped to 310°C at a rate of 10°C/min, where it was held for 10 minutes. Chemical compounds were identified and quantified by comparing their mass spectra with the NIST 05 Library database. Molecular Docking Simulation Molecular docking simulations were performed to explore the potential interactions between BAX, BCL2, p53, and NF-κB proteins with the compounds Canthaxanthin, Colchiceinamide, and Dipyridamole. The 3D structures of BAX, BCL2, p53, and NF-κB proteins were retrieved from the Protein Data Bank (PDB) ( https://www.rcsb.org/ ) with the following PDB codes: 5W62 (BAX), 2KUA (BCL2), 2IOI (p53), and 1MY5 (NF-κB). To eliminate potential interference from water molecules and heteroatoms, these were removed using Biovia Discovery Studio 2021, reducing the possibility of undesired interactions with the receptor proteins. The resulting PDB files were then converted into pdbqt format for docking analysis. The chemical structures of Canthaxanthin (CID: 5281227), Colchiceinamide (CID: 18397), and Dipyridamole (CID: 3108) were downloaded in 3D SDF format from the PubChem database ( https://pubchem.ncbi.nlm.nih.gov/ ) , minimized, and converted into pdbqt format using the PyRx tool. Docking studies were carried out using PyRx with Autodock Vina to predict the binding affinity and interaction between the proteins and compounds. The protein-ligand interactions, including hydrogen bonds, pi-alkyl bonds, hydrophobic interactions, and other relevant bonding types, were analyzed and visualized using BIOVIA Discovery Studio 2021. Statistical Analysis Experiments were performed in triplicates, and the data are presented as the mean ± standard deviation (SD). Statistical analysis was conducted using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test to compare group means. All analyses were performed using SPSS software (version 16). A p-value of < 0.05 was considered statistically significant. Results Growth inhibition of EAC cell treated with RTNSME Tumor weight reduction EAC cells growth inhibition was first determined by average tumor weight. RTNSME reduced tumor weight in EAC-bearing mice significantly. Tumor weight of untreated control EAC cells was increased over time but treatment with RTNSME at the concentration of 25, 50 and 100 mg/kg RTNSME significant reduced of tumor weight (Fig. 1 A). Cell growth inhibition Treatment with RTNSME resulted in 26.21, 38.15 and 76.05% of cell growth inhibition compared to control at 25, 50 and 100 mg/kg body weight doses respectively. Effects of RTNSME on cell growth inhibition are shown in Table 2 and Fig. 1 B. Table 2 Cell growth inhibition Name of the experiment Nature of Drug Dose in mg/kg/day body weight (i.p) No of EAC cells in mouse on Day 6 after tumour cell inoculation % of cell growth inhibition Control(EAC cell bearing mice) - (2.46 ± .12) x 10 6 d - RTNSME 25 ( 1.81 ± .12) x 10 6 c 26.21 50 (1.52 ± .12) x 10 6 b 38.15 100 (0.59 ± .20) x 10 6 a 76.05 Effect of RTNSME on EAC cell growth inhibition ( in vivo ). Data were expressed as mean ± SD (n = 3). Mean with different lowercase letters are significantly different at P < 0.05 by Duncan’s multiple-range test. Induction of cancer cell apoptosis Changes of EAC cell morphology RTNSME treated EAC cells showed condensed and fragmented DNA in nuclei as shown in Fig. 2 A whereas Morphological changes of EAC untreated cells were found round, regular in control cell whereas. Modulation of apoptotic gene expression The mRNA expressions of p53, Bcl-2, Bax were investigated by using specific primers. Red TNSME was down regulated the mRNA expression of Bcl-2 when compared with untreated control. On the other hand, the mRNA expression of Bax, p53 was up regulated when treated with RTNSME. GAPDH expression was observed equally both in RTNSME treated EAC cells and untreated control EAC cells Fig. 2 B. Inactivation of NF-κB gene expression NF-κB gene in EAC cells treated with RTNSME was determined by RT-PCR Fig. 3 . RTNSME reduced the activation of NF-κB compared to untreated control cells. This results might reduce cell growth and induction of apoptosis. Chemical composition of RTNSME GC/MS chromatogram and list of compounds revealed that RTNSME consists of 35 compounds as shown in Table 3 and Fig. 4 respectively. Table 3 Chemical composition of RTNSME analyzed by GC SL Compound Name MW Formula RT Area % 1 Benzyloxy(butyl)dimethylsilane 222 C13H22OSi 5.46 1.4 2 Isobutyl ether 130 C8H18O 6.06 6.16 3 1-(Nitromethyl)-cyclohexanol 159 C7H13NO3 5.26 2.26 4 Ethanol, 2-butoxy- 118 C6H14O2 6.39 2.50 5 1,2,3-Propanetriol, monoacetate 134 C5H10O4 8.14 1.65 6 Benzyl Alcohol 108 C7H8O 8.48 1.50 7 1,1,2-Triacetoxyethane 204 C8H12O6 8.57 3.81 8 Cyclopentasiloxane, decamethyl- 370 C10H30O5Si5 14.77 0.43 9 Cyclohexasiloxane, dodecamethyl- 444 C12H36O6Si6 17.33 0.95 10 Cyclooctasiloxane, hexadecamethyl- 592 C16H48O8Si8 18.13 3.44 11 Cycloheptasiloxane, tetradecamethyl- 518 C14H42O7Si7 19.59 0.82 12 4-Pyrimidinamine, 5-methyl-N-(trimethylsilyl)-2-[(trimethylsilyl)oxy]- 269 C11H23N3OSi2 20.47 5.04 13 Octadecamethyl-Cyclononasiloxane 666 C18H54O9Si9 20.60 6.53 14 Cyclodecasiloxane, eicosamethyl- 740 C20H60O10Si10 22.95 4.03 15 gamma.-Lumicolchicine 399 C22H25NO6 49.71 2.07 15 3,9.beta.;14,15-Diepoxypregn-16-en-20-one, 3,11.beta.,18-triacetoxy- 502 C27H34O9 50.05 4.03 16 2-Butenoic acid, 2-methyl-, 1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-9aH-cyclopropa[3,4]benz[1,2-e]azulene-9,9a-diyl ester, [1aR-[1a.alpha.,1b.beta.,4a.beta.,7a.alpha.,7b.alpha.,8.alpha.,9.beta.(E),9a.alpha.(E)]]- 528 C30H40O8 50.35 1.00 17 psi.,.psi.-Carotene, 3,4-didehydro-1,2,7',8'-tetrahydro-1-methoxy-2-oxo- 582 C41H58O2 50.52 4.17 18 9-Desoxo-9-x-acetoxy-3,8,12-tri-O-acetylingol 536 C28H40O10 51.52 2.6 19 Canthaxanthin 564 C40H52O2 51.67 5.38 20 Colchiceinamide 384 C21H24N2O5 51.76 2.67 21 Norcodeine di-TMS derivative 429 C23H35NO3Si2 51.86 3.64 22 2,4-Imidazolidinedione, 5-[3,4-bis[(trimethylsilyl)oxy]phenyl]-3-methyl-5-phenyl-1-(trimethylsilyl) 516 C25H40N2O4Si3 51.90 2.09 23 3,8,12-Triacetylingol 7-(4-methoxyphenyl)acetate 640 C35H44O11 51.97 1.52 24 6,6'-Diacetyl-7,7'-dihydroxy-2,2',4,4',5,5'-hexamethoxy-1,1'-binaphthalene 550 C30H30O10 52.08 3.95 25 D-Glucopyranosiduronic acid, 3-(5-ethylhexahydro-1,3-dimethyl-2,4,6-trioxo-5-pyrimidinyl)-1-methylbutyl 2,3,4-tris-O-(trimethylsilyl)-, methyl ester 676 C29H56N2O10Si3 52.26 3.72 26 Pregnane-11,20-dione, 3,17,21-tris[(trimethylsilyl)oxy]-, 20-[O-(phenylmethyl)oxime], (3.alpha.,5.alpha.)- 685 C37H63NO5Si3 52.35 2.24 27 Cephalotaxine, 11-(acetyloxy)-, acetate (ester), (11.alpha.)- 415 C22H25NO7 52.41 1.97 28 Silane, [[(3.beta.,5.alpha.,11.beta.,20S)-pregnane-3,11,17,20,21-pentayl]pentakis(oxy)]pentakis[trimethyl- 728 C36H76O5Si5 52.52 1.90 29 5H-Cyclopropa[3,4]benz[1,2-e]azulen-5-one, 2,4a,9,9a-tetrakis(acetyloxy)-3,[(acetyloxy)methyl]-1,1a,1b,2,3,4,4a,7a,7b,8,9,9a-dodecahydro-2,7b-dihydroxy-1,1,6,8-tetramethyl-, [1aR-(1a.alpha.,1b.beta.,2.alpha.,3.beta.,4a.beta.,7a.alpha.,7b.alpha.,8.alpha.,9.beta.,9a.alpha.)]- 608 C30H40O13 52.70 0.77 30 Dipyridamole 504 C24H40N8O4 52.82 0.70 31 6-Fluorobenzofurazane, 5-[4-(3-methoxyphenyl)piperazin-1-yl]-, 1-oxide 344 C17H17FN4O3 55.45 0.86 32 1-(3-Cyano-6-methyl-4,5,6,7-tetrahydro-thieno[2,3-c]pyridin-2-yl)-3-phenyl-thiourea 328 C16H16N4S2 57.04 0.35 33 Morphinan-6-ol, 4,5-epoxy-3-methoxy-17-methyl-, acetate (ester), (5.alpha.,6.alpha.)