Anticancer and Cancer Stem Cell Inhibitory Effects of Eleutherococcus gracilistylus Ethanol Extracts in Colorectal Cancer Models

Anticancer and Cancer Stem Cell Inhibitory Effects of Eleutherococcus gracilistylus Ethanol Extracts in Colorectal Cancer Models | 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 Anticancer and Cancer Stem Cell Inhibitory Effects of Eleutherococcus gracilistylus Ethanol Extracts in Colorectal Cancer Models Kyeoung Cheol Kim, Ji-Hyang Kim, Dong-Sun Lee, Ju-Sung Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6152350/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 Background Eleutherococcus gracilistylus (Seom-ogalpi), a rare deciduous shrub from the Araliaceae family native to Korea, is known for its antioxidant and anticancer properties. The increasing incidence of colorectal cancer, particularly among young adults in Korea, highlights the urgent need for targeted therapies. This is especially important for addressing cancer stem cells, which play a critical role in tumor progression and contribute to treatment resistance. This study investigates the anticancer effects of various parts of E. gracilistylus —leaves, branch, roots, fruits—using ethanol extracts at different concentrations to identify the most biologically active components against colorectal cancer cells and cancer stem cells. Methods E. gracilistylus was collected from Jeju Island, South Korea, and the absolute ethanol extract of fruit was fractionated into n-hexane, dichloromethane, ethyl acetate, butanol, and water fractions. HT-29 and HCT-116 colorectal cancer cell lines were cultured in DMEM with 10% FBS at 37°C, and tumorspheres were formed in ultra-low attachment plates and assessed using the NICE scan program. Cell proliferation was measured using the EZ-Cytox assay, and apoptosis was analyzed via flow cytometry. Gene expression was evaluated using RT-qPCR, and statistical significance was determined with GraphPad Prism 8 software ( p < 0.05). Results The absolute ethanol extract of E. gracilistylus , particularly from the fruit, achieved nearly 90% inhibition in the HT-29 cell line at 250 µg/ml. The ethyl acetate fraction demonstrated the highest cancer stem cell inhibition at 80%, while the butanol fraction significantly induced apoptosis, increasing the rate from 11.9–19.2%. Flow cytometry confirmed a marked reduction in ALDH 1-A1 expression and a concentration-dependent decrease in Nanog and Oct4 levels after treatment with the ethyl acetate fraction. Conclusions The absolute ethanol extract of E. gracilistylus demonstrated potent anticancer effects, especially against HT-29 cells, and significant CSC inhibition. These findings highlight its therapeutic potential in targeting colorectal cancer. Eleutherococcus gracilistylus Colorectal cancer Cancer stem cell Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Eleutherococcus gracilistylus (Seom-ogalpi) is a deciduous broadleaf shrub from the Araliaceae family, primarily distributed in Korea, China and Japan [ 1 ]. This plant is predominantly found in restricted regions of Jeju Island and Jeollanam-do, where it is recognized as a native species of the Jeju Gotjawal Forest and designated as a rare plant by the Korean Forest Service. Known for its medicinal properties, E. gracilistylus exhibits significant antioxidant and anticancer activities, which have garnered interest in its potential therapeutic applications [ 2 , 3 ]. Colorectal cancer (CRC) ranks among the most frequently diagnosed malignancies globally, with a concerning trend of increasing incidence rates. Notably, it has become the leading cancer type among young adults in Korea. Historically, CRC predominantly affected middle-aged and older populations; however, there has been a marked rise in cases among individuals under 40 years of age, with an incidence rate now reported at 12.9 per 100,000 people. This statistic positions Korea as having the highest incidence of colorectal cancer in the 20 to 40 age group, as highlighted in a recent study published in The Lancet [ 4 ]. This concerning trend is not exclusive to Korea; rather, the increasing incidence of early-onset CRC is observed globally. According to data from the American Cancer Society, approximately 20% of new colorectal cancer diagnoses in 2019 occurred in individuals under 55, indicating more than a two-fold increase compared to figures from a decade earlier. Projections suggest that the incidence of colorectal cancer will continue to rise through 2030, underscoring an urgent need for ongoing research and the development of targeted therapies to address this growing health concern [ 5 ]. Cancer stem cells (CSCs) have emerged as a focal point in oncology research due to their significant roles in tumor initiation, progression, and resistance to conventional therapies [ 6 , 7 ]. Unlike the predominant differentiated cells in tumors, CSCs possess unique capabilities for self-renewal and differentiation into various lineages within the tumor microenvironment, resembling normal stem cells but exhibiting dysregulated regulatory pathways [ 8 ]. This distinct subpopulation is implicated in promoting tumor heterogeneity, enhancing metastatic potential, and contributing to cancer recurrence following treatment. CSCs have been identified in a variety of solid tumors, including breast cancer, glioblastoma, and colorectal cancer, highlighting their critical involvement in tumor maintenance and aggressive phenotypes. Consequently, targeting CSCs is being investigated as a promising therapeutic strategy aimed at achieving more sustained clinical responses by eliminating the cells responsible for tumor regrowth and metastasis. Nonetheless, considerable challenges persist in delineating the precise molecular mechanisms that govern CSC behavior and developing selective therapies that can inhibit CSCs without adversely affecting normal stem cell populations. In this study, we aimed to evaluate the anticancer effects of various parts of E. gracilistylus , specifically focusing on the fruit, branch, root, and leaf. Ethanol extracts were prepared from each part at two different concentrations, allowing us to select the extract with the highest biological activity for subsequent experiments. Our primary focus was on colorectal cancer, for which we targeted both colorectal cancer cells and cancer stem cells. The efficacy of the different plant parts was compared against these cell types, and the most effective part was further purified to identify the specific fractions exhibiting the greatest anticancer activity. Methods Plant materials E. gracilistylus (Seom-ogalpi) different parts (leaves, branch, roots, fruits) were obtained from warm temperate and subtropical forest research center (Seogwipo, Jeju, Korea). The E. gracilistylus samples were dry and ground. The authenticator of the plant material, such as the E. gracilistylus plant identified by researcher Jin Kim (warm temperate and subtropical forest research center, national institute of forest science). Preparation and Extraction E. gracilistylus of different parts (leaves, branch, roots, fruits) were obtained by ultrasound-assisted extraction with a 70% ethanol and absolute ethanol each 30 minutes at solid-liquid ratio of 20 mg/g (ratio of the solvent volume per gram of raw material). After filtering (5A filter