The Anti-tumour effect of Aleurites moluccana on HCT-116 Cells

The Anti-tumour effect of Aleurites moluccana on HCT-116 Cells | 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 The Anti-tumour effect of Aleurites moluccana on HCT-116 Cells APPALA RAJU NEMALA, ANITHA NANDAGOPAL This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4748200/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 Aleurites moluccanus , commonly referred to as the candlenut or candleberry and also known as the Indian walnut, is a flowering tree belonging to the family Euphorbiaceae. The tree produces large, rounded (sub-globose) or slightly two-lobed fruits measuring 4–6 cm in diameter. The initial phase of this study involved phytochemical screening, revealing that the ethanol extract of Aleurites moluccana fruit contains phenolics, terpenoids, tannins, glycosides, and saponins. The presence of these compounds might be responsible for the biological activity. The anticancer activity was tested using HCT-116 cell lines and primary colon epithelial (PCE) cell. Different concentration ranging from 7.8 µg/mL to 1000 µg/mL were tested against each cell lines by MTT assay. Ethanol extract of Aleurites moluccanus fruits showed cytotoxic activity when compared to standard control, Fluorouracil (10 µg/mL). In an MTT-based cytotoxic assay evaluating anticancer activity, the ethanol extract was tested on colorectal cell lines HCT-116 and primary colon epithelial (PCE) cells. Cytotoxicity increased over time, with the highest cytotoxicity observed at 72 hours. The 72-hour treatment exhibits the IC50 of 8.54 µg/mL in HCT-116 cells and 196.72 µg/mL in PCE cells, indicating a potent and time-dependent cytotoxic effect on HCT-116 cells. Aleurites moluccanus Antitumour Cytotoxicity HCT-116 Cells and MTT Assay Figures Figure 1 Figure 2 Figure 3 Introduction Aleurites moluccana is a medium-sized tree that can grow up to 20 meters tall, with wide-spreading or hanging branches. Its bark is grey-brown and relatively smooth with fine vertical lines. The fruit is an indehiscent drupe, nearly spherical, measuring 5 cm or more in diameter, and has a thick, rough, hard shell that makes up 64–68% of the fruit. This shell is difficult to separate from the kernels and contains 1–2 hard-shelled black seeds. Aleurites moluccanus (L.) Willd, a Euphorbiaceae plant, is native to Indonesia, India, and the South Sea Islands [ 1 ]. It is known as the "Candlenut tree," "Kukui," and "Indian Walnut," and it was introduced in the early 20th century for its oil seeds, producing "tung oil," and as an ornamental plant [ 2 ]. Each tree can yield 30–80 kg of nuts. Aleurites moluccana is used in traditional medicine to treat pain, fever, asthma, hepatitis, gastric ulcers, inflammation. This species is also utilized in traditional medicine for the treatment of cardiovascular diseases, anemia, high cholesterol, hypertension, and diabetes [ 3 ]. The nut oil is applied topically for arthritis and joint pain. However, the seeds are toxic if ingested. Four new podocarpane-type tri-nor-di-terpenenes and four known diterpenes were isolated from the twigs and leaves of Aleurites moluccana [ 4 ]. All compounds were tested for cytotoxicity except for one. Only one compound, 13-O-myristyl-20-O-acetyl-12-deoxyphorbol, showed modest inhibitory action against Raji (ATCC number CCL-86) cells, with an IC50 value of 4.24 mg/ml [ 5 ]. To the best of the authors' knowledge, there has been limited comprehensive coverage on the chemical investigation, and activity assessment of A. moluccana . No scientific report has been published on the anticancer evaluation of Aleurites moluccana. Therefore, the present research aims to evaluate the anticancer activity of ethanol extracts of Aleurites moluccana fruit against colorectal cancer cells (HCT-116) in vitro using the MTT assay. Figure 1 shows the twig and fruits of Aleurites moluccana. Experimental Procurement and Identification of Plant Materials The plant Aleurites moluccana (Euphorbiaceae) was acquired from Munnalal Dawasas, Hyderabad, was meticulously examined and subsequently approved by a qualified botanist. This rigorous verification ensures the authenticity and scientific accuracy of the specimen, contributing significantly to the credibility of ongoing research and studies. Plant Ethics Approval and Consent to Participate The authors confirm that all methods were carried out in accordance with relevant guidelines and regulations of NMPB. The collection of the plants used in the study complies with local or national guidelines with no need for further affirmation. Aleurites moluccana was collected in Botanical garden Hyderabad. The plant material was identified by Dr. V. B. Reddy and a voucher specimen was deposited at Dept. of Botany, O.U, Hyderabad with voucher ID OUAS-225. Test Cancer Cells For anticancer testing, colorectal cancer cell lines, including HCT-116 and primary colon epithelial cells (PCE), were selected and obtained from KFRC, Hyderabad. Chemicals and Reagents The chemicals and reagents used include sodium hydroxide solution, dilute hydrochloric acid, 95% ethanol, concentrated hydrochloric acid, Mayer’s reagent, Wagner’s reagent, ferric chloride solution, Folin-Ciocalteu reagent, sodium carbonate solution, chloroform, concentrated sulfuric acid, dilute ammonia solution, Sudan III reagent, Molisch’s reagent, Benedict’s reagent, potassium permanganate solution, tetracycline, Dimethyl sulfoxide (DMSO), water for injection, aqueous, petroleum ether, and ethanol. Additionally, MTT solution, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and trypsin are used. General Laboratory Glassware This includes beakers, round bottom flasks, conical flasks, measuring cylinders, blenders, porcelain dishes, heating mantles, filter papers, cotton wool, test tubes, stirring glass rods, wire loops, micro hematocrit tubes, cork borers, analytical weighing balances, water baths, Bunsen burners, 10 mL serological pipettes (sterile), 96-well plates, T25 flasks, hemocytometers, Schott bottles, Eppendorf tubes, Falcon tubes, and micropipette tips. Specific Instruments This includes Soxhlet extractors, condensers, hot air ovens, desiccators, automated micropipettes, automated multichannel micropipettes, spectrophotometers, rotary evaporators, analytical thin layer chromatography (TLC) plates (Merck, India), TLC chambers, microtiter plate shakers, biological safety cabinets (BSC), incubators, Glomax Multi Detection Plate Readers (used for measuring luminescence, fluorescence, and absorbance in microplate assays), inverted microscopes (used for observing cells and organisms at the bottom of culture vessels), and centrifuges. Preparation of Plant Extracts The fruits of the plant were dried in the shade before being separated from the seeds and ground into coarse powder. This fruit powder was then extracted using a Soxhlet apparatus with ethanol as extraction solvent. Approximately 100 g of the powdered fruit was extracted with ethanol as solvent, using 200 ml of solvent in the Soxhlet extractor. The total solvent used was in a 1:2 ratios, with two parts solvent to one-part powder. The powder was placed in the main chamber of the Soxhlet extractor, above a round bottom flask filled with 200 ml of solvent, and heated on a heating mantle. The evaporated solvent passed through the sample and condensed back into the chamber, dripping onto the sample until the solvent color in the siphon tube faded. The obtained extract was filtered at the end of the process