- 343 C20H25NO4 61.02 1.35 34 Echinenone 550 C40H54O 61.50 0.43 35 2,2'-Binaphthalene]-8,8'-dicarbonitrile, 1,1'-dihydroxy-6,6',7,7'-tetramethoxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)- 568 C34H36N2O6 61.87 0.80 Molecular docking simulation Among the three compounds, no significant binding of Dipyridamole with Bax, Bcl2, p53 and NF-κB was observed as shown in Table 4 . Although a significant binding of Colchiceinamide was found only with Bcl2 with the binding energy of -7.5 kcal/mol (Fig. 5 A). Where Colchiceinamide formed two hydrogen bonds with residues ASN180 and SER81. Besides the hydrogen bonds, other types of bonding were also observed (Fig. 5 A). But Canthaxanthin exert strong binding affinity with Bax, Bcl2, p53 and NF-κB proteins with the binding energy of -7.8, -8.6, -7.0 and − 7.0 kcal/mol, respectively (Table 4 ). No hydrogen bonding was observed for Bax and Bcl2 (Fig. 5 B and C ). But a strong hydrogen bonding was observed for NF-kB (Fig. 5 D) and p53 (Fig. 5 E). Besides hydrogen bonding other type of interaction was also observed for each of the proteins as shown in the 2D picture (Fig. 5 ). Table 4 Binding energy Protein Name Compound Name Binding energy (kcal/mol) Bax Canthaxanthin -7.8 Bax Colchiceinamide -6.0 Bax Dipyridamole -5.4 Bcl2 Canthaxanthin -8.6 Bcl2 Colchiceinamide -7.5 Bcl2 Dipyridamole -6.0 p53 Canthaxanthin -7.0 p53 Colchiceinamide -5.9 p53 Dipyridamole -5.4 NFκB Canthaxanthin -7.0 NFκB Colchiceinamide -5.6 NFκB Dipyridamole -5.4 Discussion The primary objective of this study was to evaluate the growth-inhibitory and anti-proliferative effects of RTNSME in Ehrlich ascites carcinoma (EAC) cells. The findings of this study provide clear evidence that RTNSME possesses significant anticancer activity, primarily by inducing apoptosis. As demonstrated in Fig. 1 , RTNSME exhibited a dose-dependent growth inhibitory effect. Furthermore, DAPI staining of untreated EAC cells and RTNSME-treated cells revealed morphological alterations indicative of apoptosis, including membrane blebbing, cell shrinkage, and nuclear condensation (Fig. 2 A). These results suggest that RTNSME effectively inhibits cell growth by triggering apoptotic processes. Our previous study also reported apoptosis induction by MOLME in EAC cells ( 19 ). The Bcl-2 family of proteins plays a critical role in regulating cell cycle arrest and cell death ( 20 ), with Bcl-2 promoting cell survival ( 21 ). In contrast, p53 modulates apoptosis by binding to Bcl-2 family proteins, allowing Bax to be released and signal the mitochondria to initiate cell death ( 22 , 23 ). The present study suggests that RTNSME up-regulates p53, which in turn down-regulates Bcl-2 and up-regulates Bax. As a result, the Bcl-2/Bax ratio decreases, promoting apoptotic cell death (Fig. 2 B). NF-κB is a key regulator involved in cell growth, survival, and apoptosis induction ( 24 , 25 ). In cancer, NF-κB is typically up-regulated, promoting inflammation and facilitating tumorigenesis ( 26 ), while also driving metastasis ( 27 ). Activation of NF-κB generally inhibits p53 function ( 28 , 29 ), meaning that p53 and NF-κB often exhibit opposing effects in cancer cells ( 30 ). In this study, we found that RTNSME inhibited NF-κB activation (Fig. 3 ) in conjunction with growth inhibition, suggesting that NF-κB inactivation could initiate apoptotic pathways and contribute to EAC cell death. GC-MS analysis of RTNSME identified 35 compounds, many of which are known to possess biological activity. Notably, Canthaxanthin, a compound found in RTNSME, has been reported to act as an anti-tumor agent ( 31 – 34 ). Previous studies have demonstrated that Canthaxanthin inhibits the growth of murine melanoma, fibrosarcoma, and human squamous carcinoma cells in vitro ( 35 ) and can reduce cell growth by inducing apoptosis ( 36 ). Dipyridamole, also present in RTNSME, has shown antitumor activity and can enhance the sensitivity of several chemotherapy agents, including 5-fluorouracil, cisplatin, and methotrexate ( 37 ). It has also been shown to increase the sensitivity of trametinib in cancer cell lines ( 38 ). Additionally, Colchicine, an alkaloid found in RTNSME, has demonstrated anticancer potential across various cancer cell lines ( 39 ). Molecular docking, a critical tool in computational drug design, was employed in this study to investigate the interaction between RTNSME compounds and key apoptosis-regulating proteins (Bax, Bcl-2, p53, and NF-κB). Our docking results revealed a strong binding affinity between Canthaxanthin and these proteins, supporting the activation of apoptotic pathways via the modulation of these key regulatory proteins. Conclusion In conclusion, RTNSME significantly inhibited EAC cell growth by inducing apoptosis. This growth inhibitory effect is likely mediated through mitochondrial dysfunction, with reciprocal changes in the expression of Bax and Bcl-2. Furthermore, inactivation of NF-κB and up-regulation of p53 appear to play central roles in promoting EAC cell death. 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Islam MA, Kim YS, Jang WJ, et al. A mixture of trans, trans conjugated linoleic acid induces apoptosis in MCF-7 human breast cancer cells with reciprocal expression of Bax and Bcl-2. J Agric Food Chem. 2008;56(14):5970–6. 10.1021/jf8004977 . Chen J. The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. Cold Spring Harb Perspect Med . 2016;6(3):a026104. Published 2016 Mar 1. 10.1101/cshperspect.a026104 Pellegata NS, Antoniono RJ, Redpath JL, Stanbridge EJ. DNA damage and p53-mediated cell cycle arrest: a reevaluation. Proc Natl Acad Sci U S A. 1996;93(26):15209–14. 10.1073/pnas.93.26.15209 . Meyer C, Sims AH, Morgan K, et al. Transcript and protein profiling identifies signaling, growth arrest, apoptosis, and NF-κB survival signatures following GNRH receptor activation. Endocr Relat Cancer. 2013;20(1):123–36. 10.1530/ERC-12-0192 . Published 2013 Feb 18. Alonso F, Krattinger N, Mazzolai L, et al. An angiotensin II- and NF-kappaB-dependent mechanism increases connexin 43 in murine arteries targeted by renin-dependent hypertension. Cardiovasc Res. 2010;87(1):166–76. 10.1093/cvr/cvq031 . Baud V, Karin M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov. 2009;8(1):33–40. 10.1038/nrd2781 . Xia Y, Shen S, Verma IM. NF-κB, an active player in human cancers. Cancer Immunol Res. 2014;2(9):823–30. 10.1158/2326-6066.CIR-14-0112 . Dey A, Tergaonkar V, Lane DP. Double-edged swords as cancer therapeutics: simultaneously targeting p53 and NF-kappaB pathways. Nat Rev Drug Discov. 2008;7(12):1031–40. 10.1038/nrd2759 . Ak P, Levine AJ. p53 and NF-κB: different strategies for responding to stress lead to a functional antagonism. FASEB J. 2010;24(10):3643–52. 10.1096/fj.10-160549 . Carrà G, Lingua MF, Maffeo B, Taulli R, Morotti A. P53 vs NF-κB: the role of nuclear factor-kappa B in the regulation of p53 activity and vice versa. Cell Mol Life Sci. 2020;77(22):4449–58. 10.1007/s00018-020-03524-9 . Tanaka T, Makita H, Ohnishi M, Mori H, Satoh K, Hara A. Chemoprevention of rat oral carcinogenesis by naturally occurring xanthophylls, astaxanthin and canthaxanthin. Cancer Res. 1995;55(18):4059–64. Tanaka T, Kawamori T, Ohnishi M, et al. Suppression of azoxymethane-induced rat colon carcinogenesis by dietary administration of naturally occurring xanthophylls astaxanthin and canthaxanthin during the postinitiation phase. Carcinogenesis. 1995;16(12):2957–63. 10.1093/carcin/16.12.2957 . Grubbs CJ, Eto I, Juliana MM, Whitaker LM. Effect of canthaxanthin on chemically induced mammary carcinogenesis. Oncology. 1991;48(3):239–45. 10.1159/000226935 . Katsumura N, Okuno M, Onogi N, Moriwaki H, Muto Y, Kojima S. Suppression of mouse skin papilloma by canthaxanthin and beta-carotene in vivo: possibility of the regression of tumorigenesis by carotenoids without conversion to retinoic acid. Nutr Cancer. 