paper, Advantech Co., Ltd., Tokyo, Japan), E. gracilistylus from different parts 70% ethanol extracts and absolute ethanol extracts were concentrated by evaporation to remove solvent at 45℃ under reduced pressure using a rotary evaporator (Hei-VAP Precision, Heidolph, Schwabach, Germany). Ethanol was removed from a portion of the fruits ethanol extract, and it was sequentially fractionated by solvent-solvent fractionation to yield n -hexane (Hexane), dichloromethane (DCM), ethyl acetate (EtOAc), water-saturated butanol (BuOH) and residual (Aqueous) fractions respectively. Cell Lines and culture condition HT-29 and HCT-116 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM;Corning Woodland, CA). DMEM media contains 10% FBS and 1% penicillin streptomycin. Colorectal cancer cells were grown in the 5% CO 2 incubator at 37°C and cultured 2 × 10 5 cells per well. After 1 day of incubation, samples were treated for 1 day. For tumorsphere formation cells were seeded 5 × 10 4 cells per well in ultra-low attachment plates (Corning, NY, USA) using Cancer Stem Premium Media (ProMab Biotechnologies Inc., Richmond, CA, USA) for 5 days and samples were then treated for 2 days. Tumorsphere formation was evaluated using the NICE scan program [ 9 ]. Cell proliferation EZ-Cytox was used for cell proliferation assay (DoGenBio, Seoul, Korea). HT-29 and HCT-116 cells were cultured in a 96-well plate. Assays were according to the vendor’s recommendation protocol. The absorbance value was measured at 450 nm by using a FlexStation3 (Molecular Devices, Sunnyvale, CA, USA, in Bio-Health Materials Core-Facility, Jeju National University). Flow cytometric analysis for Annexin V/PI apoptosis Assay and ALDH assay We followed the manufacturer's protocol. The assay was performed by a method described previously [ 8 ]. We use annexin V/propidium iodide (PI) staining kit (BD, San Jose, CA, USA) and ALDEFUOR kit (STEMCELL Technologies). The samples were analyzed using an BD LSRFortessa cytometer (BD, NJ, USA, in Bio-Health Materials Core-Facility, Jeju National University) Gene Expression Analysis We purified total RNA using MiniBEST Universal RNA Extraction Kit (Takara, Tokyo, Japan) and RT-qPCR was performed using a one-step qRT-PCR kit (Takara, Tokyo, Japan). We followed the manufacturer's protocol and described method [ 8 ]. PCR primers were purchased from Bioneer Corp. (Daejeon, Korea). Tumorsphere formation counting We cultured CSCs in ultra-low attachment plate. After 5 days of incubation, the sample was treated for 2 days. Cells were re-cultured in 10 cm dish 1×10 5 cell/ml and counted for 3 days. Statistical analysis All data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with GraphPad Prism 8 software (GraphPad Prism Inc., San Diego, CA, USA). The data from three independent experiments were evaluated using a one-way ANOVA, with significance defined by a p -value of less than 0.05. Results Comparison of the activity of ethanol extracts at different concentrations A comparative study of extracts with 70% and absolute ethanol concentrations revealed that the extract with absolute ethanol exhibited great anticancer activity. The 70% ethanol extract showed no cytotoxic effect, while the absolute ethanol extract, particularly from the fruit part, demonstrated activity in both cell lines. In the HT-29 cell line, the fruit extract at a concentration of 250 µg/ml achieved an inhibition rate of nearly 90% compared to the control group. In the HCT-116 cell line, inhibition rates of 74%, 70%, and 88% were observed for the branch, root, and fruit extracts, respectively, at 250 µg/ml. Based on these results, further experiments were conducted using the absolute ethanol extract (Fig. 1 A). The activity of cancer stem cells was confirmed in these absolute ethanol part-specific extracts. Among the two cell lines, the fruit extract showed significant activity in the HT-29 cell line (Fig. 1 B). General solvent fractionation scheme and solvent-specific anticancer and cancer stem cell inhibition Figure 2 Schematic diagram of fractionation according to polarity of E. gracilistylus ethanol fruit extract Fruit ethanol extract was used to perform solvent fractionation. The crude ethanol extract was obtained by evaporating the ethanol under reduced pressure at 45°C. This extract was then suspended in water and sequentially fractionated into Hexane, DCM, EtOAc, BuOH, and an aqueous fraction (Fig. 2). The anticancer and cancer stem cell inhibitory activities were evaluated across five different fractions: Hexane, Dichloromethane (DCM), Ethyl Acetate (EtOAc), Butanol (BuOH), and the aqueous fraction. Among these, the Hexane, DCM, and aqueous fractions exhibited relatively weaker anticancer effects compared to the BuOH fraction. Similarly, their cancer stem cell inhibitory activity was lower than that of the EtOAc fraction, which demonstrated the most significant effect. Figure 3 presents the results of these evaluations, showing that the (A) Hexane fraction, (B) DCM fraction, and (C) aqueous fraction displayed limited anticancer and cancer stem cell inhibitory activities. These fractions were less effective in suppressing cancer cell proliferation and cancer stem cell activity compared to the BuOH and EtOAc fractions. This suggests that while the BuOH fraction may have a stronger anticancer effect, the EtOAc fraction is more potent in targeting cancer stem cells. The BuOH fraction was evaluated for its effects on both cancer stem cell activity and general anticancer properties. While this fraction did not exhibit significant cancer stem cell inhibitory activity (Fig. 4 B), it did show notable anticancer effects by reducing cell proliferation (Fig. 4 A). To further explore the mechanism underlying this anticancer activity, an apoptosis assay was conducted. The results revealed that treatment with the BuOH fraction led to a nearly two-fold increase in apoptosis, rising from 11.9–19.2%, suggesting that its anticancer effect is mediated through apoptosis induction (Fig. 4 C). Figure 4 shows these findings, where (A) represents the results of the cell proliferation assay, confirming the anticancer effect of the BuOH fraction, (B) shows its lack of significant cancer stem cell inhibition activity, and (C) demonstrates the increased apoptosis activity observed in cells treated with this fraction. These findings suggest that the anticancer properties of the BuOH fraction are primarily driven by its ability to promote apoptotic cell death rather than directly targeting cancer stem cells. Analysis of the EtOAc fraction revealed that, although its anticancer activity was lower than that of the BuOH fraction, it exhibited the strongest cancer stem cell inhibitory effect among all tested fractions. Specifically, the EtOAc fraction achieved an inhibition rate of 80%, indicating its potent ability to target cancer stem cells (Fig. 5 B). This suggests that while the BuOH fraction is more effective in reducing general cancer cell proliferation, the EtOAc fraction plays a crucial role in suppressing cancer stem cell activity, which is often linked to tumor recurrence and resistance to therapy. Figure 5 provides a visual representation of these findings. In panel (A), the cell proliferation assay demonstrates that the anticancer activity of the EtOAc fraction is less potent than that of the BuOH fraction. Panel (B) highlights its remarkable cancer stem cell inhibitory effect, with an inhibition rate of 80%, making it the most effective fraction in this regard. Meanwhile, panel (C) presents apoptosis activity data, offering further insight into its mechanism of action. These results indicate that the EtOAc fraction holds potential for specifically targeting cancer stem cells, which are associated with tumor recurrence and therapeutic resistance. Evaluation of Cancer Stem Cell Inhibitory Activity To assess the cancer stem cell inhibitory capacity of the EtOAc fraction, three experiments were conducted. First, the expression of Aldehyde Dehydrogenase 1-A1 (ALDH 1-A1), a known cancer stem cell marker, was analyzed using flow cytometry. The results indicated a significant reduction in marker expression, decreasing from 1.1–0.5%, representing more than a 50% reduction. In this experiment, diethylaminobenzaldehyde (DEAB) served as a positive control (Fig. 6A). Second, the mRNA levels of Nanog and Oct4, which are associated with cancer stem cell expression, were analyzed. The results indicated a concentration-dependent decrease in these gene expressions following treatment with the EtOAc fraction at concentrations of 100 and 200 µg/ml (Fig. 6B). The impact of the EtOAc fraction on tumorsphere growth was investigated by incorporating the fraction into tumorsphere cultures and subsequently recording the cell counts. This treatment led to cell death within the tumorspheres. Consequently, the results demonstrate a significant decrease in tumorsphere growth associated with the EtOAc fraction (Fig. 6C). The EtOAc fraction significantly reduced the protein expression of PD-L1 in tumorsphere cultures, indicating that this fraction not only inhibits the proliferation of cancer stem cells but also appears to modulate the immune checkpoint protein PD-L1, which may potentially enhance the immune system's ability to recognize and respond to the tumor. Discussions E. gracilistylus is a living organism that contains various active substances such as anti-diabetic and antioxidant properties [ 10 , 11 ]. This study demonstrates the potential of E. gracilistylus extracts, particularly those obtained with absolute ethanol, to exhibit both anticancer and cancer stem cell (CSC) inhibitory effects. Through a comparative evaluation of extracts at 70% and absolute ethanol concentrations, alongside additional fractionation of the most active fruit extract by solvent polarity, significant influences of ethanol concentration and solvent fractionation on anticancer efficacy were observed. In the process of purifying a single component, the concentration and type of solvent are important. The substances that can be separated vary depending on the solvent and concentration used [ 12 ]. The absolute ethanol fruit extract exhibited substantial anticancer activity against the HT-29 and HCT-116 cell lines, achieving nearly 90% inhibition in the HT-29 line at 250 µg/ml, whereas the 70% ethanol extract showed no cytotoxicity. This pronounced difference underscores the importance of ethanol concentration in extracting bioactive compounds with potential anti-cancer properties [ 13 ]. Subsequent fractionation of the absolute ethanol fruit extract yielded diverse activity profiles across solvent fractions. The hexane, DCM, and aqueous fractions demonstrated less than 50% inhibition in both anticancer and CSC assays, while the BuOH and EtOAc fractions exhibited notable activities, specifically for anticancer and CSC inhibition, respectively. The BuOH fraction was observed to induce apoptosis, as evidenced by an increase in apoptotic cell populations, indicating that its anticancer effect may be mediated through apoptotic pathways [ 14 ]. This finding suggests that the BuOH fraction may contain compounds with apoptosis-inducing properties, which are of particular interest in cancer treatment. Moreover, the EtOAc fraction demonstrated the highest CSC inhibitory activity, achieving an 80% inhibition rate. Although its anticancer activity was comparatively lower than that of the BuOH fraction, the selective CSC inhibition of the EtOAc fraction may hold therapeutic relevance for addressing tumor recurrence and treatment resistance. This specificity implies that the EtOAc fraction may contain compounds capable of selectively targeting CSCs, thereby offering a potential strategy for the prevention of tumor relapse and overcoming therapeutic resistance. The observed differential activities across fractions highlight the importance of solvent fractionation [ 15 ] as an approach to isolate and evaluate compounds with targeted anticancer and CSC inhibitory effects. Future studies should aim to isolate and characterize these bioactive compounds to facilitate the development of new therapeutic agents that can simultaneously target bulk cancer cells and CSCs, potentially reducing relapse rates and improving patient outcomes. In summary, this study underscores the efficacy of E. gracilistylus fruit extracts, particularly when extracted with absolute ethanol, as a valuable source of compounds with anticancer and CSC inhibitory properties. The distinct activity profiles identified across various solvent fractions support the utility of polarity-based fractionation in biological activity studies and provide insight into strategies for the development of targeted anticancer therapies.0 Conclusions In conclusion, the current study highlights the potent anticancer properties of E. gracilistylus , particularly noting that the absolute ethanol extract derived from its fruit achieved nearly 90% inhibition in the HT-29 colorectal cancer cell line. The extract's efficacy against cancer stem cells further establishes its potential as a therapeutic agent, especially considering the alarming rise in colorectal cancer cases among young adults in Korea. Solvent fractionation revealed that the EtOAc fraction exhibited the most substantial cancer stem cell inhibition, reaching 80%, while the BuOH fraction was notably effective in inducing apoptosis in colorectal cancer cells. Flow cytometry analysis corroborated these findings, showing significant reductions in ALDH 1-A1 expression and concentration-dependent decreases in the expression levels of key stem cell markers, Nanog and Oct4, following treatment with the EtOAc fraction. These results underscore the relevance of E. gracilistylus as a valuable source of bioactive compounds that could be harnessed for the development of targeted therapies against colorectal cancer and its stem cell populations. The study contributes to the expanding evidence base supporting the use of natural products in oncology, emphasizing the importance of further research into the underlying mechanisms that mediate these anticancer effects. Subsequent investigations should focus on isolating and characterizing specific active components from E. gracilistylus to better understand their roles in inhibiting colorectal cancer and enhancing therapeutic efficacy. Abbreviations CRC Colorectal cancer CSCs Cancer stem cells Hexane n -Hexane BuOH n -Butanol DCM Dichloromethane EtOAc Ethyl acetate DMEM Dulbecco’s modified Eagle’s medium SD Standard deviation TFE Tumorsphere formation efficiency ALDH 1-A1 Aldehyde Dehydrogenase 1-A1 DEAB Diethylaminobenzaldehyde Declarations Ethics approval and consent to participate This research did not involve human study, the informed consent form was thus not used. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding No external funding was received for this study. Author contributions Ju-Sung Kim and Dong-Sun Lee designed the research. Kyeoung Cheol Kim and Ji-Hyang Kim wrote the manuscript; Kyeoung Cheol Kim and Ji-Hyang Kim performed the research; Ju-Sung Kim and Kyeoung Cheol Kim analyzed the data. Ju-Sung Kim and Dong-Sun Lee developed methodology and supervised the study. All authors read and approved the final manuscript. References Li L, Zhang J, Zhong LW, Gu R, Zhong SH, editors. Eleutherococcus giraldii (Harms) Nakai: a review of botany, traditional uses, phytochemistry, pharmacology and quality control. Phytochem Rev. 2025;24:1027–1055. Lim SH, Park YH, Kwon CJ, Ham HJ, Jeong HN, Kim KH, Ahn YS. Anti-diabetic and hypoglycemic effect of Eleutherococcus spp. J Korean Soc Food Sci Nutr. 2010;39(12):1761–8. Załuski D, Smolarz HD, Szpilewska M. Eleutherosides in aerial parts of Eleutherococcus species cultivated in Poland. J AOAC Int. 2011;94(5):1422–6. 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Azmir J, Zaidul ISM, Rahman MM, Sharif KM, Mohamed A, Sahena F, Jahurul MHA, Ghafoor K, Norulaini NAN, Omar AKM. Techniques for extraction of bioactive compounds from plant materials: A review. J Food Eng. 2013;117(4):426–36. Pfeffer CM, Singh AT. Apoptosis: a target for anticancer therapy. Int J Mol Sci. 2018;19(2):448. Azwanida NN. A review on the extraction methods use in medicinal plants, principle, strength and limitation. MAP. 2015;4(196):2167–0412. 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. 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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-6152350","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":427749948,"identity":"1f340d14-cf88-4041-81e8-ed6cc60f026e","order_by":0,"name":"Kyeoung Cheol Kim","email":"","orcid":"","institution":"Bio-Health Materials Core-Facility Center, Jeju National University","correspondingAuthor":false,"prefix":"","firstName":"Kyeoung","middleName":"Cheol","lastName":"Kim","suffix":""},{"id":427749949,"identity":"c7785a5f-6487-4fac-a5e7-2f82d2719489","order_by":1,"name":"Ji-Hyang Kim","email":"","orcid":"","institution":"Bio-Health Materials Core-Facility Center, Jeju National University","correspondingAuthor":false,"prefix":"","firstName":"Ji-Hyang","middleName":"","lastName":"Kim","suffix":""},{"id":427749950,"identity":"a739ea4b-614a-4d1a-9047-48d25548916c","order_by":2,"name":"Dong-Sun Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIie3NMUvDQBTA8VcC6fLarhcofoYLB1k6+FWSpV1UhIAfIV2uzvZbxG9w8MAsZ+cMUuNSEBzORRpw8Kx00V7EreD94bjH8X4cgM93jKmvawwQKA747bmL2N0w/TNB/vP5UMNqQc9t8YCjvjSXZkwXXAVPBvTaSSK9mk4GxQYjeV/GN0g5V6FgUOdOwuuzRPQKQl6flwKRslJBAmDSThK3lpw+vjTifUf6b78R0Qw+f2EIAnYE7S+1m0RaJwGuCJme8ljiLFsS5izVbjKspHjdXtHJaE4b3spJdl3Nb425cxNbyPYD70l7BfZ0Arti9kMD2+5Vn8/n+599ADjNW+KS6bY2AAAAAElFTkSuQmCC","orcid":"","institution":"Bio-Health Materials Core-Facility Center, Jeju National University","correspondingAuthor":true,"prefix":"","firstName":"Dong-Sun","middleName":"","lastName":"Lee","suffix":""},{"id":427749951,"identity":"2bded41f-0366-4a10-b9d1-79e4a6a68c2f","order_by":3,"name":"Ju-Sung Kim","email":"","orcid":"","institution":"Major in Plant Resource and Environment College of Agriculture and Life Science, Jeju National University","correspondingAuthor":false,"prefix":"","firstName":"Ju-Sung","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-03-04 08:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6152350/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6152350/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78691963,"identity":"b539b5ee-2cc4-4443-8c85-88781d827fca","added_by":"auto","created_at":"2025-03-17 16:22:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":923905,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of activity of ethanol extracts from different parts. 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(B) Tumorsphere formation efficiency(TFE) experiment of absolute ethanol extracts 125μg/ml from each part.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6152350/v1/06ecc072925db49824c1b8b4.png"},{"id":78690812,"identity":"7be59449-18d8-4ca2-a3cf-2f5898370608","added_by":"auto","created_at":"2025-03-17 16:14:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69673,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of fractionation according to polarity of \u003cem\u003eE. gracilistylus\u003c/em\u003e ethanol fruit extract\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6152350/v1/f93356d22d0cf24e293c56d1.png"},{"id":78690813,"identity":"a7838caf-df73-4d71-9ca9-5cf963814071","added_by":"auto","created_at":"2025-03-17 16:14:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":551484,"visible":true,"origin":"","legend":"\u003cp\u003eThe anticancer and cancer stem cell inhibitory activities. (A) Hexane fraction, (B) DCM fraction, (C) Aqueous fraction\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6152350/v1/360a7fa090900e34a11a6351.png"},{"id":78691964,"identity":"2b5e0dd5-28bc-43b4-8908-1946034c7103","added_by":"auto","created_at":"2025-03-17 16:22:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":478302,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of BuOH fraction, (A) Cell proliferation assay (B) Cancer stem cell inhibition activity (C) Apoptosis activity in the BuOH fraction.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6152350/v1/7cd9c5a958d98180fe2362ed.png"},{"id":78691971,"identity":"27a6e6d3-ed52-4b24-9fc0-cef4d6a11e88","added_by":"auto","created_at":"2025-03-17 16:22:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":466824,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of EtOAc fraction, (A) Cell proliferation assay (B) Cancer stem cell inhibition activity (C) Apoptosis activity in the BuOH fraction.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6152350/v1/942624fb6798ee956d3a3fdf.png"},{"id":78690815,"identity":"7e9d9aee-08d0-41ec-900a-0e13d00b3818","added_by":"auto","created_at":"2025-03-17 16:14:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":306481,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of the EtOAc Fraction on Cancer Stem Cell Loads in Colorectal Cancer. (A) Aldehyde Dehydrogenase 1-A1 (ALDH 1-A1) assay. (B) The transcriptional levels of Oct4 and Nanog. (C) Tumorsphere counting assay with EtOAc fraction. (D) \u0026nbsp;The protein expression of PD-L1 were determined in Tumorsphere with antibodies to PD-L1, and β-actin. EtOAc fraction decreased the protein level of PD-L1 in Tumorsphere.