Anticancer Activity and Cytotoxic Effect of Rhizome Extract Against MCF-7 Breast cancer Cell Line – an In Vitro study

Anticancer Activity and Cytotoxic Effect of Rhizome Extract Against MCF-7 Breast cancer Cell Line – an In Vitro study | 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 Article Anticancer Activity and Cytotoxic Effect of Rhizome Extract Against MCF-7 Breast cancer Cell Line – an In Vitro study Bassam S.M. Al Kazman, Emhemed Abukhattala, Marwa Brakat, Ayman Balla Mustafa, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6875241/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 Many factors stimulate breast cancer in women, including genetic, chemical, metabolic, environmental, and physical elements. Currently, a diet rich in antioxidants is considered an effective means of controlling cancer growth and spread. Therefore, cytotoxic and anticancer compounds derived from medicinal plants are promising therapeutic agents, as they induce apoptosis pathways. Ginger is one of the medicinal herbs, and its rhizome extracts are used as an anticancer treatment for many types of malignancies. In this study, the viability of human breast cancer cell (MCF-7) was evaluated after treatment with ethanolic ginger extract (GEE) at concentrations of 0.0, 7.8, 15.62, 31.25, 62.50, 125, 250, 500, and 1000 µg/mL for 24, 48 and 72 h using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. GEE significantly reduced the viability of treated cells and increased their inhibition compared to the control group with increasing dose and incubation duration. GEE had significant toxic activity on treated cells (p < 0.05), with IC₅₀ values based on incubation periods of 24, 48 and 72 hours (420.04 ± 16.68, 124.23 ± 8.91 and 60.74 ± 3.04 µg/mL), respectively. Using inverted microscopy, significant morphological changes were observed in MCF-7 cells compared to the control group. The treated cells became smaller and more rounded with a proportional increase in abnormal and dead cells; the morphological changes were most pronounced at a concentration of 500 µg/mL after 72 h of incubation. Flow cytometry measurements revealed a significant induction of apoptosis and cell cycle arrest in the G2/M phase after exposing MCF-7 cells to ginger extract at doses of 100, 200, and 500 µg/mL for 24, 48, and 72 h, compared to the control group (p < 0.05). Meanwhile, no significant change was observed in the G1 phase of cells. Biological sciences/Cancer/Breast cancer Biological sciences/Cancer/Cancer therapy Health sciences/Oncology/Cancer Biological sciences/Cancer Health sciences/Oncology Health sciences/Medical research Health sciences/Medical research/Biomarkers Health sciences/Medical research/Experimental models of disease Health sciences/Medical research/Stem cell research Ginger extract MCF-7 cell line MTT Cell cycle Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Medicinal plants are used as therapeutic agents in resource-limited communities due to their abundance and low cost. They are a natural source of biologically active and therapeutic components that play an important role in disease prevention and improving human health (Berida et al., 2024 ). Medicinal plants and their compounds, especially phenolics, can scavenge free radicals and protect cells from oxidative damage (Sun and Shahrajabian, 2023 ), by interacting with oxidation products derived from fatty acids to prevent oxidative stress and improve food quality and flavor (Ziarno et al., 2021 ). Therefore, medicinal plants and their compounds and extracts are the main source of many secondary metabolites that exhibit therapeutic properties (Gang et al., 2023 ). Among many medicinal plants, researchers and nutritionists have been particularly interested in ginger (Zingiber officinale), which is widely used in low-income communities as both a spice and for medicinal purposes (Yadav et al., 2020 ; Berida et al., 2024 ) as an additional drug for the treatment of various types of cancer (Ziarno et al., 2021 ). Ginger contains volatile oils, anthocyanins, tannins, phenolic compounds, and sesquiterpenes as major components, in addition to terpenes and non-volatile pungent substances such as oleoresins called gingerols (Hosseinzadeh et al., 2017 ; Bobde, 2022). Several studies have provided evidence that ginger and its pungent organic compounds have anti-inflammatory and antitumor activities through free radical scavenging, antioxidant pathways, gene expression modification, and apoptosis induction, which contribute to reducing tumor initiation and progression (Dhanik et al., 2017 ; Shahrajabian et al., 2019 ; Ziarno et al., 2021 ; Berida et al., 2024 ). Breast cancer is a common type of cancer among women. It has a high metastatic potential and can spread to different parts of the