to remove impurities. The crude extract was then concentrated using a rotary evaporator under reduced pressure at appropriate temperatures at 79°C (175 mbar) for ethanol. The concentrated extract was placed in a desiccator containing calcium chloride to prevent moisture and stored in airtight containers at below 4°C to prevent microbial growth and protect from direct sunlight. Preliminary Phytochemical Analysis Plant extracts of Aleurites moluccana fruits were tested to identify the presence of various phytochemical compounds. The screening was done to qualitatively detect flavonoids, alkaloids, phenolic compounds, terpenoids, tannins, glycosides, saponins, fixed oils and fats, carbohydrates, and proteins, all of which have potential pharmacological effects. Anticancer Screening: Plant Extract Dilution: Ethanolic plant extract contained 2000 µg was dissolved in 2.0 mL of phosphate buffered saline. The concentrations listed below were prepared using a two-fold dilution method: The concentrations used were 1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.2 µg/mL, 15.6 µg/mL, and 7.8 µg/mL. Preparation of culture medium: Gathered RPMI 1640 medium, FBS (pre-warmed to 56°C for 30 minutes to inactivate the complement), Penicillin-streptomycin, and a sterile 250 mL Schott bottle. Poured 250 mL of RPMI 1640 into the sterile Schott bottle. Using a sterile serological pipette, carefully pipetted 25 mL (10%) of FBS into the RPMI 1640 medium, ensuring the pipette tip did not touch any surfaces to maintain sterility. Discarded the pipette if contamination occurred. Using a new sterile serological pipette, added 2.5 mL (1%) of Penicillin-streptomycin to the medium. Gently stirred the Schott bottle to mix the supplements thoroughly with the medium. Adjusted the total volume of the medium to 250 mL. Labeled the prepared 250 mL medium and stored in a refrigerator at 4°C, ready for use. Cell Culture: The colorectal cancer cells HCT-116 and PCE were grown in RPMI 1640 media with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin [ 6 ]. The cultures were kept in T25 flasks and incubated for 48 hours at 37°C in a humidified environment with 5% CO 2 . Cell Seeding Cells were seeded when they reached approximately 80–90% confluency. First, the growth medium was aspirated and the cells were washed three times with PBS solution to remove any residual medium. Next, the cells were treated with trypsin for 5 minutes to detach them from the culture surface. The trypsinized cells were then collected into a Falcon tube and centrifuged for 5 minutes. After centrifugation, the supernatant (medium) was carefully discarded, leaving the cell pellet at the bottom of the tube. Fresh medium was added to the Falcon tube containing the cell pellet, and the mixture was gently pipetted to re-suspend the cells. Exponentially growing colorectal cancer cells (HCT-116) and PCE were seeded at a density of 1000 cells per well into 96-well plates and incubated for 48 hours. The number of viable cells per well was determined using a hemocytometer [ 7 ]. Cell Treatment Various concentrations of extracts were prepared (1000, 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 µg/mL). The growth medium was aspirated from the wells of a 96-well plate, and the prepared extracts were added to the wells. The plates were then placed in an incubator and maintained for 24, 48, and 72 hours under standard cell culture conditions. As a positive control, 500 µg/mL of 5-fluorouracil was included in separate wells alongside the extracts [ 8 ]. MTT Assay [ 9 ] Following treatment periods of 24, 48, and 72 hours, MTT solution was added to the wells of the 96-well plates to assess the cytotoxicity of the extracts. The MTT solution, at a concentration of 5 mg/mL, was added to each well, and the plates were then placed in an incubator at 37°C for 4 hours. After 4-hour incubation period, the cell suspension in each well was carefully removed using a micropipette. Subsequently, 100 µL of dimethyl sulfoxide (DMSO) was added to each well, and the plates were covered with aluminum foil. The plates were then placed on a microtiter plate shaker and shaken for 15 minutes to ensure thorough mixing. Quantification of MTT Cell Proliferation Assay The 96-well plate was placed into a Glomax microplate reader to measure absorbance readings at a wavelength of 570 nm, with a reference wavelength of 630 nm. The percentage of cell viability was calculated based on the dose-response relationship, which illustrates how the cells respond to different concentrations of the tested compounds (extract). The IC 50 value, representing the concentration at which 50% of cell growth is inhibited, was determined by plotting a graph of percentage cell viability (y-axis) against the concentration of the compounds (x-axis). Results and Discussion Soxhlet Extraction of Aleurites moluccana Fruits A total of 100 g of coarse powder from air-dried fruits was subjected to Soxhlet extraction using ethanol. The percentage yield of the fruit extraction was calculated using the formula: The characteristics of ethanolic extract of Aleurites moluccana fruit was studied. A mass of 15.01 grams of extract was yielded, resulting in a percentage yield of 15%. The extract exhibited a dark brown color and had a semi-solid consistency. Phytochemical screening: The preliminary phytochemical analysis of Aleurites moluccana fruit extracts aimed to identify alkaloids, flavonoids, terpenoids, phenolic compounds, tannins, glycosides, steroids, saponins, fixed oils and fats, carbohydrates, and proteins. Table 1 summarizes the findings from this screening. The qualitative analysis revealed that the ethanol extract of Aleurites moluccana fruits contained significant amounts of alkaloids, phenolic compounds, triterpenoids, tannins, glycosides, saponins, and carbohydrates, indicating potential bioactive properties [ 10 ]. Table 1 Phytochemical analysis of Ethanolic extracts of Aleurites moluccana. Phytochemical Identification test Ethanolic Extract Flavonoid Shinoda test - Alkaloid Mayer’s reagent - Wagner’s reagent - Phenolic Ferric chloride + Folin-Ciocalteau + Triterpenoid Salkowski + Tannin Ferric chloride + Diluted KMnO 4 + Glycoside Modified Borntrager (C-glycoside) + Saponin Foam + Fats and oils Sudan Red III - Carbohydrates Molisch’s reagent - Benedict’s reagent - Protein Xanthoprotein - Anticancer Evaluation: The HCT-116 colorectal cancer cells and primary colon epithelial (PCE) cells were used to assess the anticancer activity of ethanol extracts from Aleurites moluccana (EEAM) fruits [ 11 ]. The proportion of cell inhibition and IC50 values were calculated using plots in which the percentage of cell inhibition (y-axis) was plotted against extract concentration (x-axis). These graphs enabled a visual comparison of the extracts' cytotoxic effects on HCT-116 and PCE cells during three different treatment periods: 24, 48, and 72 hours. The outcomes demonstrated the extracts' varying degrees of cytotoxicity over time, offering important new information on their potential as anticancer treatments. Cell Viability Assessment Samples in triplicate were prepared in a 96-well plate and analyzed using a Glomax microplate reader to determine cell viability (%). The formula used for calculation was [ 12 ]: For HCT-116 cancer cells, the percentage of cell viability was measured at 24, 48, and 72 hours of incubation with a positive control of Fluorouracil at 10 µg/mL. Results were tabulated in Table 2. Similarly, for primary colon