1996;26(2):203–8. 10.1080/01635589609514476 . Huang DS, Odeleye OE, Watson RR. Inhibitory effects of canthaxanthin on in vitro growth of murine tumor cells. Cancer Lett. 1992;65(3):209–13. 10.1016/0304-3835(92)90233-l . Palozza P, Maggiano N, Calviello G, et al. Canthaxanthin induces apoptosis in human cancer cell lines. Carcinogenesis. 1998;19(2):373–6. 10.1093/carcin/19.2.373 . Ge SM, Zhan DL, Zhang SH, Song LQ, Han WW. Reverse screening approach to identify potential anti-cancer targets of dipyridamole. Am J Transl Res. 2016;8(12):5187–98. Published 2016 Dec 15. Zhou S, Xu H, Tang Q, Xia H, Bi F. Dipyridamole Enhances the Cytotoxicities of Trametinib against Colon Cancer Cells through Combined Targeting of HMGCS1 and MEK Pathway. Mol Cancer Ther. 2020;19(1):135–46. 10.1158/1535-7163.MCT-19-0413 . Kumar A, Sharma PR, Mondhe DM. Potential anticancer role of colchicine-based derivatives: an overview. Anticancer Drugs. 2017;28(3):250–62. 10.1097/CAD.0000000000000464 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-6950088","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488973098,"identity":"ad0be3c8-fbdc-4ef4-bb6e-ff208f802192","order_by":0,"name":"Saharia Yeasmin Asha","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Saharia","middleName":"Yeasmin","lastName":"Asha","suffix":""},{"id":488973102,"identity":"f818b42f-9b38-4d2c-91e8-e08fc9753208","order_by":1,"name":"Mst. Ayesha Siddika","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Mst.","middleName":"Ayesha","lastName":"Siddika","suffix":""},{"id":488973103,"identity":"662d3341-03a9-4fce-95a0-f11889d33b23","order_by":2,"name":"Mahmud Ismail","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Mahmud","middleName":"","lastName":"Ismail","suffix":""},{"id":488973104,"identity":"b9e43ce2-23e4-4cdb-baf7-f1597da2af9a","order_by":3,"name":"Arifur Rahman","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Arifur","middleName":"","lastName":"Rahman","suffix":""},{"id":488973105,"identity":"e5a4aa3c-1ccb-4e5a-9a2d-e6e586d16a0e","order_by":4,"name":"Maksudul Haque","email":"","orcid":"","institution":"University of Science and Technology Chitagong","correspondingAuthor":false,"prefix":"","firstName":"Maksudul","middleName":"","lastName":"Haque","suffix":""},{"id":488973109,"identity":"0febac4c-72f5-4572-813b-7eb031a4014a","order_by":5,"name":"A. R.M. Tareq","email":"","orcid":"","institution":"Atomic Energy Centre","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"R.M.","lastName":"Tareq","suffix":""},{"id":488973113,"identity":"ff2a4a88-7cfb-408a-af34-78ba8adeece5","order_by":6,"name":"Farhadul Islam","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Farhadul","middleName":"","lastName":"Islam","suffix":""},{"id":488973114,"identity":"7da789b2-911d-4415-a91c-09e057c03c7e","order_by":7,"name":"Syed Rashel Kabir","email":"","orcid":"","institution":"University of Rajshahi","correspondingAuthor":false,"prefix":"","firstName":"Syed","middleName":"Rashel","lastName":"Kabir","suffix":""},{"id":488973115,"identity":"6705f2a4-625c-47be-b3d6-05e69f98b1f0","order_by":8,"name":"Md. Abdur Rakib","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAkUlEQVRIiWNgGAWjYHACxgcQmodBglgtzAYka2GTIE2LbvvZZ9WVbQzy/A28B28QpcXsTLrZzbNtDIYzDvAlWxCn5UAa283GNgbGDQw8ZsQ5zOz8M7ZCoBZ7ErTcSGNjBGpJJEXLM2bJhnMSyTMOE+2X82mMHxvKbGz723uJDDEoADqJmRT1o2AUjIJRMArwAwCQzSe9Ng006QAAAABJRU5ErkJggg==","orcid":"","institution":"University of Rajshahi","correspondingAuthor":true,"prefix":"","firstName":"Md.","middleName":"Abdur","lastName":"Rakib","suffix":""}],"badges":[],"createdAt":"2025-06-22 14:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6950088/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6950088/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87434180,"identity":"9d75b5f4-27aa-4999-acf2-b476abafb044","added_by":"auto","created_at":"2025-07-23 18:19:40","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75062,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of RTNSME on \u003cstrong\u003e(A)\u003c/strong\u003e Tumor weight of EAC bearing mice and \u003cstrong\u003e(B)\u003c/strong\u003e EAC cell growth inhibition. Results are shown as mean ± SD (standard deviation) (n=4). Mean with different lowercase letters are significantly different at P \u0026lt; 0.05 by Duncan’s multiple-range test.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6950088/v1/0e72faa6e6798b04c6f7e423.jpeg"},{"id":87434778,"identity":"1304f03e-ba46-4b21-a59a-89fd8cc78309","added_by":"auto","created_at":"2025-07-23 18:27:40","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":400254,"visible":true,"origin":"","legend":"\u003cp\u003eInduction of apoptosis in EAC cells treated with RTNSME. \u003cstrong\u003e(A)\u003c/strong\u003e DAPI staining of EAC cells treated with RTNSME followed by fluorescence microscopic analysis of cells. \u003cstrong\u003e(B)\u003c/strong\u003e Expression of P53, Bax and Bcl-2 genes in EAC cells treated with RTNSME. UV-trans illuminator visualizations of P53, Bcl-2, Bax and GAPDH. Here, M: Marker, C: EAC cells and T: EAC cells treated with RTNSME.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6950088/v1/75c1c1435e6cec27b7e539b3.jpeg"},{"id":87434179,"identity":"69b23b63-b4a4-4b5d-a9f0-3f019defd772","added_by":"auto","created_at":"2025-07-23 18:19:40","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109432,"visible":true,"origin":"","legend":"\u003cp\u003eInactivation of NF-𝜅B gene expression in EAC cells treated with RTNSME. UV-trans illuminator visualizations of NF-𝜅B and GAPDH. Here, M: Marker, C: EAC cells and T: EAC cells treated with RTNSME.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6950088/v1/3576e55fbfebdd1bfb870c5d.jpeg"},{"id":87433945,"identity":"e146ed1c-d028-4f07-85da-55d979e6bf1f","added_by":"auto","created_at":"2025-07-23 18:11:40","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":382804,"visible":true,"origin":"","legend":"\u003cp\u003eGC/MS chromatogram of RTNSME.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6950088/v1/4acd161a676de679117e8528.jpeg"},{"id":87435268,"identity":"6fe0c506-502d-49d4-a68d-be94de0f8c71","added_by":"auto","created_at":"2025-07-23 18:35:40","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":82450,"visible":true,"origin":"","legend":"\u003cp\u003eBinding affinity between Dipyridamole, Colchiceinamide, and Canthaxanthin with different proteins. \u003cstrong\u003e(A)\u003c/strong\u003e Represents binding of Colchiceinamide with BCL-2. While \u003cstrong\u003e(B)\u003c/strong\u003e, \u003cstrong\u003e(C)\u003c/strong\u003e, \u003cstrong\u003e(D)\u003c/strong\u003e and \u003cstrong\u003e(E)\u003c/strong\u003e represents interaction between Canthaxanthin with BAX, BCL2, p53, and NFkB, respectively.