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6152350/v1/104456d65a40ba0c24b5851c.png"},{"id":79096661,"identity":"419ce23b-9130-45de-b75d-e74f5aeede00","added_by":"auto","created_at":"2025-03-24 11:09:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3474568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6152350/v1/4cd78742-523c-4772-9fb2-9031aef577b0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anticancer and Cancer Stem Cell Inhibitory Effects of Eleutherococcus gracilistylus Ethanol Extracts in Colorectal Cancer Models","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eEleutherococcus gracilistylus\u003c/em\u003e (Seom-ogalpi) is a deciduous broadleaf shrub from the Araliaceae family, primarily distributed in Korea, China and Japan [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This plant is predominantly found in restricted regions of Jeju Island and Jeollanam-do, where it is recognized as a native species of the Jeju Gotjawal Forest and designated as a rare plant by the Korean Forest Service. Known for its medicinal properties, \u003cem\u003eE. gracilistylus\u003c/em\u003e exhibits significant antioxidant and anticancer activities, which have garnered interest in its potential therapeutic applications [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eColorectal cancer (CRC) ranks among the most frequently diagnosed malignancies globally, with a concerning trend of increasing incidence rates. Notably, it has become the leading cancer type among young adults in Korea. Historically, CRC predominantly affected middle-aged and older populations; however, there has been a marked rise in cases among individuals under 40 years of age, with an incidence rate now reported at 12.9 per 100,000 people. This statistic positions Korea as having the highest incidence of colorectal cancer in the 20 to 40 age group, as highlighted in a recent study published in \u003cem\u003eThe Lancet\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis concerning trend is not exclusive to Korea; rather, the increasing incidence of early-onset CRC is observed globally. According to data from the American Cancer Society, approximately 20% of new colorectal cancer diagnoses in 2019 occurred in individuals under 55, indicating more than a two-fold increase compared to figures from a decade earlier. Projections suggest that the incidence of colorectal cancer will continue to rise through 2030, underscoring an urgent need for ongoing research and the development of targeted therapies to address this growing health concern [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCancer stem cells (CSCs) have emerged as a focal point in oncology research due to their significant roles in tumor initiation, progression, and resistance to conventional therapies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Unlike the predominant differentiated cells in tumors, CSCs possess unique capabilities for self-renewal and differentiation into various lineages within the tumor microenvironment, resembling normal stem cells but exhibiting dysregulated regulatory pathways [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This distinct subpopulation is implicated in promoting tumor heterogeneity, enhancing metastatic potential, and contributing to cancer recurrence following treatment. CSCs have been identified in a variety of solid tumors, including breast cancer, glioblastoma, and colorectal cancer, highlighting their critical involvement in tumor maintenance and aggressive phenotypes. Consequently, targeting CSCs is being investigated as a promising therapeutic strategy aimed at achieving more sustained clinical responses by eliminating the cells responsible for tumor regrowth and metastasis. Nonetheless, considerable challenges persist in delineating the precise molecular mechanisms that govern CSC behavior and developing selective therapies that can inhibit CSCs without adversely affecting normal stem cell populations.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to evaluate the anticancer effects of various parts of \u003cem\u003eE. gracilistylus\u003c/em\u003e, specifically focusing on the fruit, branch, root, and leaf. Ethanol extracts were prepared from each part at two different concentrations, allowing us to select the extract with the highest biological activity for subsequent experiments. Our primary focus was on colorectal cancer, for which we targeted both colorectal cancer cells and cancer stem cells. The efficacy of the different plant parts was compared against these cell types, and the most effective part was further purified to identify the specific fractions exhibiting the greatest anticancer activity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. gracilistylus\u003c/em\u003e (Seom-ogalpi) different parts (leaves, branch, roots, fruits) were obtained from warm temperate and subtropical forest research center (Seogwipo, Jeju, Korea). The \u003cem\u003eE. gracilistylus\u003c/em\u003e samples were dry and ground. The authenticator of the plant material, such as the \u003cem\u003eE. gracilistylus\u003c/em\u003e plant identified by researcher Jin Kim (warm temperate and subtropical forest research center, national institute of forest science).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation and Extraction\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eE. gracilistylus\u003c/em\u003e of different parts (leaves, branch, roots, fruits) were obtained by ultrasound-assisted extraction with a 70% ethanol and absolute ethanol each 30 minutes at solid-liquid ratio of 20 mg/g (ratio of the solvent volume per gram of raw material). After filtering (5A filter paper, Advantech Co., Ltd., Tokyo, Japan), \u003cem\u003eE. gracilistylus\u003c/em\u003e from different parts 70% ethanol extracts and absolute ethanol extracts were concentrated by evaporation to remove solvent at 45℃ under reduced pressure using a rotary evaporator (Hei-VAP Precision, Heidolph, Schwabach, Germany). Ethanol was removed from a portion of the fruits ethanol extract, and it was sequentially fractionated by solvent-solvent fractionation to yield \u003cem\u003en\u003c/em\u003e-hexane (Hexane), dichloromethane (DCM), ethyl acetate (EtOAc), water-saturated butanol (BuOH) and residual (Aqueous) fractions respectively.\u003c/p\u003e\n\u003ch3\u003eCell Lines and culture condition\u003c/h3\u003e\n\u003cp\u003eHT-29 and HCT-116 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM;Corning Woodland, CA). DMEM media contains 10% FBS and 1% penicillin streptomycin. Colorectal cancer cells were grown in the 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C and cultured 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. After 1 day of incubation, samples were treated for 1 day. For tumorsphere formation cells were seeded 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in ultra-low attachment plates (Corning, NY, USA) using Cancer Stem Premium Media (ProMab Biotechnologies Inc., Richmond, CA, USA) for 5 days and samples were then treated for 2 days. Tumorsphere formation was evaluated using the NICE scan program [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCell proliferation\u003c/h3\u003e\n\u003cp\u003eEZ-Cytox was used for cell proliferation assay (DoGenBio, Seoul, Korea). HT-29 and HCT-116 cells were cultured in a 96-well plate. Assays were according to the vendor\u0026rsquo;s recommendation protocol. The absorbance value was measured at 450 nm by using a FlexStation3 (Molecular Devices, Sunnyvale, CA, USA, in Bio-Health Materials Core-Facility, Jeju National University).