body (Torre et al., 2015 ). Although there are different treatments available for breast cancer, including chemotherapy, radiation, hormonal and surgical (Cardoso, 2019) and human epidermal growth factor receptor-2 (HER2) targeted therapy (Florido, 2017), these treatments are associated with drug resistance, toxicity and side effects (Al-Kishawi et al., 2016 ). Therefore, nowadays, medicinal plant and herbal therapy is resorted to, as their polyphenolic extracts inhibit human cancer cell lines, including colon (HT-29), oral (KB and CAL-27), breast (MCF-7), and prostate (LNCaP and DU-145) cancers (Zhang et al., 2008 ; Thirthalli et al., 2016 ; Bahrami and Tafrihi, 2023 ). Ginger extract is characterized by containing compounds such as 6-shogaol and 6-gingerol, which exhibit anticancer properties (Lindler et al., 2020 ) as they inhibit the proliferation of cancer cells and induce cell death in vitro (Anusha et al., 2023 ). Lee et al. ( 2008 ) found that 6-gingerol has a direct effect on adhesion, invasion, motility, and inhibition of metastasis by reducing the activities and expression of matrix metallopeptidase (MMP-2 and MMP-9) in cultured human breast cancer cells. While Tuntiwechapikul et al. ( 2010 ) found that 10-gingerol inhibited the expression of two prominent cancer molecular targets, c-Myc and human telomerase reverse transcriptase (hTERT). One study showed that treatment with ethanolic ginger extract at doses of 1.0 and 0.2 mg/mL significantly suppressed proliferation, colony formation and modified colony size in MCF-7 and MDA-MB-231 breast cancer cell lines compared to the normal breast cell line MCF-10A (Elkady et al., 2012 ). The cell division cycle is a series of events controlled by proteins and kinases (Zadorozhna & Mangieri, 2021 ) that occurs in a cell leading to its division into two daughter cells (Sun et al., 2021 ). However, cell division in the absence or dysfunction of the control mechanism leads to random divisions that cause cancer and many genetic diseases (Zadorozhna & Mangieri, 2021 ). Cancer cells are characterized by their ability to pass cell division checkpoints by acquiring certain genetic molecular changes that activate or inactivate key components of the cycle to support the abnormal proliferation of cancer cells (Liu et al., 2021 ). Therefore, the cell division cycle is important for maintaining cell proliferation and tissue integrity (Koboldt et al., 2012 ). Experimental studies have shown that ginger derivatives and extracts are able to modulate cell cycle progression as part of its protective mechanism (Girisa et al., 2021 ). For example, when breast cancer cell lines (MCF-7 and MDA-MB-231) were treated with 6-shogaol, it induced cell cycle arrest in the G2/M phase in both monolayers and cancer stem cell-like spheres, and interfered with the stem cell self-renewal pathway (Ray et al., 2015 ). In another study, 10-gingerol treatment was observed to inhibit cell proliferation and subsequent induction of G1 phase arrest and inhibition of cell invasion in response to cellular stimulation by inhibiting cell cycle regulatory proteins such as protein kinase (Akt), mitogen-activated protein kinase (p38MAPK) and suppressing the expression of epidermal growth factor receptor (EGFR) (Joo et al., 2016 ). Bernard et al. ( 2017 ) found that 10-gingerol, 8-gingerol and 6-gingerol, were able to inhibit the growth of human and mouse breast cancer cells through an inhibitory effect on the growth of the MDA-MB-231 cell line by reducing the number of cell divisions the cell cycle in the S phase, in addition to inducing apoptosis. In a study by Abdullah et al. ( 2010 ), ginger extract arrested the cell cycle in the G0/G1 phases in colon cell lines (HT29 and HCT116) due to an inhibitory effect on cyclin-dependent kinase (CDKs). From these experiments, ginger extracts appear to interfere with cancer cell proliferation and cell cycle by arresting them in G0/G1 or G2/M phase, significantly reducing the expression of cyclin and increasing the expression of cyclin-dependent kinase inhibitor (Pei et al., 2020 ). Our present study aims to evaluate the cytotoxic and anticancer activity, cell morphological changes, apoptosis-inducing effect, and cell cycle analysis of the ethanolic extract of ginger rhizome against the human breast cancer cell line MCF-7. 2. Materials and Methods 2.1. Culture Medium Human breast cancer cell line MCF-7, obtained from American College of Human Cancer Collection (ATCC, Rockvile, MD), were cultured in flask with RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich, USA) and 50 µg/mL gentamicin (Sigma-Aldrich, USA). Cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. The culture medium was changed every 2 days and cells were re-cultured 2–3 times per week. Cells were then washed with phosphate-buffered saline (PBS), incubated with 2 mL of trypsin (Gibco, USA), centrifuged, and resuspended in culture medium for plating. Only cells that had grown to exponential phase and reached 70% confluence density were used in all experiments. 