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6950088/v1/268adbe3af4b19ffe7092bc3.jpeg"},{"id":88894403,"identity":"8b80aef3-6852-474f-a1a2-7cda4fe799aa","added_by":"auto","created_at":"2025-08-12 13:02:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2135449,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6950088/v1/b279463d-b2e3-4d3e-8b68-c895bba22cfe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGrowth inhibitory action of Red Trapa natans shell methanolic extract (RTNSME) in Ehrlich ascites carcinoma (EAC) cells in Swiss Albino Mice by inducing apoptosis through inactivation of NFkB\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer remains one of the deadliest diseases worldwide, claiming the lives of over six million people annually (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). While both synthetic drugs and natural compounds with anticancer properties have shown promise in cancer treatment (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), current chemotherapeutic agents are often expensive and associated with severe side effects. Therefore, the development of cost-effective and safer therapeutic alternatives is a critical priority in cancer research (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Due to their lower toxicity, wide availability, and bioactivity, natural compounds are increasingly being explored for their anticancer potential (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eTrapa natans\u003c/em\u003e L., commonly known as water chestnut or water caltrop, is an annual aquatic floating herb belonging to the family Trapaceae (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). It typically grows in lakes, ponds, ditches, and other stagnant water bodies, and is widely cultivated in tropical, subtropical, and temperate regions of the world (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Two primary varieties of \u003cem\u003eTrapa natans\u003c/em\u003e are recognized. Various extracts of its fruit have been reported to exhibit a broad spectrum of pharmacological properties, including antimicrobial (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), analgesic (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), anti-inflammatory (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), antidiabetic (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), antioxidant, antiulcer, and antifungal activities (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious studies have also demonstrated the anticancer potential of \u003cem\u003eTrapa natans\u003c/em\u003e in several human cancer cell lines, such as human colon cancer (Colo-205), human ductal breast carcinoma (T47D), and human breast adenocarcinoma (MCF-7) cells (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, its anticancer activity against Ehrlich ascites carcinoma (EAC) cells has not yet been investigated.\u003c/p\u003e\u003cp\u003eOur earlier study revealed that the methanolic extract of red \u003cem\u003eTrapa natans\u003c/em\u003e shell (RTNSME) contains a high concentration of phytochemicals and exhibits strong antioxidant activity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Building on these findings, the present study aims to evaluate the anti-proliferative effects of RTNSME on EAC cells and to elucidate its underlying molecular mechanisms, with a focus on apoptosis and NF-κB signaling.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003ePreparation of Red\u003c/b\u003e \u003cb\u003eTrapa natans\u003c/b\u003e \u003cb\u003eShell Methanolic Extract (RTNSME)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMature \u003cem\u003eTrapa natans\u003c/em\u003e fruits were procured from a local market in Rajshahi, Bangladesh. The species was taxonomically authenticated by a specialist in the Department of Botany, University of Rajshahi. The red-colored fruits were thoroughly washed with distilled water to remove surface impurities. The shells were manually separated, shade-dried, and then ground into a fine powder using a mechanical grinder. The powdered shell material was stored in an airtight container at room temperature until extraction.\u003c/p\u003e\u003cp\u003eThe extraction process followed the method described by Luqman A. Olayaki et al. (2014) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), with slight modifications. Approximately 150 grams of the powdered shell were soaked in 500 mL of methanol and kept under continuous stirring for 24 hours at room temperature. After initial extraction, the mixture was filtered through Whatman No. 1 filter paper, and an additional 100 mL of methanol was added to the residue for re-extraction. The combined filtrates were concentrated using a rotary evaporator at 37\u0026deg;C to remove the solvent. The resulting dried extract (RTNSME) was stored at 4\u0026deg;C in a refrigerator for further use in experiments.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eSwiss albino mice (6 to 8 weeks old, weighing 25\u0026thinsp;\u0026plusmn;\u0026thinsp;4 g) were obtained from the Department of Pharmacy, Jahangirnagar University, Bangladesh. The animals were housed in standard laboratory conditions with a 12-hour light/dark cycle and maintained at room temperature with proper ventilation. Six mice were kept per cage. All animals were provided with standard laboratory chow and drinking water \u003cem\u003ead libitum\u003c/em\u003e throughout the experimental period.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEthical Clearance\u003c/h3\u003e\n\u003cp\u003e All experimental procedures involving animals were conducted in accordance with ethical guidelines and approved by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) for experimentation on Animals, Humans, Microbes, and Living Natural Sources, Institute of Biological Sciences, University of Rajshahi, Bangladesh (Approval No. 117/320 (47)/IAMEBBC/IBSc).\u003c/p\u003e\n\u003ch3\u003eCell Line\u003c/h3\u003e\n\u003cp\u003eEhrlich ascites carcinoma (EAC) cells were kindly provided by the Department of Pharmacy, Jahangirnagar University, Bangladesh. All procedures involving biosafety and animal handling were conducted in compliance with the guidelines of the Institute of Biological Sciences, University of Rajshahi, Bangladesh.\u003c/p\u003e\n\u003ch3\u003eEstimation of Tumor Weight\u003c/h3\u003e\n\u003cp\u003eTumor weight was estimated following the method described by (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Experimental mice were divided into groups, each consisting of five mice. On day 0, each mouse was intraperitoneally inoculated with 1.0 \u0026times; 10⁶ EAC cells. After 24 hours, the mice were treated with RTNSME at doses of 25, 50, and 100 mg/kg body weight, respectively, for 20 consecutive days. Tumor weight was measured every 48 hours throughout the treatment period.\u003c/p\u003e\n\u003ch3\u003eCell Growth Inhibition Determination\u003c/h3\u003e\n\u003cp\u003eCell growth inhibition was assessed as previously described (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). On day 0, mice were intraperitoneally inoculated with 1 \u0026times; 10⁶ EAC cells. After 24 hours, RTNSME was administered at doses of 25, 50, and 100 mg/kg body weight per mouse per day to the respective treatment groups for five consecutive days. A control group received only normal saline. On day 6, all animals were sacrificed, and the ascitic fluid was collected. EAC cells were harvested and counted using a hemocytometer to evaluate the extent of cell growth inhibition.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDAPI Staining for Morphological Appearance and Nuclear Damage of EAC Cells\u003c/h2\u003e\u003cp\u003eThe induction of apoptosis in EAC cells was assessed as previously described (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Both treated and untreated control EAC cells were analyzed using a fluorescence inverted microscope (Olympus IX71, Japan). Initially, the cells were collected and stained with 25 \u0026micro;L of DAPI solution (1 mg/mL) at 37\u0026deg;C for 20 minutes. After staining, the cells were washed three times with PBS in the dark to remove excess dye. Morphological changes, including nuclear condensation and fragmentation, were observed under the fluorescence microscope to assess nuclear damage and apoptotic characteristics.