\u003c/p\u003e\n\u003ch3\u003eFlow cytometric analysis for Annexin V/PI apoptosis Assay and ALDH assay\u003c/h3\u003e\n\u003cp\u003eWe followed the manufacturer's protocol. The assay was performed by a method described previously [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. We use annexin V/propidium iodide (PI) staining kit (BD, San Jose, CA, USA) and ALDEFUOR kit (STEMCELL Technologies). The samples were analyzed using an BD LSRFortessa cytometer (BD, NJ, USA, in Bio-Health Materials Core-Facility, Jeju National University)\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Analysis\u003c/h2\u003e \u003cp\u003eWe purified total RNA using MiniBEST Universal RNA Extraction Kit (Takara, Tokyo, Japan) and RT-qPCR was performed using a one-step qRT-PCR kit (Takara, Tokyo, Japan). We followed the manufacturer's protocol and described method [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. PCR primers were purchased from Bioneer Corp. (Daejeon, Korea).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTumorsphere formation counting\u003c/h3\u003e\n\u003cp\u003eWe cultured CSCs in ultra-low attachment plate. After 5 days of incubation, the sample was treated for 2 days. Cells were re-cultured in 10 cm dish 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cell/ml and counted for 3 days.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed with GraphPad Prism 8 software (GraphPad Prism Inc., San Diego, CA, USA). The data from three independent experiments were evaluated using a one-way ANOVA, with significance defined by a \u003cem\u003ep\u003c/em\u003e-value of less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComparison of the activity of ethanol extracts at different concentrations\u003c/h2\u003e \u003cp\u003eA comparative study of extracts with 70% and absolute ethanol concentrations revealed that the extract with absolute ethanol exhibited great anticancer activity. The 70% ethanol extract showed no cytotoxic effect, while the absolute ethanol extract, particularly from the fruit part, demonstrated activity in both cell lines. In the HT-29 cell line, the fruit extract at a concentration of 250 \u0026micro;g/ml achieved an inhibition rate of nearly 90% compared to the control group. In the HCT-116 cell line, inhibition rates of 74%, 70%, and 88% were observed for the branch, root, and fruit extracts, respectively, at 250 \u0026micro;g/ml. Based on these results, further experiments were conducted using the absolute ethanol extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The activity of cancer stem cells was confirmed in these absolute ethanol part-specific extracts. Among the two cell lines, the fruit extract showed significant activity in the HT-29 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneral solvent fractionation scheme and solvent-specific anticancer and cancer stem cell inhibition\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;2 Schematic diagram of fractionation according to polarity of \u003cem\u003eE. gracilistylus\u003c/em\u003e ethanol fruit extract\u003c/p\u003e \u003cp\u003eFruit ethanol extract was used to perform solvent fractionation. The crude ethanol extract was obtained by evaporating the ethanol under reduced pressure at 45\u0026deg;C. This extract was then suspended in water and sequentially fractionated into Hexane, DCM, EtOAc, BuOH, and an aqueous fraction (Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThe anticancer and cancer stem cell inhibitory activities were evaluated across five different fractions: Hexane, Dichloromethane (DCM), Ethyl Acetate (EtOAc), Butanol (BuOH), and the aqueous fraction. Among these, the Hexane, DCM, and aqueous fractions exhibited relatively weaker anticancer effects compared to the BuOH fraction. Similarly, their cancer stem cell inhibitory activity was lower than that of the EtOAc fraction, which demonstrated the most significant effect. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the results of these evaluations, showing that the (A) Hexane fraction, (B) DCM fraction, and (C) aqueous fraction displayed limited anticancer and cancer stem cell inhibitory activities. These fractions were less effective in suppressing cancer cell proliferation and cancer stem cell activity compared to the BuOH and EtOAc fractions. This suggests that while the BuOH fraction may have a stronger anticancer effect, the EtOAc fraction is more potent in targeting cancer stem cells.\u003c/p\u003e \u003cp\u003eThe BuOH fraction was evaluated for its effects on both cancer stem cell activity and general anticancer properties. While this fraction did not exhibit significant cancer stem cell inhibitory activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), it did show notable anticancer effects by reducing cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To further explore the mechanism underlying this anticancer activity, an apoptosis assay was conducted. The results revealed that treatment with the BuOH fraction led to a nearly two-fold increase in apoptosis, rising from 11.9\u0026ndash;19.2%, suggesting that its anticancer effect is mediated through apoptosis induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows these findings, where (A) represents the results of the cell proliferation assay, confirming the anticancer effect of the BuOH fraction, (B) shows its lack of significant cancer stem cell inhibition activity, and (C) demonstrates the increased apoptosis activity observed in cells treated with this fraction. These findings suggest that the anticancer properties of the BuOH fraction are primarily driven by its ability to promote apoptotic cell death rather than directly targeting cancer stem cells.\u003c/p\u003e \u003cp\u003eAnalysis of the EtOAc fraction revealed that, although its anticancer activity was lower than that of the BuOH fraction, it exhibited the strongest cancer stem cell inhibitory effect among all tested fractions. Specifically, the EtOAc fraction achieved an inhibition rate of 80%, indicating its potent ability to target cancer stem cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This suggests that while the BuOH fraction is more effective in reducing general cancer cell proliferation, the EtOAc fraction plays a crucial role in suppressing cancer stem cell activity, which is often linked to tumor recurrence and resistance to therapy. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e provides a visual representation of these findings. In panel (A), the cell proliferation assay demonstrates that the anticancer activity of the EtOAc fraction is less potent than that of the BuOH fraction. Panel (B) highlights its remarkable cancer stem cell inhibitory effect, with an inhibition rate of 80%, making it the most effective fraction in this regard. Meanwhile, panel (C) presents apoptosis activity data, offering further insight into its mechanism of action. These results indicate that the EtOAc fraction holds potential for specifically targeting cancer stem cells, which are associated with tumor recurrence and therapeutic resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Cancer Stem Cell Inhibitory Activity\u003c/h2\u003e \u003cp\u003eTo assess the cancer stem cell inhibitory capacity of the EtOAc fraction, three experiments were conducted. First, the expression of Aldehyde Dehydrogenase 1-A1 (ALDH 1-A1), a known cancer stem cell marker, was analyzed using flow cytometry. The results indicated a significant reduction in marker expression, decreasing from 1.1\u0026ndash;0.5%, representing more than a 50% reduction. In this experiment, diethylaminobenzaldehyde (DEAB) served as a positive control (Fig.