epithelial (PCE) cells, representing the normal cell line, the cell viability percentage was calculated using the same formula and tabulated in Table 2 across the three treatment periods. Based on Table 2 , graphs were plotted showing the percentage of cell viability against the concentration of EEAM (ethanol extracts of Aleurites moluccana fruits). Figures 2 and 3 depict different treatment durations (24, 48, and 72 hours) for each cell line (HCT-116 and PCE), except for Figs. 2 and 3 , which combine data from all three treatment periods. These graphs illustrate the varying effects of EEAM extracts on cell viability over time for both cancerous and normal cell lines. The Fig. 2 depicted the cytotoxic impact of EEAM on HCT-116 cell lines across concentrations ranging from 7.8 µg/mL to 1000 µg/mL, following a two-fold serial dilution, over a 24-hour treatment period. Analysis of the graph data indicated that EEAM exerted potent cytotoxic effects against HCT-116 cells. Notably, a significant reduction in cell viability was observed, with an IC50 value of 14.99 µg/mL. Increasing concentrations of EEAM from 15.6 µg/mL to 1000 µg/mL led to progressively lower percentages of cell viability: 48.65%, 46.99%, 45.64%, 43.16%, 40.31%, 38.43%, and 28.09%, respectively. This concentration-dependent response indicates that higher concentrations of EEAM resulted in greater suppression of cell viability among HCT-116 cells. Comparatively, EEAM exhibited superior cytotoxic efficacy compared to the standard chemotherapeutic drug (positive control). At a concentration of 15.6 µg/mL, EEAM achieved a cell viability percentage of 48.65%. This trend persisted across higher concentrations, with EEAM consistently demonstrating lower cell viability percentages than the positive control drug. In summary, the results underscore the concentration-dependent cytotoxicity of EEAM against HCT-116 cells, highlighting its potential as a promising therapeutic agent in cancer treatment research. Figure 2 illustrates the cytotoxic impact of EEAM on HCT-116 cell lines across concentrations ranging from 7.8 µg/mL to 1000 µg/mL after a 48-hour treatment period. The graph clearly shows that EEAM exerted a significant cytotoxic effect against HCT-116 cells. An IC50 value of 8.97 µg/mL was determined, indicating the concentration at which 50% of the cells were inhibited. As the concentration of EEAM increased beyond the IC50 value, there was a corresponding decrease in cell viability: 38.60%, 34.52%, 32.52%, 29.48%, 25.84%, 23.40%, and 18.84%, respectively. Compared to Fluorouracil at 10 µg/mL, EEAM demonstrated a stronger cytotoxic effect. While the cell viability with Fluorouracil was 48.50%, the viability with EEAM ranged lower than 48.50% across concentrations from 15.63 µg/mL to 1000 µg/mL. These findings highlight the concentration-dependent cytotoxicity of EEAM against HCT-116 cells, suggesting its potential as an effective agent in cancer treatment. Figure 2 illustrates the cytotoxic effects of EEAM on HCT-116 cell lines after a 72-hour treatment period. EEAM demonstrated strong cytotoxic activity against HCT-116 cells, with an IC50 value observed at 8.54 µg/mL. The lowest percentage of cell viability, 17.87%, was recorded at the highest concentration tested, 1000 µg/mL. In comparison to the standard control, EEAM exhibited greater cytotoxic effects than the positive control only at concentrations of 250 µg/mL and above. These results highlight the potent concentration-dependent cytotoxicity of EEAM against HCT-116 cells, suggesting its potential as an effective therapeutic agent for cancer treatment. The cytotoxicity observed followed a decreasing trend, with the lowest viability seen after the 72-hour treatment period compared to 24 and 48 hours. This indicates that longer exposure to EEAM resulted in higher cytotoxicity. Specifically, the 72-hour treatment period demonstrated the most pronounced cytotoxic effects, with an IC50 value as low as 8.54 µg/mL, which was lower than those observed at 24 and 48 hours. These findings underscore the potent and time-dependent cytotoxic potential of EEAM against HCT-116 cells. The table-2 and Figure-3 presents the percentage viability of PCE cells at 24, 48, and 72-hour intervals. Initially, the cell viability is 100% across all time points, indicating the control or untreated cells. As treatment progresses, there is a notable decrease in cell viability at each interval. At 24 hours, the viability drops from 100% to as low as 41.01%. This decline continues at 48 hours, with viability decreasing further to 32.36%. By 72 hours, the viability reaches its lowest point, with a minimum of 30.71%. These observations highlight a clear trend: the viability of PCE cells progressively decreases over time, demonstrating that the cytotoxic effect of the treatment intensifies with longer exposure durations. Conclusion The phytochemical screening was the initial phase of this study. The ethanol extract of Aleurites moluccana fruit showed the presence of most constituents, including phenolics, terpenoids, tannins, glycosides, and saponins. In the evaluation of anticancer activity of Aleurites moluccana fruits using an MTT-based cytotoxic assay, ethanol extract was tested on colorectal cell lines HCT-116 and primary colon epithelial (PCE) cells. Cytotoxicity followed a decreasing trend, with the lowest viability observed at 72 hours compared to 24 and 48 hours. The 72-hour treatment showed the highest cytotoxicity with an IC50 of 8.54 µg/mL in HCT-116 and IC50 of 8.54 µg/mL in PCE-cells was found to be 196.72 µg/mL. These findings highlight the potent and time-dependent cytotoxic effect of EEAM on HCT-116 cells. Table-2: Cell viability of EEAM on HCT-116 Cells and PCE-Cells after 24, 48, and 72 hours of treatment EEAM Extract (µg/mL) % Viability of HCT-116-Cells % Viability of PCE Cells 24 hrs 48 hrs 72 hrs 24 hrs 48 hrs 72 hrs Negative Control 100.00 100.00 100.00 100.00 100.00 100.00 Positive Control 59.63 48.50 29.95 59.49 47.83 30.71 7.8 65.29 52.02 50.93 83.21 61.07 71.52 15.63 48.65 38.60 41.14 72.01 58.12 69.54 31.25 46.99 34.52 37.44 70.48 57.30 68.04 62.5 45.64 32.52 35.14 62.93 54.31 65.29 125 43.16 29.48 31.28 55.99 52.75 61.51 250 40.31 25.84 27.90 49.26 48.52 41.45 500 38.43 23.40 22.22 45.11 34.13 37.33 1000 28.09 18.84 17.87 41.01 32.36 32.09 The table shows the effects of different EEAM Extract concentrations (µg/mL) on HCT-116 and PCE cell viability over 24, 48, and 72 hours. Negative control maintains 100% viability, while positive control shows a decrease, with HCT-116 cells at 59.63%, 48.50%, and 29.95%, and PCE cells at 59.49%, 47.83%, and 30.71%. At 7.8 µg/mL, HCT-116 cell viability is moderately reduced, but drops significantly at 1000 µg/mL to 28.09%, 18.84%, and 17.87%. PCE cells also show decreased viability, less pronounced than HCT-116 cells. Higher EEAM concentrations result in lower viability, more significantly in HCT-116 cells. Declarations Author Contribution Both authors, Dr. Nemala Appala Raju and Dr. Anitha Nandagopal, contributed equally to this work. Dr. Nemala Appala Raju and Dr. Anitha Nandagopal were involved in the conceptualization, methodology, formal analysis, and investigation of the research. They jointly handled resources and data curation. The original draft was written by both authors, and both contributed to the review and editing process. Visualization of the results was performed by Dr. Nemala Appala Raju and Dr. Anitha Nandagopal. Supervision and project administration were shared responsibilities between the authors. Both authors have read and agreed to the published version of the manuscript. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request References Shaah MA, Allafi F, Hossain MS, Alsaedi A, Ismail N, Kadir MOA, Ahmad MI. Candlenut oil: review on oil properties and future liquid biofuel prospects. Int J Energy Res. 2021; 45(0):17057–79. Zakaria M, Hawa LC, Djoyowasito G. Effect of NaOH concentration and immersion of ice water on physical and mechanical characteristics of candlenut seeds ( Aleurites moluccana L Willd). Indones Green Technol J. 2019; 8(1):22–30. Hidayat S, Zuhud EA, Widyatmoko D, Bahruni B, Batubara I. The commercial potential of forest trees as medicinal and health ingredients. Biodivers J Bio Divers. 2021; 22(7): 2795–804. Liu HY, Li SJ, Zhao Yi, Ni W, Hao XJ, Li JZ, Hua Y, Xie BB, Qing C, Chen CX. Four new podocarpane-type trinorditerpenes from Aleurites moluccana . Helv Chim Acta. 2007; 90: 2017–23. Sun CY, Liu XY, Chen Y, Liu F, Wang Y. Experimental study on anticancer effect of curcumin on Raji cells in vitro. Zhongguo Zhong Xi Yi Jie He Za Zhi. 2004; 24(11):1003–6. Xu Y, Zhang L, Wang Q, Zheng M. Comparison of different colorectal cancer with liver metastases models using six colorectal cancer cell lines. Pathol. Oncol. Res. 2020; 26: 2177–2183. Betul AY. Anti-cancer, antiproliferative activity of active anionic H2O8 oxygen solution on HCT-116 cancer cell. W J Adv Res and Rev. 2021; 12(02): 179–184. Erdoğan MK, Agca CA, Askin H. Enhanced antiproliferative and apoptotic effects of 5-fluorouracil by combined with Pistacia eurycarpa extracts on human colorectal cancer cells. Bio Di Con. 2019; 12: 27–38. Shen W, Zhao Y, Chen H, Zhang T, Wu S, Liu P. M3, a natural lignan xyloside, exhibits potent anticancer activity in HCT116 cells. Oncol Lett. 2019;17(2):2117–22. Ayouaz S, Fibri DLN, Arab R, Mouhoubi K, Madani K. Phytochemistry and biological-pharmacological profile of Aleurites moluccanus : A critical review. 2023; 4(2): 310–16. Guon TE, Chung H. Induction of apoptosis with Moringa oleifera fruits in HCT116 human colon cancer cells via intrinsic pathway. Nat Prod Sci. 2017; 23: 227–34. Agudelo C, Arango VS, Cortacs-Mancera F, Rojano B, Maldonado ME. Antiproliferative and pro-apoptotic effects of Andean berry juice (Vaccinium meridionale Swartz) on human colon adenocarcinoma SW480 cells. J Med Plants Res. 2017; 11(24): 393–402. 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-4748200","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":342671496,"identity":"9a5ce1cb-ccea-44be-88dc-ba420f82d56a","order_by":0,"name":"APPALA RAJU NEMALA","email":"","orcid":"","institution":"Sultan ul Uloom College of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"APPALA","middleName":"RAJU","lastName":"NEMALA","suffix":""},{"id":342671497,"identity":"971af7f6-86ce-4d2c-ac23-c78ed6224638","order_by":1,"name":"ANITHA NANDAGOPAL","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYFACxgZmBgYbOJeHGC2NzQwMaSRpYWAEajlMgrP4Zze3Py6oOW9vcPzsAYYfNQwy5oS0SNw52Ng849jtxA1n8hIYe44x8Fg2ENJzI7GxmYftdoLBDR4DBt4GBh6DAwR0yIO1/DtnD9LC+JcYLQYgLbxtBxg3ALUwE2WLIVDLbN6+5MSZQL8cljkmQViL3I30B595vtnZ8x0/e/Dhmxobe4JakAAPA1CxBPHqGYiM91EwCkbBKBiJAADlfkA8W3aLOQAAAABJRU5ErkJggg==","orcid":"","institution":"Sultan ul Uloom College of Pharmacy","correspondingAuthor":true,"prefix":"","firstName":"ANITHA","middleName":"","lastName":"NANDAGOPAL","suffix":""}],"badges":[],"createdAt":"2024-07-16 08:29:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4748200/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4748200/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62948710,"identity":"78b45a96-ad66-4811-b83e-482bf9d484c4","added_by":"auto","created_at":"2024-08-21 10:51:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAleurites moluccana\u003c/em\u003e twig, Fruit and kernel\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4748200/v1/7c1548ec49ab8914a783ebf1.jpg"},{"id":62949404,"identity":"5c955a04-e66a-44e2-b700-4b769671da4b","added_by":"auto","created_at":"2024-08-21 10:59:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxic activity of EEAM extract against HCT-116 Cells\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4748200/v1/791a4a1e4f788ee63aff9040.jpg"},{"id":62948711,"identity":"07b2fcd9-c6d4-4c1c-822f-02f1cef216c5","added_by":"auto","created_at":"2024-08-21 10:51:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxic activity of EEAM extract against HCT-116 Cells\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4748200/v1/cae19fd318cf82bfdf70fc3a.jpg"},{"id":68801496,"identity":"2b7d9297-a2d0-4bd8-830b-271ef0802fe7","added_by":"auto","created_at":"2024-11-12 07:24:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":715302,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4748200/v1/28c1d058-f108-4966-8af3-79e824c5bd72.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Anti-tumour effect of Aleurites moluccana on HCT-116 Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAleurites moluccana\u003c/em\u003e is a medium-sized tree that can grow up to 20 meters tall, with wide-spreading or hanging branches. Its bark is grey-brown and relatively smooth with fine vertical lines. The fruit is an indehiscent drupe, nearly spherical, measuring 5 cm or more in diameter, and has a thick, rough, hard shell that makes up 64\u0026ndash;68% of the fruit. This shell is difficult to separate from the kernels and contains 1\u0026ndash;2 hard-shelled black seeds. \u003cem\u003eAleurites moluccanus\u003c/em\u003e (L.) Willd, a Euphorbiaceae plant, is native to Indonesia, India, and the South Sea Islands [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is known as the \"Candlenut tree,\" \"Kukui,\" and \"Indian Walnut,\" and it was introduced in the early 20th century for its oil seeds, producing \"tung oil,\" and as an ornamental plant [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Each tree can yield 30\u0026ndash;80 kg of nuts. \u003cem\u003eAleurites moluccana\u003c/em\u003e is used in traditional medicine to treat pain, fever, asthma, hepatitis, gastric ulcers, inflammation. This species is also utilized in traditional medicine for the treatment of cardiovascular diseases, anemia, high cholesterol, hypertension, and diabetes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The nut oil is applied topically for arthritis and joint pain. However, the seeds are toxic if ingested. Four new podocarpane-type tri-nor-di-terpenenes and four known diterpenes were isolated from the twigs and leaves of \u003cem\u003eAleurites moluccana\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. All compounds were tested for cytotoxicity except for one. Only one compound, 13-O-myristyl-20-O-acetyl-12-deoxyphorbol, showed modest inhibitory action against Raji (ATCC number CCL-86) cells, with an IC50 value of 4.24 mg/ml [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To the best of the authors' knowledge, there has been limited comprehensive coverage on the chemical investigation, and activity assessment of \u003cem\u003eA. moluccana\u003c/em\u003e. No scientific report has been published on the anticancer evaluation of \u003cem\u003eAleurites moluccana.\u003c/em\u003e Therefore, the present research aims to evaluate the anticancer activity of ethanol extracts of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruit against colorectal cancer cells (HCT-116) in vitro using the MTT assay. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the twig and fruits of \u003cem\u003eAleurites moluccana.