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReverse Transcriptase Polymerase Chain Reaction (RT-PCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from cells using an RNA extraction kit according to the manufacturer\u0026rsquo;s instructions. cDNA was synthesized via reverse transcription PCR following the protocol provided with the kit. The primers used for the experiment are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The PCR products were subjected to electrophoresis on a 1.0% agarose gel, which was then stained with ethidium bromide. The gel was visualized under a UV transilluminator to assess the amplification of target genes.\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\u003eThe sequence of primers used for PCR amplification.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer sequence references\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward: (5\u0026prime;-GTGGAAGGACTCATGACCACAG-3\u0026prime;)\u003c/p\u003e\u003cp\u003eReverse: (5\u0026prime;-CTGGTGCTCAGTGTAGCCCAG-3\u0026prime;)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward: (5\u0026prime;-CACAAAAACAGGTTAAACCCAG-3\u0026prime;)\u003c/p\u003e\u003cp\u003eReverse: (5\u0026prime;-AGCACATAGGAGGCAGAGAC-3\u0026prime;)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBcl-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward: (5\u0026prime;-GTGGAGGAGCTCTTCAGGGA-3\u0026prime;)\u003c/p\u003e\u003cp\u003eReverse: (5\u0026prime;-AGGCACCCAGGGTGATGCAA-3\u0026prime;)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBax\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward: (5'-GGCCCACCAGCTCTGAGCAGA-3')\u003c/p\u003e\u003cp\u003eReverse: (3'-GCCACGTGGGCGTCCCAAAGT-5')\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNF-kB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward: (5'-AACAAAATGCCCCACGGTTA-3')\u003c/p\u003e\u003cp\u003eReverse: (3'-GGGACGATGCAATGGACTGT-5')\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eGas Chromatography-Mass Spectrometry (GC-MS) Analysis\u003c/h3\u003e\n\u003cp\u003eGC-MS analysis of RTNSME was performed using a Varian GC spectrophotometer (Model CP-3800, USA) coupled with a Varian Saturn-2200 mass spectrometer. The system was equipped with a flame ionization detector and a VF-5 ms capillary column (30 m \u0026times; 0.25 mm, 0.25 \u0026micro;m). The instrument was operated in electron impact mode at 70 eV ionization voltage, with the injector temperature set at 250\u0026deg;C and the detector temperature set at 280\u0026deg;C. A 1 \u0026micro;L sample was injected with helium as the carrier gas at a flow rate of 1 mL/min. The column temperature was initially set to 40\u0026deg;C for 1 minute, then ramped to 310\u0026deg;C at a rate of 10\u0026deg;C/min, where it was held for 10 minutes. Chemical compounds were identified and quantified by comparing their mass spectra with the NIST 05 Library database.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMolecular Docking Simulation\u003c/h2\u003e\u003cp\u003eMolecular docking simulations were performed to explore the potential interactions between BAX, BCL2, p53, and NF-κB proteins with the compounds Canthaxanthin, Colchiceinamide, and Dipyridamole. The 3D structures of BAX, BCL2, p53, and NF-κB proteins were retrieved from the Protein Data Bank (PDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e with the following PDB codes: 5W62 (BAX), 2KUA (BCL2), 2IOI (p53), and 1MY5 (NF-κB). To eliminate potential interference from water molecules and heteroatoms, these were removed using Biovia Discovery Studio 2021, reducing the possibility of undesired interactions with the receptor proteins. The resulting PDB files were then converted into pdbqt format for docking analysis.\u003c/p\u003e\u003cp\u003eThe chemical structures of Canthaxanthin (CID: 5281227), Colchiceinamide (CID: 18397), and Dipyridamole (CID: 3108) were downloaded in 3D SDF format from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, minimized, and converted into pdbqt format using the PyRx tool. Docking studies were carried out using PyRx with Autodock Vina to predict the binding affinity and interaction between the proteins and compounds. The protein-ligand interactions, including hydrogen bonds, pi-alkyl bonds, hydrophobic interactions, and other relevant bonding types, were analyzed and visualized using BIOVIA Discovery Studio 2021.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eExperiments were performed in triplicates, and the data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was conducted using one-way analysis of variance (ANOVA), followed by Duncan\u0026rsquo;s multiple range test to compare group means. All analyses were performed using SPSS software (version 16). A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGrowth inhibition of EAC cell treated with RTNSME\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003eTumor weight reduction\u003c/h2\u003e\u003cp\u003eEAC cells growth inhibition was first determined by average tumor weight. RTNSME reduced tumor weight in EAC-bearing mice significantly. Tumor weight of untreated control EAC cells was increased over time but treatment with RTNSME at the concentration of 25, 50 and 100 mg/kg RTNSME significant reduced of tumor weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCell growth inhibition\u003c/h2\u003e\u003cp\u003eTreatment with RTNSME resulted in 26.21, 38.15 and 76.05% of cell growth inhibition compared to control at 25, 50 and 100 mg/kg body weight doses respectively. Effects of RTNSME on cell growth inhibition are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCell growth inhibition\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName of the experiment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNature of Drug\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDose in mg/kg/day body weight (i.p)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo of EAC cells in mouse on Day 6 after tumour cell inoculation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e% of cell\u003c/p\u003e\u003cp\u003egrowth\u003c/p\u003e\u003cp\u003einhibition\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl(EAC cell bearing mice)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;.12) x 10\u003csup\u003e6 d\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eRTNSME\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e( 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;.12) x 10\u003csup\u003e6 c\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;.12) x 10\u003csup\u003e6 b\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;.20) x 10\u003csup\u003e6 a\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e76.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eEffect of RTNSME on EAC cell growth inhibition (\u003cem\u003ein vivo\u003c/em\u003e). Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). Mean with different lowercase letters are significantly different at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by Duncan\u0026rsquo;s multiple-range test.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eInduction of cancer cell apoptosis\u003c/h2\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003eChanges of EAC cell morphology\u003c/h2\u003e\u003cp\u003eRTNSME treated EAC cells showed condensed and fragmented DNA in nuclei as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA whereas Morphological changes of EAC untreated cells were found round, regular in control cell whereas.