\u0026nbsp;6A). Second, the mRNA levels of Nanog and Oct4, which are associated with cancer stem cell expression, were analyzed. The results indicated a concentration-dependent decrease in these gene expressions following treatment with the EtOAc fraction at concentrations of 100 and 200 \u0026micro;g/ml (Fig.\u0026nbsp;6B). The impact of the EtOAc fraction on tumorsphere growth was investigated by incorporating the fraction into tumorsphere cultures and subsequently recording the cell counts. This treatment led to cell death within the tumorspheres. Consequently, the results demonstrate a significant decrease in tumorsphere growth associated with the EtOAc fraction (Fig.\u0026nbsp;6C). The EtOAc fraction significantly reduced the protein expression of PD-L1 in tumorsphere cultures, indicating that this fraction not only inhibits the proliferation of cancer stem cells but also appears to modulate the immune checkpoint protein PD-L1, which may potentially enhance the immune system's ability to recognize and respond to the tumor.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cp\u003e \u003cem\u003eE. gracilistylus\u003c/em\u003e is a living organism that contains various active substances such as anti-diabetic and antioxidant properties [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This study demonstrates the potential of \u003cem\u003eE. gracilistylus\u003c/em\u003e extracts, particularly those obtained with absolute ethanol, to exhibit both anticancer and cancer stem cell (CSC) inhibitory effects. Through a comparative evaluation of extracts at 70% and absolute ethanol concentrations, alongside additional fractionation of the most active fruit extract by solvent polarity, significant influences of ethanol concentration and solvent fractionation on anticancer efficacy were observed. In the process of purifying a single component, the concentration and type of solvent are important. The substances that can be separated vary depending on the solvent and concentration used [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The absolute ethanol fruit extract exhibited substantial anticancer activity against the HT-29 and HCT-116 cell lines, achieving nearly 90% inhibition in the HT-29 line at 250 \u0026micro;g/ml, whereas the 70% ethanol extract showed no cytotoxicity. This pronounced difference underscores the importance of ethanol concentration in extracting bioactive compounds with potential anti-cancer properties [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Subsequent fractionation of the absolute ethanol fruit extract yielded diverse activity profiles across solvent fractions. The hexane, DCM, and aqueous fractions demonstrated less than 50% inhibition in both anticancer and CSC assays, while the BuOH and EtOAc fractions exhibited notable activities, specifically for anticancer and CSC inhibition, respectively. The BuOH fraction was observed to induce apoptosis, as evidenced by an increase in apoptotic cell populations, indicating that its anticancer effect may be mediated through apoptotic pathways [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This finding suggests that the BuOH fraction may contain compounds with apoptosis-inducing properties, which are of particular interest in cancer treatment. Moreover, the EtOAc fraction demonstrated the highest CSC inhibitory activity, achieving an 80% inhibition rate. Although its anticancer activity was comparatively lower than that of the BuOH fraction, the selective CSC inhibition of the EtOAc fraction may hold therapeutic relevance for addressing tumor recurrence and treatment resistance. This specificity implies that the EtOAc fraction may contain compounds capable of selectively targeting CSCs, thereby offering a potential strategy for the prevention of tumor relapse and overcoming therapeutic resistance. The observed differential activities across fractions highlight the importance of solvent fractionation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] as an approach to isolate and evaluate compounds with targeted anticancer and CSC inhibitory effects. Future studies should aim to isolate and characterize these bioactive compounds to facilitate the development of new therapeutic agents that can simultaneously target bulk cancer cells and CSCs, potentially reducing relapse rates and improving patient outcomes. In summary, this study underscores the efficacy of \u003cem\u003eE. gracilistylus\u003c/em\u003e fruit extracts, particularly when extracted with absolute ethanol, as a valuable source of compounds with anticancer and CSC inhibitory properties. The distinct activity profiles identified across various solvent fractions support the utility of polarity-based fractionation in biological activity studies and provide insight into strategies for the development of targeted anticancer therapies.0\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, the current study highlights the potent anticancer properties of \u003cem\u003eE. gracilistylus\u003c/em\u003e, particularly noting that the absolute ethanol extract derived from its fruit achieved nearly 90% inhibition in the HT-29 colorectal cancer cell line. The extract's efficacy against cancer stem cells further establishes its potential as a therapeutic agent, especially considering the alarming rise in colorectal cancer cases among young adults in Korea. Solvent fractionation revealed that the EtOAc fraction exhibited the most substantial cancer stem cell inhibition, reaching 80%, while the BuOH fraction was notably effective in inducing apoptosis in colorectal cancer cells. Flow cytometry analysis corroborated these findings, showing significant reductions in ALDH 1-A1 expression and concentration-dependent decreases in the expression levels of key stem cell markers, Nanog and Oct4, following treatment with the EtOAc fraction. These results underscore the relevance of \u003cem\u003eE. gracilistylus\u003c/em\u003e as a valuable source of bioactive compounds that could be harnessed for the development of targeted therapies against colorectal cancer and its stem cell populations. The study contributes to the expanding evidence base supporting the use of natural products in oncology, emphasizing the importance of further research into the underlying mechanisms that mediate these anticancer effects. Subsequent investigations should focus on isolating and characterizing specific active components from \u003cem\u003eE. gracilistylus\u003c/em\u003e to better understand their roles in inhibiting colorectal cancer and enhancing therapeutic efficacy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCRC Colorectal cancer\u003c/p\u003e \u003cp\u003eCSCs Cancer stem cells\u003c/p\u003e \u003cp\u003eHexane \u003cem\u003en\u003c/em\u003e-Hexane\u003c/p\u003e \u003cp\u003eBuOH \u003cem\u003en\u003c/em\u003e-Butanol\u003c/p\u003e \u003cp\u003eDCM Dichloromethane\u003c/p\u003e \u003cp\u003eEtOAc Ethyl acetate\u003c/p\u003e \u003cp\u003eDMEM Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e \u003cp\u003eSD Standard deviation\u003c/p\u003e \u003cp\u003eTFE Tumorsphere formation efficiency\u003c/p\u003e \u003cp\u003eALDH 1-A1 Aldehyde Dehydrogenase 1-A1\u003c/p\u003e \u003cp\u003eDEAB Diethylaminobenzaldehyde\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not involve human study, the informed consent form was thus not used.