2.2. Ginger Extract Ginger rhizomes weighing 250 g, purchased from Zagazig market in Egypt, were used in all experiments of this research. To prepare the ethanolic ginger extract (GEE), rhizomes were dried and ground, then extracted using 70% aqueous ethanol for 72 h. With the help of a rotary evaporator (Buchi R-210, Switzerland) at 45°C and 175 mbar pressure, ginger ethanol extract was filtered and concentrated. The resulting 4 g of brown extract was stored in the refrigerator at 4°C until use. To prepare GEE stock solution, dimethyl sulfoxide (DMSO) was used, then the solution was diluted to obtain different concentrations (Osman et al., 2020 ). 2.3. Cell Treatments To study the effect of ethanolic ginger extract on cell viability using MTT assay, MCF-7 cells were seeded and allowed to adhere for 24 h. The cells were then incubated for 24, 48 and 72 h with ginger extract at the following concentrations: 0, 7.8, 15.6, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL. Morphological changes were studied using inverted microscopy and crystal violet-stained cells. Cell cycle progression and apoptosis were analyzed using flow cytometry to investigate the effects of GEE on cell lineage. 2.3.1. MTT Assay Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Merck) MTT assay, the cytotoxic effect of ethanolic ginger extract was investigated. This assay is based on the conversion of MTT to formazan crystals by living cells, which determines mitochondrial activity. Cancer cell lines were cultured in medium in Corning® 96-well tissue culture plates at a concentration of 5×10⁴ cells/well in 100 µL medium, incubated for 24 h. After cell adhesion to the plates, the culture medium was removed and ethanolic ginger extract was added at different concentrations of 7.8, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL in three replicates for each concentration. As a control, 0.5% DMSO was used in each of the 96-well plates. All were incubated for 24, 48, and 72 h. After the incubation period, the media were removed and replaced with 100 µL of RPMI-1640 medium and 10 µL of MTT stock solution (5 mg MTT in 1 mL PBS). They were incubated at 37°C and 5% CO₂ for 4 h. The blue formazan crystals were dissolved in 50 µL of DMSO, mixed well, and incubated at 37°C for 10 min. The optical density of the dissolved formazan was then measured at 570 nm with a reference wavelength of 630 nm using a microplate reader (SunRise, TECAN, Inc., USA) to determine the number of viable cells, and the percentage of viability was calculated as follows: (ODt/ODc) × 100% Where: ODt is the average optical density of the treated wells, and ODc is the average optical density of the controls. The 50% half-maximal inhibitory concentration (IC₅₀), the concentration required to cause toxic effects in 50% of healthy cells, was estimated by graphing the dose-response curve for each concentration using GraphPad Prism software (version 8.0, GraphPad Software Inc., USA). The relationship between surviving cells and drug concentration was plotted to obtain the survival curve for each tumor cell line after treatment (Mossman, 1983). 2.3.2. Morphological Changes To study the morphological changes in the treated cells compared with the control cells, ethanolic ginger extract at concentrations of 100, 200 and 500 µg/mL was added to the MCF-7 cell line, and each concentration was incubated for 24, 48 and 72 h. After that, the plates were inverted to remove the medium, and the wells were washed three times with 300 µL of phosphate-buffered saline pH 7.2. 10% formalin was used for 15 min at room temperature to fix the cells, then the cells were stained with 100 µL of 0.25% crystal violet for 20 min. Finally, the plates were rinsed with deionized water to remove excess dye and allowed to dry for observing the pathological cellular effects microscopically at 200× using an inverted microscope (CKX41; Olympus, Japan) equipped with a digital microscope camera for image capture (Rezano et al., 2021 ). 2.3.3. Apoptosis and Cell Cycle Analysis 2.3.3.1. Apoptosis Analysis Dead cells were analyzed using BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) and an Annexin V-FITC detection kit. MCF-7 and control cells were cultured in a confluent monolayer and then treated with ethanolic ginger extract at concentrations of 100, 200, and 500 µg/mL. After incubation for 24, 48, and 72 h, cells were harvested and washed twice in phosphate-buffered saline (PBS) for 20 min each time. Cells were centrifuged for 5 min and resuspended in 500 µL of binding buffer (BD, Franklin Lakes, NJ). They were then resuspended in 100 µL of binding buffer with 1 µL of FITC-Annexin V (Becton Dickinson BD PharmingenTM, Heidelberg, Germany) and incubated for 40 min at 4°C. The cells were then washed and resuspended in 150 µL of binding buffer supplemented with 1 µL of DAPI (1 µg/mL in PBS) (Invitrogen, Life Technologies, Darmstadt, Germany) to make the cells ready for analysis and detection of early and late apoptosis and necrosis (Jamalzadeh et al., 2017 ). 