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eModulation of apoptotic gene expression\u003c/h2\u003e\u003cp\u003eThe mRNA expressions of p53, Bcl-2, Bax were investigated by using specific primers. Red TNSME was down regulated the mRNA expression of Bcl-2 when compared with untreated control. On the other hand, the mRNA expression of Bax, p53 was up regulated when treated with RTNSME. GAPDH expression was observed equally both in RTNSME treated EAC cells and untreated control EAC cells Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eInactivation of NF-κB gene expression\u003c/h2\u003e\u003cp\u003eNF-κB gene in EAC cells treated with RTNSME was determined by RT-PCR Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. RTNSME reduced the activation of NF-κB compared to untreated control cells. This results might reduce cell growth and induction of apoptosis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eChemical composition of RTNSME\u003c/h2\u003e\u003cp\u003eGC/MS chromatogram and list of compounds revealed that RTNSME consists of 35 compounds as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e respectively.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChemical composition of RTNSME analyzed by GC\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompound Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMW\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFormula\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eArea %\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBenzyloxy(butyl)dimethylsilane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e222\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC13H22OSi\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIsobutyl ether\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC8H18O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1-(Nitromethyl)-cyclohexanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e159\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC7H13NO3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEthanol, 2-butoxy-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e118\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC6H14O2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1,2,3-Propanetriol, monoacetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC5H10O4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBenzyl Alcohol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC7H8O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1,1,2-Triacetoxyethane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e204\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC8H12O6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCyclopentasiloxane, decamethyl-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e370\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC10H30O5Si5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCyclohexasiloxane, dodecamethyl-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e444\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC12H36O6Si6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCyclooctasiloxane, hexadecamethyl-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e592\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC16H48O8Si8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e18.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCycloheptasiloxane, tetradecamethyl-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e518\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC14H42O7Si7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e19.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4-Pyrimidinamine, 5-methyl-N-(trimethylsilyl)-2-[(trimethylsilyl)oxy]-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC11H23N3OSi2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOctadecamethyl-Cyclononasiloxane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e666\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC18H54O9Si9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCyclodecasiloxane, eicosamethyl-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e740\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC20H60O10Si10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e22.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003egamma.-Lumicolchicine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e399\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC22H25NO6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e49.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3,9.beta.;14,15-Diepoxypregn-16-en-20-one, 3,11.beta.,18-triacetoxy-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e502\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC27H34O9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e50.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2-Butenoic acid, 2-methyl-, 1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-9aH-cyclopropa[3,4]benz[1,2-e]azulene-9,9a-diyl ester, [1aR-[1a.alpha.,1b.beta.,4a.beta.,7a.alpha.,7b.alpha.,8.alpha.,9.beta.(E),9a.alpha.(E)]]-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e528\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC30H40O8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e50.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epsi.,.psi.-Carotene, 3,4-didehydro-1,2,7',8'-tetrahydro-1-methoxy-2-oxo-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e582\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC41H58O2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e50.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e18\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9-Desoxo-9-x-acetoxy-3,8,12-tri-O-acetylingol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e536\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC28H40O10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCanthaxanthin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e564\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC40H52O2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColchiceinamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e384\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC21H24N2O5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e21\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNorcodeine di-TMS derivative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e429\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC23H35NO3Si2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e22\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2,4-Imidazolidinedione, 5-[3,4-bis[(trimethylsilyl)oxy]phenyl]-3-methyl-5-phenyl-1-(trimethylsilyl)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC25H40N2O4Si3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e23\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3,8,12-Triacetylingol 7-(4-methoxyphenyl)acetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC35H44O11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6,6'-Diacetyl-7,7'-dihydroxy-2,2',4,4',5,5'-hexamethoxy-1,1'-binaphthalene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC30H30O10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e52.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eD-Glucopyranosiduronic acid, 3-(5-ethylhexahydro-1,3-dimethyl-2,4,6-trioxo-5-pyrimidinyl)-1-methylbutyl 2,3,4-tris-O-(trimethylsilyl)-, methyl ester\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e676\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC29H56N2O10Si3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e52.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e3.72\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e26\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePregnane-11,20-dione, 3,17,21-tris[(trimethylsilyl)oxy]-, 20-[O-(phenylmethyl)oxime], (3.alpha.,5.alpha.)