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProcurement and Identification of Plant Materials\u003c/h2\u003e \u003cp\u003eThe plant \u003cem\u003eAleurites moluccana\u003c/em\u003e (Euphorbiaceae) was acquired from Munnalal Dawasas, Hyderabad, was meticulously examined and subsequently approved by a qualified botanist. This rigorous verification ensures the authenticity and scientific accuracy of the specimen, contributing significantly to the credibility of ongoing research and studies.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003ePlant Ethics Approval and Consent to Participate\u003c/h2\u003e \u003cp\u003eThe authors confirm that all methods were carried out in accordance with relevant guidelines and regulations of NMPB. The collection of the plants used in the study complies with local or national guidelines with no need for further affirmation. \u003cem\u003eAleurites moluccana\u003c/em\u003e was collected in Botanical garden Hyderabad. The plant material was identified by Dr. V. B. Reddy and a voucher specimen was deposited at Dept. of Botany, O.U, Hyderabad with voucher ID OUAS-225.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTest Cancer Cells\u003c/h2\u003e \u003cp\u003eFor anticancer testing, colorectal cancer cell lines, including HCT-116 and primary colon epithelial cells (PCE), were selected and obtained from KFRC, Hyderabad.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Reagents\u003c/h2\u003e \u003cp\u003eThe chemicals and reagents used include sodium hydroxide solution, dilute hydrochloric acid, 95% ethanol, concentrated hydrochloric acid, Mayer\u0026rsquo;s reagent, Wagner\u0026rsquo;s reagent, ferric chloride solution, Folin-Ciocalteu reagent, sodium carbonate solution, chloroform, concentrated sulfuric acid, dilute ammonia solution, Sudan III reagent, Molisch\u0026rsquo;s reagent, Benedict\u0026rsquo;s reagent, potassium permanganate solution, tetracycline, Dimethyl sulfoxide (DMSO), water for injection, aqueous, petroleum ether, and ethanol. Additionally, MTT solution, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and trypsin are used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGeneral Laboratory Glassware\u003c/h2\u003e \u003cp\u003eThis includes beakers, round bottom flasks, conical flasks, measuring cylinders, blenders, porcelain dishes, heating mantles, filter papers, cotton wool, test tubes, stirring glass rods, wire loops, micro hematocrit tubes, cork borers, analytical weighing balances, water baths, Bunsen burners, 10 mL serological pipettes (sterile), 96-well plates, T25 flasks, hemocytometers, Schott bottles, Eppendorf tubes, Falcon tubes, and micropipette tips.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSpecific Instruments\u003c/h2\u003e \u003cp\u003eThis includes Soxhlet extractors, condensers, hot air ovens, desiccators, automated micropipettes, automated multichannel micropipettes, spectrophotometers, rotary evaporators, analytical thin layer chromatography (TLC) plates (Merck, India), TLC chambers, microtiter plate shakers, biological safety cabinets (BSC), incubators, Glomax Multi Detection Plate Readers (used for measuring luminescence, fluorescence, and absorbance in microplate assays), inverted microscopes (used for observing cells and organisms at the bottom of culture vessels), and centrifuges.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003ePreparation of Plant Extracts\u003c/h2\u003e \u003cp\u003eThe fruits of the plant were dried in the shade before being separated from the seeds and ground into coarse powder. This fruit powder was then extracted using a Soxhlet apparatus with ethanol as extraction solvent.\u003c/p\u003e \u003cp\u003eApproximately 100 g of the powdered fruit was extracted with ethanol as solvent, using 200 ml of solvent in the Soxhlet extractor. The total solvent used was in a 1:2 ratios, with two parts solvent to one-part powder. The powder was placed in the main chamber of the Soxhlet extractor, above a round bottom flask filled with 200 ml of solvent, and heated on a heating mantle. The evaporated solvent passed through the sample and condensed back into the chamber, dripping onto the sample until the solvent color in the siphon tube faded.\u003c/p\u003e \u003cp\u003eThe obtained extract was filtered at the end of the process to remove impurities. The crude extract was then concentrated using a rotary evaporator under reduced pressure at appropriate temperatures at 79\u0026deg;C (175 mbar) for ethanol. The concentrated extract was placed in a desiccator containing calcium chloride to prevent moisture and stored in airtight containers at below 4\u0026deg;C to prevent microbial growth and protect from direct sunlight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003ePreliminary Phytochemical Analysis\u003c/h2\u003e \u003cp\u003ePlant extracts of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruits were tested to identify the presence of various phytochemical compounds. The screening was done to qualitatively detect flavonoids, alkaloids, phenolic compounds, terpenoids, tannins, glycosides, saponins, fixed oils and fats, carbohydrates, and proteins, all of which have potential pharmacological effects.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnticancer Screening:\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003ePlant Extract Dilution:\u003c/h2\u003e \u003cp\u003eEthanolic plant extract contained 2000 \u0026micro;g was dissolved in 2.0 mL of phosphate buffered saline. The concentrations listed below were prepared using a two-fold dilution method: The concentrations used were 1000 \u0026micro;g/mL, 500 \u0026micro;g/mL, 250 \u0026micro;g/mL, 125 \u0026micro;g/mL, 62.5 \u0026micro;g/mL, 31.2 \u0026micro;g/mL, 15.6 \u0026micro;g/mL, and 7.8 \u0026micro;g/mL.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of culture medium:\u003c/h2\u003e \u003cp\u003eGathered RPMI 1640 medium, FBS (pre-warmed to 56\u0026deg;C for 30 minutes to inactivate the complement), Penicillin-streptomycin, and a sterile 250 mL Schott bottle. Poured 250 mL of RPMI 1640 into the sterile Schott bottle. Using a sterile serological pipette, carefully pipetted 25 mL (10%) of FBS into the RPMI 1640 medium, ensuring the pipette tip did not touch any surfaces to maintain sterility. Discarded the pipette if contamination occurred. Using a new sterile serological pipette, added 2.5 mL (1%) of Penicillin-streptomycin to the medium. Gently stirred the Schott bottle to mix the supplements thoroughly with the medium. Adjusted the total volume of the medium to 250 mL. Labeled the prepared 250 mL medium and stored in a refrigerator at 4\u0026deg;C, ready for use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture:\u003c/h2\u003e \u003cp\u003eThe colorectal cancer cells HCT-116 and PCE were grown in RPMI 1640 media with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The cultures were kept in T25 flasks and incubated for 48 hours at 37\u0026deg;C in a humidified environment with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell Seeding\u003c/h2\u003e \u003cp\u003eCells were seeded when they reached approximately 80\u0026ndash;90% confluency. First, the growth medium was aspirated and the cells were washed three times with PBS solution to remove any residual medium.