2.3.3.2. Cell Cycle Analysis Cell cycle analysis was performed using the CycleTESTTM PLUS DNA Detection Kit (Becton Dickinson Immune Cytometry Systems, San Jose, CA) to determine the effect of active cultures on the cell cycle distribution of the MCF-7 cell line. Treated and control cells were stained with propidium iodide after RNase treatment, following treatment with ethanolic ginger extract at concentrations of 100, 200, and 500 µg/mL and incubating them for 24, 48, and 72 h. Cell cycle distribution was calculated using Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA) (Eldenha et al., 2017; Wagdy et al., 2018 ). 2.4. Statistical Analysis Statistical analysis of the experiments was performed using the SPSS statistical analysis program (version 25.0, IBM Corp., USA), and the data were expressed as mean ± standard deviation for experiments conducted in triplicate. The independent t-test was used to compare the treated and control cells, with p < 0.05 considered statistically significant. 3. Results This section presents the experimental findings on the anticancer and cytotoxic effects of ethanolic ginger extract (GEE) on MCF-7 breast cancer cells. The results are organized into three main categories: (1) cell viability and inhibition effects, (2) morphological changes, and (3) apoptosis and cell cycle analysis. 3.1. Effect of Ethanolic Ginger Extract on MCF-7 Cell Viability and Inhibition The MTT assay demonstrated that GEE exerted significant dose- and time-dependent cytotoxic effects on MCF-7 breast cancer cells. As shown in Table 1 and Fig. 1 , cell viability decreased progressively with increasing GEE concentration and incubation time. After 24 hours of treatment, significant inhibition (p < 0.05) began at 62.5 µg/mL (1.21 ± 0.65%), reaching maximum inhibition at 1000 µg/mL (87.26 ± 0.92%). For 48-hour treatment, inhibition started at a lower concentration of 31.25 µg/mL (1.72 ± 0.64%) and reached 94.68 ± 0.68% at 1000 µg/mL. The 72-hour treatment showed the most potent effect, with inhibition beginning at the lowest tested concentration of 7.8 µg/mL (4.31 ± 0.43%) and achieving near-complete inhibition at 1000 µg/mL (97.04 ± 0.42%). The half-maximal inhibitory concentration (IC₅₀) values, representing the concentration required to inhibit 50% of cell growth, decreased significantly with longer incubation periods (Fig. 2 ). The IC₅₀ values were 420.04 ± 16.68, 124.23 ± 8.91, and 60.74 ± 3.04 µg/mL for 24, 48, and 72 hours of treatment, respectively. This inverse relationship between incubation time and IC₅₀ values indicates that longer exposure to GEE substantially enhances its cytotoxic efficacy against MCF-7 cells. Table 1 Effect of ethanolic ginger extract on MCF-7 cell viability and inhibition at different concentrations and incubation periods. Incubation Time 7.8 µg/mL 15.6 µg/mL 31.25 µg/mL 62.5 µg/mL 125 µg/mL 250 µg/mL 500 µg/mL 1000 µg/mL 24 hours Viability % 100.00 100.00 100.00 100.00 98.79 94.13 75.02 38.17 12.74 Inhibition % 0.00 0.00 0.00 0.00 1.21 5.87 24.98 61.83 87.26 48 hours Viability % 100.00 100.00 100.00 98.28 88.64 49.52 30.61 17.58 5.32 Inhibition % 0.00 0.00 0.00 1.72 11.36 50.48 69.39 82.42 94.68 72 hours Viability % 100.00 95.69 91.43 79.05 48.27 36.59 21.48 8.73 2.96 Inhibition % 0.00 4.31 8.57 20.95 51.73 63.41 78.52 91.27 97.04 3.2. Morphological Changes in MCF-7 Cells Induced by Ethanolic Ginger Extract Inverted microscopy revealed significant morphological alterations in MCF-7 cells treated with GEE at concentrations of 100, 200, and 500 µg/mL for 24, 48, and 72 hours. The observed changes were both dose- and time-dependent, with the most pronounced effects at 500 µg/mL after 72 hours of treatment. Compared to the control cells, which displayed typical epithelial morphology with regular shapes and intact cell membranes, the treated cells exhibited characteristic apoptotic features, including: Cell shrinkage and rounding Membrane blebbing and formation of apoptotic bodies Detachment from the cultural surface Decreased cell density and increased intercellular spaces Nuclear condensation and fragmentation These morphological changes are consistent with the induction of apoptosis and correlate with the cytotoxicity results from the MTT assay. The progressive nature of these changes with increasing concentration and incubation time further supports the dose- and time-dependent cytotoxic effects of GEE on MCF-7 cells. 