-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC37H63NO5Si3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e52.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e27\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCephalotaxine, 11-(acetyloxy)-, acetate (ester), (11.alpha.)-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e415\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC22H25NO7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e52.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e28\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSilane, [[(3.beta.,5.alpha.,11.beta.,20S)-pregnane-3,11,17,20,21-pentayl]pentakis(oxy)]pentakis[trimethyl-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e728\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC36H76O5Si5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e52.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e29\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5H-Cyclopropa[3,4]benz[1,2-e]azulen-5-one, 2,4a,9,9a-tetrakis(acetyloxy)-3,[(acetyloxy)methyl]-1,1a,1b,2,3,4,4a,7a,7b,8,9,9a-dodecahydro-2,7b-dihydroxy-1,1,6,8-tetramethyl-, [1aR-(1a.alpha.,1b.beta.,2.alpha.,3.beta.,4a.beta.,7a.alpha.,7b.alpha.,8.alpha.,9.beta.,9a.alpha.)]-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e608\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC30H40O13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e52.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDipyridamole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e504\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC24H40N8O4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e52.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6-Fluorobenzofurazane, 5-[4-(3-methoxyphenyl)piperazin-1-yl]-, 1-oxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e344\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC17H17FN4O3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e55.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1-(3-Cyano-6-methyl-4,5,6,7-tetrahydro-thieno[2,3-c]pyridin-2-yl)-3-phenyl-thiourea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e328\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC16H16N4S2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e57.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMorphinan-6-ol, 4,5-epoxy-3-methoxy-17-methyl-, acetate (ester), (5.alpha.,6.alpha.)-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e343\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC20H25NO4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e61.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEchinenone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC40H54O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e61.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2,2'-Binaphthalene]-8,8'-dicarbonitrile, 1,1'-dihydroxy-6,6',7,7'-tetramethoxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e568\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC34H36N2O6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e61.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.80\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=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eMolecular docking simulation\u003c/h2\u003e\u003cp\u003eAmong the three compounds, no significant binding of Dipyridamole with Bax, Bcl2, p53 and NF-κB was observed as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Although a significant binding of Colchiceinamide was found only with Bcl2 with the binding energy of -7.5 kcal/mol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Where Colchiceinamide formed two hydrogen bonds with residues ASN180 and SER81. Besides the hydrogen bonds, other types of bonding were also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). But Canthaxanthin exert strong binding affinity with Bax, Bcl2, p53 and NF-κB proteins with the binding energy of -7.8, -8.6, -7.0 and \u0026minus;\u0026thinsp;7.0 kcal/mol, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). No hydrogen bonding was observed for Bax and Bcl2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u003cb\u003eand C\u003c/b\u003e). But a strong hydrogen bonding was observed for NF-kB (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Besides hydrogen bonding other type of interaction was also observed for each of the proteins as shown in the 2D picture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBinding energy\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProtein Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompound Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBinding energy (kcal/mol)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBax\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCanthaxanthin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-7.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBax\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColchiceinamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-6.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBax\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDipyridamole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-5.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBcl2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCanthaxanthin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-8.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBcl2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColchiceinamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-7.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBcl2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDipyridamole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-6.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCanthaxanthin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-7.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColchiceinamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-5.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ep53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDipyridamole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-5.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNFκB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCanthaxanthin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-7.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNFκB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColchiceinamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-5.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNFκB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDipyridamole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-5.4\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"},{"header":"Discussion","content":"\u003cp\u003eThe primary objective of this study was to evaluate the growth-inhibitory and anti-proliferative effects of RTNSME in Ehrlich ascites carcinoma (EAC) cells. The findings of this study provide clear evidence that RTNSME possesses significant anticancer activity, primarily by inducing apoptosis. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, RTNSME exhibited a dose-dependent growth inhibitory effect. Furthermore, DAPI staining of untreated EAC cells and RTNSME-treated cells revealed morphological alterations indicative of apoptosis, including membrane blebbing, cell shrinkage, and nuclear condensation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results suggest that RTNSME effectively inhibits cell growth by triggering apoptotic processes.