\u003c/p\u003e \u003cp\u003eNext, the cells were treated with trypsin for 5 minutes to detach them from the culture surface. The trypsinized cells were then collected into a Falcon tube and centrifuged for 5 minutes. After centrifugation, the supernatant (medium) was carefully discarded, leaving the cell pellet at the bottom of the tube. Fresh medium was added to the Falcon tube containing the cell pellet, and the mixture was gently pipetted to re-suspend the cells.\u003c/p\u003e \u003cp\u003eExponentially growing colorectal cancer cells (HCT-116) and PCE were seeded at a density of 1000 cells per well into 96-well plates and incubated for 48 hours. The number of viable cells per well was determined using a hemocytometer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell Treatment\u003c/h2\u003e \u003cp\u003eVarious concentrations of extracts were prepared (1000, 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 \u0026micro;g/mL). The growth medium was aspirated from the wells of a 96-well plate, and the prepared extracts were added to the wells. The plates were then placed in an incubator and maintained for 24, 48, and 72 hours under standard cell culture conditions. As a positive control, 500 \u0026micro;g/mL of 5-fluorouracil was included in separate wells alongside the extracts [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMTT Assay\u003c/b\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eFollowing treatment periods of 24, 48, and 72 hours, MTT solution was added to the wells of the 96-well plates to assess the cytotoxicity of the extracts. The MTT solution, at a concentration of 5 mg/mL, was added to each well, and the plates were then placed in an incubator at 37\u0026deg;C for 4 hours.\u003c/p\u003e \u003cp\u003eAfter 4-hour incubation period, the cell suspension in each well was carefully removed using a micropipette. Subsequently, 100 \u0026micro;L of dimethyl sulfoxide (DMSO) was added to each well, and the plates were covered with aluminum foil. The plates were then placed on a microtiter plate shaker and shaken for 15 minutes to ensure thorough mixing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of MTT Cell Proliferation Assay\u003c/h2\u003e \u003cp\u003eThe 96-well plate was placed into a Glomax microplate reader to measure absorbance readings at a wavelength of 570 nm, with a reference wavelength of 630 nm.\u003c/p\u003e \u003cp\u003eThe percentage of cell viability was calculated based on the dose-response relationship, which illustrates how the cells respond to different concentrations of the tested compounds (extract). The IC\u003csub\u003e50\u003c/sub\u003e value, representing the concentration at which 50% of cell growth is inhibited, was determined by plotting a graph of percentage cell viability (y-axis) against the concentration of the compounds (x-axis).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eSoxhlet Extraction of\u003c/b\u003e \u003cb\u003eAleurites moluccana\u003c/b\u003e \u003cb\u003eFruits\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 100 g of coarse powder from air-dried fruits was subjected to Soxhlet extraction using ethanol. The percentage yield of the fruit extraction was calculated using the formula:\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"274\" height=\"58\"\u003e\u003c/p\u003e\u003cp\u003eThe characteristics of ethanolic extract of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruit was studied. A mass of 15.01 grams of extract was yielded, resulting in a percentage yield of 15%. The extract exhibited a dark brown color and had a semi-solid consistency.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePhytochemical screening:\u003c/h2\u003e \u003cp\u003eThe preliminary phytochemical analysis of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruit extracts aimed to identify alkaloids, flavonoids, terpenoids, phenolic compounds, tannins, glycosides, steroids, saponins, fixed oils and fats, carbohydrates, and proteins. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the findings from this screening. The qualitative analysis revealed that the ethanol extract of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruits contained significant amounts of alkaloids, phenolic compounds, triterpenoids, tannins, glycosides, saponins, and carbohydrates, indicating potential bioactive properties [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhytochemical analysis of Ethanolic extracts of \u003cem\u003eAleurites moluccana.\u003c/em\u003e\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhytochemical\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIdentification test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEthanolic Extract\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShinoda test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAlkaloid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMayer\u0026rsquo;s reagent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWagner\u0026rsquo;s reagent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePhenolic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFerric chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFolin-Ciocalteau\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriterpenoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSalkowski\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTannin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFerric chloride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiluted KMnO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eModified Borntrager\u003c/p\u003e \u003cp\u003e(C-glycoside)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaponin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFoam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFats and oils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSudan Red III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCarbohydrates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolisch\u0026rsquo;s reagent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBenedict\u0026rsquo;s reagent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXanthoprotein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\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=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnticancer Evaluation:\u003c/h2\u003e \u003cp\u003eThe HCT-116 colorectal cancer cells and primary colon epithelial (PCE) cells were used to assess the anticancer activity of ethanol extracts from \u003cem\u003eAleurites moluccana\u003c/em\u003e (EEAM) fruits [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The proportion of cell inhibition and IC50 values were calculated using plots in which the percentage of cell inhibition (y-axis) was plotted against extract concentration (x-axis).\u003c/p\u003e \u003cp\u003eThese graphs enabled a visual comparison of the extracts' cytotoxic effects on HCT-116 and PCE cells during three different treatment periods: 24, 48, and 72 hours. The outcomes demonstrated the extracts' varying degrees of cytotoxicity over time, offering important new information on their potential as anticancer treatments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCell Viability Assessment\u003c/h2\u003e \u003cp\u003eSamples in triplicate were prepared in a 96-well plate and analyzed using a Glomax microplate reader to determine cell viability (%). The formula used for calculation was [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]:\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"385\" height=\"59\"\u003e\u003c/p\u003e \u003cp\u003eFor HCT-116 cancer cells, the percentage of cell viability was measured at 24, 48, and 72 hours of incubation with a positive control of Fluorouracil at 10 \u0026micro;g/mL. Results were tabulated in Table\u0026nbsp;2. Similarly, for primary colon epithelial (PCE) cells, representing the normal cell line, the cell viability percentage was calculated using the same formula and tabulated in Table\u0026nbsp;2 across the three treatment periods.