3.3. Effects of Ethanolic Ginger Extract on Apoptosis and Cell Cycle Progression 3.3.1. Apoptosis Analysis Flow cytometry analysis using Annexin V-FITC/DAPI staining revealed that GEE induced significant apoptosis in MCF-7 cells in a concentration- and time-dependent manner (Table 2 , Fig. 3 ). After 24 hours of treatment, early apoptosis was most prominent at 100 µg/mL (18.73%), while late apoptosis and necrosis were highest at 100 µg/mL (6.18%) and 500 µg/mL (9.91%), respectively. At 48 hours, early apoptosis peaked at 200 µg/mL (19.61%), late apoptosis at 100 µg/mL (16.02%), and necrosis at 500 µg/mL (21.62%). After 72 hours, early apoptosis increased with concentration, reaching 15.05% at 500 µg/mL, while late apoptosis was highest at 200 µg/mL (7.69%) and necrosis at 500 µg/mL (4.41%). All treated groups showed significantly higher rates of apoptosis compared to the control group (p < 0.05), which exhibited minimal apoptosis (early: 0.53%, late: 0.21%) and necrosis (1.09%). Table 2 Apoptosis assessment of MCF-7 cells treated with different concentrations of ethanolic ginger extract for 24, 48, and 72 hours. Incubation Time Control 100 µg/mL 200 µg/mL 500 µg/mL 24 hours 0.53 18.73 11.03 14.39 0.21 6.18 3.29 2.61 1.09 7.25 9.12 9.91 48 hours 0.53 8.11 19.61 15.36 0.21 16.02 2.61 5.77 1.09 3.41 8.81 21.62 72 hours 0.53 11.51 14.52 15.05 0.21 3.68 7.69 2.49 1.09 2.27 1.30 4.41 3.3.2. Cell Cycle Analysis Cell cycle analysis by flow cytometry revealed that GEE treatment induced significant alterations in cell cycle distribution of MCF-7 cells (Table 3 , Fig. 4 ). The most notable effect was the accumulation of cells in the G2/M phase, particularly at higher concentrations and longer incubation periods. After 48 hours of treatment with 500 µg/mL GEE, the percentage of cells in G2/M phase reached 31.09%, compared to 20.43% in the control group. Similarly, after 72 hours of treatment with 500 µg/mL, G2/M phase accumulation was 27.51%. This G2/M phase arrest was accompanied by a corresponding decrease in the G0/G1 phase population, particularly at 500 µg/mL after 48 hours (44.93% vs. 55.41% in control). The S phase population also showed concentration-dependent changes, with the highest percentage (31.55%) observed at 200 µg/mL after 48 hours of treatment. These results indicate that GEE induces cell cycle arrest primarily at the G2/M phase, preventing the completion of mitosis and contributing to the observed cytotoxic effects. The cell cycle arrest was most pronounced at higher concentrations (500 µg/mL) and longer incubation periods (48 and 72 hours), correlating with the cytotoxicity and apoptosis data. Table 3 Cell cycle distribution of MCF-7 cells treated with different concentrations of ethanolic ginger extract for 24, 48, and 72 hours. Incubation Time Control 100 µg/mL 200 µg/mL 500 µg/mL 24 hours 55.41 52.26 47.39 51.36 24.16 27.85 28.74 22.23 20.43 19.89 23.87 26.41 48 hours 55.41 51.07 49.03 44.93 24.16 25.36 31.55 23.98 20.43 23.57 19.42 31.09 72 hours 55.41 57.20 55.47 48.33 24.16 19.62 21.35 24.16 20.43 23.18 23.18 27.51 In summary, our results demonstrate that ethanolic ginger extract exerts potent anticancer effects on MCF-7 breast cancer cells through multiple mechanisms: (1) dose- and time-dependent reduction in cell viability, (2) induction of characteristic apoptotic morphological changes, (3) activation of both early and late apoptotic pathways, and (4) cell cycle arrest primarily at the G2/M phase. These effects were most pronounced at higher concentrations (200–500 µg/mL) and longer incubation periods (48–72 hours), suggesting that both dose and duration of exposure are critical factors in the anticancer activity of ginger extract against breast cancer cells. 4. Discussion Recently, many anticancer drugs have been derived from natural sources, such as plants and herbs, as well as their compounds (Abdallah et al., 2010; Chunarkar-Patil et al., 2024 ). Numerous studies have demonstrated that ginger exhibits antioxidant and anti-inflammatory properties (Nile and Park, 2015 ; Zhang et al., 2016 ). Research has also revealed the anticancer effect of ginger on various human cancer cells (Habib et al., 2008 ; Afshin et al., 2022 ). In vitro studies have also shown that the flavonoid and polyphenol components present in ginger rhizome are capable of suppressing cancerous tumors (Mao et al., 2019 ; Asuzu et al., 2022 ; Anusha et al., 2023 ). In this study, the MCF-7 human breast cancer cell line served as a model to evaluate ethanolic ginger extract's effects on cytotoxicity, viability, morphology, apoptosis, and cell cycle progression (Al-Kishawi et al., 2016 ; Abdallah et al., 2010). Our MTT assay