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo external funding was received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJu-Sung Kim and Dong-Sun Lee designed the research. Kyeoung Cheol Kim and Ji-Hyang Kim wrote the manuscript; Kyeoung Cheol Kim and Ji-Hyang Kim performed the research; Ju-Sung Kim and Kyeoung Cheol Kim analyzed the data. Ju-Sung Kim and Dong-Sun Lee developed methodology and supervised the study. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi L, Zhang J, Zhong LW, Gu R, Zhong SH, editors. \u003cem\u003eEleutherococcus giraldii\u003c/em\u003e (Harms) Nakai: a review of botany, traditional uses, phytochemistry, pharmacology and quality control. Phytochem Rev. 2025;24:1027\u0026ndash;1055.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim SH, Park YH, Kwon CJ, Ham HJ, Jeong HN, Kim KH, Ahn YS. Anti-diabetic and hypoglycemic effect of \u003cem\u003eEleutherococcus\u003c/em\u003e spp. J Korean Soc Food Sci Nutr. 2010;39(12):1761\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZałuski D, Smolarz HD, Szpilewska M. Eleutherosides in aerial parts of \u003cem\u003eEleutherococcus\u003c/em\u003e species cultivated in Poland. J AOAC Int. 2011;94(5):1422\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel SG, Karlitz JJ, Yen T, Lieu CH, Boland CR. The rising tide of early-onset colorectal cancer: a comprehensive review of epidemiology, clinical features, biology, risk factors, prevention, and early detection. Lancet Gastroenterol Hepatol. 2022;7(3):262\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller KD, Nogueira L, Devasia T, Mariotto AB, Yabroff KR, Jemal A, Kramer J, Siegel RL. Cancer treatment and survivorship statistics, 2022. CA Cancer J Clin. 2022;72(5):409\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, Rosen JM. WNT/β-catenin mediates radiation resistance of mouse mammary progenitor cells. PNAS. 2007;104(2):618\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi HS, Kim JH, Kim SL, Deng HY, Lee D, Kim CS, Yun BS, Lee DS. Catechol derived from aronia juice through lactic acid bacteria fermentation inhibits breast cancer stem cell formation via modulation Stat3/IL-6 signaling pathway. Mol Carcinog. 2018;57(11):1467\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JH, Choi HS, Kim SL, Lee DS. The PAK1-Stat3 signaling pathway activates IL-6 gene transcription and human breast cancer stem cell formation. Cancers. 2019;11(10):1527.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SL, Choi HS, Lee DS. BRD4/nuclear PD-L1/RelB circuit is involved in the stemness of breast cancer cells. CCS. 2023;21(1):315.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu MX, Yang Y, Zou QP, Luo J, Zhang BB, Liu XQ, Hwang EH. Anti-diabetic effects of Acankoreagenin from the leaves of Acanthopanax gracilistylus herb in RIN-m5F cells via suppression of NF-κB activation. Molecules. 2018;23(4):958.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZałuski D, Smolarz HD, Gawlik-Dziki U. Bioactive compounds and antioxidative, antileukemic and anti-MMPs activity of \u003cem\u003eEleutherococcus\u003c/em\u003e species cultivated in Poland. Nat Prod Commun. 2012;7(11):1934578X1200701118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHikmawanti NPE, Fatmawati S, Asri AW. The effect of ethanol concentrations as the extraction solvent on antioxidant activity of Katuk (Sauropus androgynus (L.) Merr.) leaves extracts. IOP Conf Ser Earth Environ Sci. 2021;755(1):012060.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzmir J, Zaidul ISM, Rahman MM, Sharif KM, Mohamed A, Sahena F, Jahurul MHA, Ghafoor K, Norulaini NAN, Omar AKM. Techniques for extraction of bioactive compounds from plant materials: A review. J Food Eng. 2013;117(4):426\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfeffer CM, Singh AT. Apoptosis: a target for anticancer therapy. Int J Mol Sci. 2018;19(2):448.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzwanida NN. A review on the extraction methods use in medicinal plants, principle, strength and limitation. MAP. 2015;4(196):2167\u0026ndash;0412.\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":"Eleutherococcus gracilistylus, Colorectal cancer, Cancer stem cell, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-6152350/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6152350/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEleutherococcus gracilistylus\u003c/em\u003e (Seom-ogalpi), a rare deciduous shrub from the Araliaceae family native to Korea, is known for its antioxidant and anticancer properties. The increasing incidence of colorectal cancer, particularly among young adults in Korea, highlights the urgent need for targeted therapies. This is especially important for addressing cancer stem cells, which play a critical role in tumor progression and contribute to treatment resistance. This study investigates the anticancer effects of various parts of \u003cem\u003eE. gracilistylus\u003c/em\u003e\u0026mdash;leaves, branch, roots, fruits\u0026mdash;using ethanol extracts at different concentrations to identify the most biologically active components against colorectal cancer cells and cancer stem cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE. gracilistylus\u003c/em\u003e was collected from Jeju Island, South Korea, and the absolute ethanol extract of fruit was fractionated into n-hexane, dichloromethane, ethyl acetate, butanol, and water fractions. HT-29 and HCT-116 colorectal cancer cell lines were cultured in DMEM with 10% FBS at 37\u0026deg;C, and tumorspheres were formed in ultra-low attachment plates and assessed using the NICE scan program. Cell proliferation was measured using the EZ-Cytox assay, and apoptosis was analyzed via flow cytometry. Gene expression was evaluated using RT-qPCR, and statistical significance was determined with GraphPad Prism 8 software (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe absolute ethanol extract of \u003cem\u003eE. gracilistylus\u003c/em\u003e, particularly from the fruit, achieved nearly 90% inhibition in the HT-29 cell line at 250 \u0026micro;g/ml. The ethyl acetate fraction demonstrated the highest cancer stem cell inhibition at 80%, while the butanol fraction significantly induced apoptosis, increasing the rate from 11.9\u0026ndash;19.2%. Flow cytometry confirmed a marked reduction in ALDH 1-A1 expression and a concentration-dependent decrease in Nanog and Oct4 levels after treatment with the ethyl acetate fraction.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe absolute ethanol extract of \u003cem\u003eE. gracilistylus\u003c/em\u003e demonstrated potent anticancer effects, especially against HT-29 cells, and significant CSC inhibition. These findings highlight its therapeutic potential in targeting colorectal cancer.\u003c/p\u003e","manuscriptTitle":"Anticancer and Cancer Stem Cell Inhibitory Effects of Eleutherococcus gracilistylus Ethanol Extracts in Colorectal Cancer Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 16:14:51","doi":"10.21203/rs.3.rs-6152350/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":"08d75c1d-7193-4c77-ab5a-b4e39df6309d","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T11:08:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-17 16:14:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6152350","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6152350","identity":"rs-6152350","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}