\u003c/p\u003e \u003cp\u003eBased on \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e, graphs were plotted showing the percentage of cell viability against the concentration of EEAM (ethanol extracts of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruits). Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e depict different treatment durations (24, 48, and 72 hours) for each cell line (HCT-116 and PCE), except for Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, which combine data from all three treatment periods. These graphs illustrate the varying effects of EEAM extracts on cell viability over time for both cancerous and normal cell lines.\u003c/p\u003e \u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicted the cytotoxic impact of EEAM on HCT-116 cell lines across concentrations ranging from 7.8 \u0026micro;g/mL to 1000 \u0026micro;g/mL, following a two-fold serial dilution, over a 24-hour treatment period. Analysis of the graph data indicated that EEAM exerted potent cytotoxic effects against HCT-116 cells. Notably, a significant reduction in cell viability was observed, with an IC50 value of 14.99 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eIncreasing concentrations of EEAM from 15.6 \u0026micro;g/mL to 1000 \u0026micro;g/mL led to progressively lower percentages of cell viability: 48.65%, 46.99%, 45.64%, 43.16%, 40.31%, 38.43%, and 28.09%, respectively. This concentration-dependent response indicates that higher concentrations of EEAM resulted in greater suppression of cell viability among HCT-116 cells.\u003c/p\u003e \u003cp\u003eComparatively, EEAM exhibited superior cytotoxic efficacy compared to the standard chemotherapeutic drug (positive control). At a concentration of 15.6 \u0026micro;g/mL, EEAM achieved a cell viability percentage of 48.65%. This trend persisted across higher concentrations, with EEAM consistently demonstrating lower cell viability percentages than the positive control drug.\u003c/p\u003e \u003cp\u003eIn summary, the results underscore the concentration-dependent cytotoxicity of EEAM against HCT-116 cells, highlighting its potential as a promising therapeutic agent in cancer treatment research.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the cytotoxic impact of EEAM on HCT-116 cell lines across concentrations ranging from 7.8 \u0026micro;g/mL to 1000 \u0026micro;g/mL after a 48-hour treatment period. The graph clearly shows that EEAM exerted a significant cytotoxic effect against HCT-116 cells. An IC50 value of 8.97 \u0026micro;g/mL was determined, indicating the concentration at which 50% of the cells were inhibited.\u003c/p\u003e \u003cp\u003eAs the concentration of EEAM increased beyond the IC50 value, there was a corresponding decrease in cell viability: 38.60%, 34.52%, 32.52%, 29.48%, 25.84%, 23.40%, and 18.84%, respectively. Compared to Fluorouracil at 10 \u0026micro;g/mL, EEAM demonstrated a stronger cytotoxic effect. While the cell viability with Fluorouracil was 48.50%, the viability with EEAM ranged lower than 48.50% across concentrations from 15.63 \u0026micro;g/mL to 1000 \u0026micro;g/mL. These findings highlight the concentration-dependent cytotoxicity of EEAM against HCT-116 cells, suggesting its potential as an effective agent in cancer treatment.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the cytotoxic effects of EEAM on HCT-116 cell lines after a 72-hour treatment period. EEAM demonstrated strong cytotoxic activity against HCT-116 cells, with an IC50 value observed at 8.54 \u0026micro;g/mL. The lowest percentage of cell viability, 17.87%, was recorded at the highest concentration tested, 1000 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003eIn comparison to the standard control, EEAM exhibited greater cytotoxic effects than the positive control only at concentrations of 250 \u0026micro;g/mL and above. These results highlight the potent concentration-dependent cytotoxicity of EEAM against HCT-116 cells, suggesting its potential as an effective therapeutic agent for cancer treatment.\u003c/p\u003e \u003cp\u003eThe cytotoxicity observed followed a decreasing trend, with the lowest viability seen after the 72-hour treatment period compared to 24 and 48 hours. This indicates that longer exposure to EEAM resulted in higher cytotoxicity.\u003c/p\u003e \u003cp\u003eSpecifically, the 72-hour treatment period demonstrated the most pronounced cytotoxic effects, with an IC50 value as low as 8.54 \u0026micro;g/mL, which was lower than those observed at 24 and 48 hours. These findings underscore the potent and time-dependent cytotoxic potential of EEAM against HCT-116 cells.\u003c/p\u003e \u003cp\u003eThe table-2 and Figure-3 presents the percentage viability of PCE cells at 24, 48, and 72-hour intervals. Initially, the cell viability is 100% across all time points, indicating the control or untreated cells. As treatment progresses, there is a notable decrease in cell viability at each interval. At 24 hours, the viability drops from 100% to as low as 41.01%. This decline continues at 48 hours, with viability decreasing further to 32.36%. By 72 hours, the viability reaches its lowest point, with a minimum of 30.71%. These observations highlight a clear trend: the viability of PCE cells progressively decreases over time, demonstrating that the cytotoxic effect of the treatment intensifies with longer exposure durations.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe phytochemical screening was the initial phase of this study. The ethanol extract of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruit showed the presence of most constituents, including phenolics, terpenoids, tannins, glycosides, and saponins. In the evaluation of anticancer activity of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruits using an MTT-based cytotoxic assay, ethanol extract was tested on colorectal cell lines HCT-116 and primary colon epithelial (PCE) cells. Cytotoxicity followed a decreasing trend, with the lowest viability observed at 72 hours compared to 24 and 48 hours. The 72-hour treatment showed the highest cytotoxicity with an IC50 of 8.54 \u0026micro;g/mL in HCT-116 and IC50 of 8.54 \u0026micro;g/mL in PCE-cells was found to be 196.72 \u0026micro;g/mL. These findings highlight the potent and time-dependent cytotoxic effect of EEAM on HCT-116 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTable-2: Cell viability of EEAM on HCT-116 Cells and PCE-Cells after 24, 48, and 72 hours of treatment\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" 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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEEAM Extract (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e% Viability of HCT-116-Cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e% Viability of PCE Cells\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48 hrs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e72 hrs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e30.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e61.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e71.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e58.