demonstrated that ginger extract significantly reduced MCF-7 cell viability in a dose- and time-dependent manner (Elkady et al., 2012 ). Inhibition increased with higher concentrations (ranging from 0 to 1000 µg/mL) and longer incubation periods (24, 48, and 72 h), as shown in Table 1 and Fig. 1 . There was a significant difference in cell viability between treated cells and the control group (p < 0.05). The total activity values of ginger extract IC₅₀ (the concentration causing toxic effects in 50% of healthy cells) for 24, 48, and 72 h of treatment were 420.04 ± 16.68, 124.23 ± 8.91, and 60.74 ± 3.04 µg/mL, respectively (Churiyah et al., 2020 ). In one study also using MTT assay, Afshina et al. ( 2022 ) observed that the viability of MCF-7 cells decreased significantly with increasing concentration of ginger extract (2.5, 5, and 10 µg/mL) for a 48 h incubation period compared to the control group. 5. Conclusions and Recommendations Ginger is well known for its uses as a spice and medicinal remedy due to the diverse biological activities of its extracts. From our research, we conclude that ethanolic ginger extract induces time-dependent cell cycle arrest in G2/M phase, which leads to decreased or impaired autophagy and promotes apoptosis in breast cancer cells. In addition, ginger extract was able to inhibit the proliferation, metastasis, and invasion of MCF-7 breast cancer cells depending on the dose concentration and incubation period. The anticancer effects of ginger extract may arise from its ability to induce changes in multiple cellular processes, including cell division, cell death, and differentiation. Based on these promising results, we recommend conducting extensive in vivo studies to investigate the therapeutic potential of specific ginger extract components against various cancer types, particularly focusing on the mechanisms of cell cycle arrest and apoptosis induction. Declarations The study was conducted according to the guidelines of the Declaration of the Libyan Academy and approved by the Ethics Committee of the School of Life Sciences, Libyan Academy - Misurata (SLS-LAM Protocol No. 2024:12, approved on July 21, 2024). Authors' contributions EA and MA carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. BSMA and ARE carried out the immunoassays. AA, AIA, and RAA participated in the sequence alignment. ES participated in the design of the study and performed the statistical analysis. ABM and NIA conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.” Informed consent statement There is no informed consent because the study was conducted on MCF-7 cells. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflict of interest The authors declare that there is no conflict of interest. Funding This research received no external funding. Acknowledgments This research was supported by the Faculty of Pharmacy, University of Jazan, KSA. The authors would like to thank the technical staff of the Cell Culture Laboratory for their assistance with the experiments. References Abdullah S, Abidin SAZ, Murad NA, Makpol S, Ngah WZW, Yusof YAM. Ginger extract (Zingiber officinale) triggers apoptosis and G0/G1 cells arrest in HCT 116 and HT 29 colon cancer cell lines. Afr J Biochem Res 2010;4:134-42. Afshin A, Khosravi A, Mansouri K, Hasanvand A, Amiri F, Razzaghi-Asl N, et al. 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Ali","email":"","orcid":"","institution":"Majmaah University","correspondingAuthor":false,"prefix":"","firstName":"Nagi","middleName":"I.","lastName":"Ali","suffix":""}],"badges":[],"createdAt":"2025-06-12 00:08:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6875241/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6875241/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86417454,"identity":"a60d9fd1-e92b-4680-9211-f81d89f37e3b","added_by":"auto","created_at":"2025-07-10 11:55:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":169089,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent effects of ethanolic ginger extract on MCF-7 cell viability (A) and inhibition (B) after 24, 48, and 72 hours of treatment. Values represent the mean of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6875241/v1/a311a3c774994ee2c2ea4c7d.png"},{"id":86417459,"identity":"a747798b-0334-4d01-a788-3b9f59394b4d","added_by":"auto","created_at":"2025-07-10 11:55:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102910,"visible":true,"origin":"","legend":"\u003cp\u003eIC₅₀ values of ethanolic ginger extract on MCF-7 cells after 24, 48, and 72 hours of treatment. Values represent mean ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6875241/v1/1fbca72ce2c09e0a8d7cc1bb.png"},{"id":86417458,"identity":"73de9955-445a-4714-b35d-4c2ac3c87046","added_by":"auto","created_at":"2025-07-10 