\u003c/p\u003e\u003cp\u003eOur previous study also reported apoptosis induction by MOLME in EAC cells (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The Bcl-2 family of proteins plays a critical role in regulating cell cycle arrest and cell death (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), with Bcl-2 promoting cell survival (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In contrast, p53 modulates apoptosis by binding to Bcl-2 family proteins, allowing Bax to be released and signal the mitochondria to initiate cell death (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The present study suggests that RTNSME up-regulates p53, which in turn down-regulates Bcl-2 and up-regulates Bax. As a result, the Bcl-2/Bax ratio decreases, promoting apoptotic cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eNF-κB is a key regulator involved in cell growth, survival, and apoptosis induction (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In cancer, NF-κB is typically up-regulated, promoting inflammation and facilitating tumorigenesis (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), while also driving metastasis (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Activation of NF-κB generally inhibits p53 function (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), meaning that p53 and NF-κB often exhibit opposing effects in cancer cells (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In this study, we found that RTNSME inhibited NF-κB activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) in conjunction with growth inhibition, suggesting that NF-κB inactivation could initiate apoptotic pathways and contribute to EAC cell death.\u003c/p\u003e\u003cp\u003eGC-MS analysis of RTNSME identified 35 compounds, many of which are known to possess biological activity. Notably, Canthaxanthin, a compound found in RTNSME, has been reported to act as an anti-tumor agent (\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Previous studies have demonstrated that Canthaxanthin inhibits the growth of murine melanoma, fibrosarcoma, and human squamous carcinoma cells in vitro (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) and can reduce cell growth by inducing apoptosis (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Dipyridamole, also present in RTNSME, has shown antitumor activity and can enhance the sensitivity of several chemotherapy agents, including 5-fluorouracil, cisplatin, and methotrexate (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). It has also been shown to increase the sensitivity of trametinib in cancer cell lines (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Additionally, Colchicine, an alkaloid found in RTNSME, has demonstrated anticancer potential across various cancer cell lines (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMolecular docking, a critical tool in computational drug design, was employed in this study to investigate the interaction between RTNSME compounds and key apoptosis-regulating proteins (Bax, Bcl-2, p53, and NF-κB). Our docking results revealed a strong binding affinity between Canthaxanthin and these proteins, supporting the activation of apoptotic pathways via the modulation of these key regulatory proteins.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, RTNSME significantly inhibited EAC cell growth by inducing apoptosis. This growth inhibitory effect is likely mediated through mitochondrial dysfunction, with reciprocal changes in the expression of Bax and Bcl-2. Furthermore, inactivation of NF-κB and up-regulation of p53 appear to play central roles in promoting EAC cell death. Our findings suggest that RTNSME down-regulates NF-κB, which in turn up-regulates p53, leading to apoptosis in EAC cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNo specific fund provided for this study. Department of Biochemistry and Molecular Biology, University of Rajshahi, supported the study in partial.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.Y.A., M.A.S. design the study, performed the experiments and drafted the manuscript. M.I., A.R., M.H. analyzed the data and generated figures and tables, A.R.M.T. performed GC-MS, S.R.K. supported docking experiments, M.A.R., F.I. planned, supervised the project and edited the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdullaev FI, Rivera-Luna R, Roitenburd-Belacortu V, Espinosa-Aguirre J. Pattern of childhood cancer mortality in Mexico. Arch Med Res. 2000;31(5):526\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0188-4409(00)00094-1\u003c/span\u003e\u003cspan address=\"10.1016/s0188-4409(00)00094-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIslam F, Gopalan V, Lam AKY, Kabir SR. Kaempferia rotunda tuberous rhizome lectin induces apoptosis and growth inhibition of colon cancer cells in vitro. 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Anticancer Drugs. 2017;28(3):250\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/CAD.0000000000000464\u003c/span\u003e\u003cspan address=\"10.1097/CAD.0000000000000464\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Trapa natans L, apoptosis, NF-kB, GC/MS, molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-6950088/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6950088/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eTrapa natans\u003c/em\u003e L., commonly known as water chestnut, is a small aquatic herb belonging to the family \u003cem\u003eTrapaceae\u003c/em\u003e. Its fruit is valued for both nutritional and pharmaceutical properties. This study investigates the anticancer potential of the methanolic extract of red \u003cem\u003eTrapa natans\u003c/em\u003e shell (RTNSME) against Ehrlich ascites carcinoma (EAC) cells. RTNSME demonstrated significant anticancer activity, inhibiting EAC cell growth by 76.05% compared to the untreated control. Fluorescence microscopy of DAPI-stained cells revealed classic apoptotic features such as DNA fragmentation, nuclear condensation, cell shrinkage, and membrane alterations in RTNSME-treated cells. These findings were supported by RT-PCR analysis of apoptosis-related genes. RTNSME upregulated the expression of \u003cem\u003ep53\u003c/em\u003e and \u003cem\u003eBax\u003c/em\u003e, while downregulating the anti-apoptotic gene \u003cem\u003eBcl-2\u003c/em\u003e. Additionally, RTNSME suppressed the activity of the nuclear factor kappa B (NF-κB), further indicating its role in promoting apoptosis. GC-MS analysis of RTNSME identified 35 phytochemical constituents, including known bioactive and anticancer compounds such as canthaxanthin, dipyridamole, and colchiceinamide. Molecular docking studies suggested that canthaxanthin exhibits the strongest binding affinity and is likely the most potent compound against EAC cells. Overall, this study demonstrates that RTNSME exerts significant anticancer effects on EAC cells, primarily through NF-κB inactivation and induction of apoptosis.\u003c/p\u003e","manuscriptTitle":"Growth inhibitory action of Red Trapa natans shell methanolic extract (RTNSME) in Ehrlich ascites carcinoma (EAC) cells in Swiss Albino Mice by inducing apoptosis through inactivation of NFkB","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 18:11:36","doi":"10.21203/rs.3.rs-6950088/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e16318b-a433-4e8f-8099-50f5c5c3c179","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-12T12:54:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 18:11:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6950088","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6950088","identity":"rs-6950088","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}