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e69.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e46.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e57.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e62.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e54.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e65.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e55.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e52.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e61.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e48.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e41.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e34.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e41.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e32.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe table shows the effects of different EEAM Extract concentrations (\u0026micro;g/mL) on HCT-116 and PCE cell viability over 24, 48, and 72 hours. Negative control maintains 100% viability, while positive control shows a decrease, with HCT-116 cells at 59.63%, 48.50%, and 29.95%, and PCE cells at 59.49%, 47.83%, and 30.71%. At 7.8 \u0026micro;g/mL, HCT-116 cell viability is moderately reduced, but drops significantly at 1000 \u0026micro;g/mL to 28.09%, 18.84%, and 17.87%. PCE cells also show decreased viability, less pronounced than HCT-116 cells. Higher EEAM concentrations result in lower viability, more significantly in HCT-116 cells.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBoth authors, Dr. Nemala Appala Raju and Dr. Anitha Nandagopal, contributed equally to this work. Dr. Nemala Appala Raju and Dr. Anitha Nandagopal were involved in the conceptualization, methodology, formal analysis, and investigation of the research. They jointly handled resources and data curation. The original draft was written by both authors, and both contributed to the review and editing process. Visualization of the results was performed by Dr. Nemala Appala Raju and Dr. Anitha Nandagopal. Supervision and project administration were shared responsibilities between the authors. Both authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShaah MA, Allafi F, Hossain MS, Alsaedi A, Ismail N, Kadir MOA, Ahmad MI. Candlenut oil: review on oil properties and future liquid biofuel prospects. Int J Energy Res. 2021; 45(0):17057\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZakaria M, Hawa LC, Djoyowasito G. Effect of NaOH concentration and immersion of ice water on physical and mechanical characteristics of candlenut seeds (\u003cem\u003eAleurites moluccana\u003c/em\u003e L Willd). Indones Green Technol J. 2019; 8(1):22\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHidayat S, Zuhud EA, Widyatmoko D, Bahruni B, Batubara I. The commercial potential of forest trees as medicinal and health ingredients. Biodivers J Bio Divers. 2021; 22(7): 2795\u0026ndash;804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu HY, Li SJ, Zhao Yi, Ni W, Hao XJ, Li JZ, Hua Y, Xie BB, Qing C, Chen CX. Four new podocarpane-type trinorditerpenes from \u003cem\u003eAleurites moluccana\u003c/em\u003e. Helv Chim Acta. 2007; 90: 2017\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun CY, Liu XY, Chen Y, Liu F, Wang Y. Experimental study on anticancer effect of curcumin on Raji cells in vitro. Zhongguo Zhong Xi Yi Jie He Za Zhi. 2004; 24(11):1003\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Y, Zhang L, Wang Q, Zheng M. Comparison of different colorectal cancer with liver metastases models using six colorectal cancer cell lines. Pathol. Oncol. Res. 2020; 26: 2177\u0026ndash;2183.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBetul AY. Anti-cancer, antiproliferative activity of active anionic H2O8 oxygen solution on HCT-116 cancer cell. W J Adv Res and Rev. 2021; 12(02): 179\u0026ndash;184.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErdoğan MK, Agca CA, Askin H. Enhanced antiproliferative and apoptotic effects of 5-fluorouracil by combined with \u003cem\u003ePistacia eurycarpa\u003c/em\u003e extracts on human colorectal cancer cells. Bio Di Con. 2019; 12: 27\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen W, Zhao Y, Chen H, Zhang T, Wu S, Liu P. M3, a natural lignan xyloside, exhibits potent anticancer activity in HCT116 cells. Oncol Lett. 2019;17(2):2117\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAyouaz S, Fibri DLN, Arab R, Mouhoubi K, Madani K. Phytochemistry and biological-pharmacological profile of \u003cem\u003eAleurites moluccanus\u003c/em\u003e: A critical review. 2023; 4(2): 310\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuon TE, Chung H. Induction of apoptosis with \u003cem\u003eMoringa oleifera\u003c/em\u003e fruits in HCT116 human colon cancer cells via intrinsic pathway. Nat Prod Sci. 2017; 23: 227\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgudelo C, Arango VS, Cortacs-Mancera F, Rojano B, Maldonado ME. Antiproliferative and pro-apoptotic effects of Andean berry juice (Vaccinium meridionale Swartz) on human colon adenocarcinoma SW480 cells. J Med Plants Res. 2017; 11(24): 393\u0026ndash;402.\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":"Aleurites moluccanus, Antitumour, Cytotoxicity, HCT-116 Cells and MTT Assay","lastPublishedDoi":"10.21203/rs.3.rs-4748200/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4748200/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eAleurites moluccanus\u003c/b\u003e, commonly referred to as the candlenut or candleberry and also known as the Indian walnut, is a flowering tree belonging to the family Euphorbiaceae. The tree produces large, rounded (sub-globose) or slightly two-lobed fruits measuring 4\u0026ndash;6 cm in diameter. The initial phase of this study involved phytochemical screening, revealing that the ethanol extract of \u003cem\u003eAleurites moluccana\u003c/em\u003e fruit contains phenolics, terpenoids, tannins, glycosides, and saponins. The presence of these compounds might be responsible for the biological activity. The anticancer activity was tested using HCT-116 cell lines and primary colon epithelial (PCE) cell. Different concentration ranging from 7.8 \u0026micro;g/mL to 1000 \u0026micro;g/mL were tested against each cell lines by MTT assay. Ethanol extract of \u003cb\u003eAleurites moluccanus\u003c/b\u003e fruits showed cytotoxic activity when compared to standard control, Fluorouracil (10 \u0026micro;g/mL). In an MTT-based cytotoxic assay evaluating anticancer activity, the ethanol extract was tested on colorectal cell lines HCT-116 and primary colon epithelial (PCE) cells. Cytotoxicity increased over time, with the highest cytotoxicity observed at 72 hours. The 72-hour treatment exhibits the IC50 of 8.54 \u0026micro;g/mL in HCT-116 cells and 196.72 \u0026micro;g/mL in PCE cells, indicating a potent and time-dependent cytotoxic effect on HCT-116 cells.\u003c/p\u003e","manuscriptTitle":"The Anti-tumour effect of Aleurites moluccana on HCT-116 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-21 10:51:02","doi":"10.21203/rs.3.rs-4748200/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":"e288b3c4-c3eb-4adb-8371-6623b8603dae","owner":[],"postedDate":"August 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-12T07:23:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-21 10:51:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4748200","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4748200","identity":"rs-4748200","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}