11:55:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":330337,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis analysis of MCF-7 cells treated with different concentrations of ethanolic ginger extract for 24 hours (A), 48 hours (B), and 72 hours (C). Values represent the mean of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6875241/v1/74266820940277c392a30bf8.png"},{"id":86417455,"identity":"ee9ceb55-3e68-4462-b5cf-bb826bf0c267","added_by":"auto","created_at":"2025-07-10 11:55:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":318011,"visible":true,"origin":"","legend":"\u003cp\u003eCell cycle distribution of MCF-7 cells treated with different concentrations of ethanolic ginger extract for 24 hours (A), 48 hours (B), and 72 hours (C). Values represent the mean of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6875241/v1/172c7388300acd43f11446d0.png"},{"id":91565908,"identity":"55d338e7-d0a5-43c7-aced-25d3152abde4","added_by":"auto","created_at":"2025-09-17 19:31:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1715455,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6875241/v1/e986c986-7dc3-4fbb-90dd-6f554c06665d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anticancer Activity and Cytotoxic Effect of Rhizome Extract Against MCF-7 Breast cancer Cell Line – an In Vitro study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMedicinal plants are used as therapeutic agents in resource-limited communities due to their abundance and low cost. They are a natural source of biologically active and therapeutic components that play an important role in disease prevention and improving human health (Berida et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Medicinal plants and their compounds, especially phenolics, can scavenge free radicals and protect cells from oxidative damage (Sun and Shahrajabian, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), by interacting with oxidation products derived from fatty acids to prevent oxidative stress and improve food quality and flavor (Ziarno et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, medicinal plants and their compounds and extracts are the main source of many secondary metabolites that exhibit therapeutic properties (Gang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among many medicinal plants, researchers and nutritionists have been particularly interested in ginger (Zingiber officinale), which is widely used in low-income communities as both a spice and for medicinal purposes (Yadav et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Berida et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) as an additional drug for the treatment of various types of cancer (Ziarno et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ginger contains volatile oils, anthocyanins, tannins, phenolic compounds, and sesquiterpenes as major components, in addition to terpenes and non-volatile pungent substances such as oleoresins called gingerols (Hosseinzadeh et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bobde, 2022). Several studies have provided evidence that ginger and its pungent organic compounds have anti-inflammatory and antitumor activities through free radical scavenging, antioxidant pathways, gene expression modification, and apoptosis induction, which contribute to reducing tumor initiation and progression (Dhanik et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shahrajabian et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ziarno et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Berida et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBreast cancer is a common type of cancer among women. It has a high metastatic potential and can spread to different parts of the body (Torre et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Although there are different treatments available for breast cancer, including chemotherapy, radiation, hormonal and surgical (Cardoso, 2019) and human epidermal growth factor receptor-2 (HER2) targeted therapy (Florido, 2017), these treatments are associated with drug resistance, toxicity and side effects (Al-Kishawi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, nowadays, medicinal plant and herbal therapy is resorted to, as their polyphenolic extracts inhibit human cancer cell lines, including colon (HT-29), oral (KB and CAL-27), breast (MCF-7), and prostate (LNCaP and DU-145) cancers (Zhang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Thirthalli et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bahrami and Tafrihi, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Ginger extract is characterized by containing compounds such as 6-shogaol and 6-gingerol, which exhibit anticancer properties (Lindler et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) as they inhibit the proliferation of cancer cells and induce cell death in vitro (Anusha et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Lee et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) found that 6-gingerol has a direct effect on adhesion, invasion, motility, and inhibition of metastasis by reducing the activities and expression of matrix metallopeptidase (MMP-2 and MMP-9) in cultured human breast cancer cells. While Tuntiwechapikul et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) found that 10-gingerol inhibited the expression of two prominent cancer molecular targets, c-Myc and human telomerase reverse transcriptase (hTERT). One study showed that treatment with ethanolic ginger extract at doses of 1.0 and 0.2 mg/mL significantly suppressed proliferation, colony formation and modified colony size in MCF-7 and MDA-MB-231 breast cancer cell lines compared to the normal breast cell line MCF-10A (Elkady et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe cell division cycle is a series of events controlled by proteins and kinases (Zadorozhna \u0026amp; Mangieri, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) that occurs in a cell leading to its division into two daughter cells (Sun et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, cell division in the absence or dysfunction of the control mechanism leads to random divisions that cause cancer and many genetic diseases (Zadorozhna \u0026amp; Mangieri, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cancer cells are characterized by their ability to pass cell division checkpoints by acquiring certain genetic molecular changes that activate or inactivate key components of the cycle to support the abnormal proliferation of cancer cells (Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, the cell division cycle is important for maintaining cell proliferation and tissue integrity (Koboldt et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExperimental studies have shown that ginger derivatives and extracts are able to modulate cell cycle progression as part of its protective mechanism (Girisa et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, when breast cancer cell lines (MCF-7 and MDA-MB-231) were treated with 6-shogaol, it induced cell cycle arrest in the G2/M phase in both monolayers and cancer stem cell-like spheres, and interfered with the stem cell self-renewal pathway (Ray et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In another study, 10-gingerol treatment was observed to inhibit cell proliferation and subsequent induction of G1 phase arrest and inhibition of cell invasion in response to cellular stimulation by inhibiting cell cycle regulatory proteins such as protein kinase (Akt), mitogen-activated protein kinase (p38MAPK) and suppressing the expression of epidermal growth factor receptor (EGFR) (Joo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Bernard et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) found that 10-gingerol, 8-gingerol and 6-gingerol, were able to inhibit the growth of human and mouse breast cancer cells through an inhibitory effect on the growth of the MDA-MB-231 cell line by reducing the number of cell divisions the cell cycle in the S phase, in addition to inducing apoptosis. In a study by Abdullah et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), ginger extract arrested the cell cycle in the G0/G1 phases in colon cell lines (HT29 and HCT116) due to an inhibitory effect on cyclin-dependent kinase (CDKs). From these experiments, ginger extracts appear to interfere with cancer cell proliferation and cell cycle by arresting them in G0/G1 or G2/M phase, significantly reducing the expression of cyclin and increasing the expression of cyclin-dependent kinase inhibitor (Pei et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our present study aims to evaluate the cytotoxic and anticancer activity, cell morphological changes, apoptosis-inducing effect, and cell cycle analysis of the ethanolic extract of ginger rhizome against the human breast cancer cell line MCF-7.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Culture Medium\u003c/h2\u003e\u003cp\u003eHuman breast cancer cell line MCF-7, obtained from American College of Human Cancer Collection (ATCC, Rockvile, MD), were cultured in flask with RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich, USA) and 50 \u0026micro;g/mL gentamicin (Sigma-Aldrich, USA). Cells were maintained in a humidified atmosphere with 5% CO₂ at 37\u0026deg;C. The culture medium was changed every 2 days and cells were re-cultured 2\u0026ndash;3 times per week. Cells were then washed with phosphate-buffered saline (PBS), incubated with 2 mL of trypsin (Gibco, USA), centrifuged, and resuspended in culture medium for plating. Only cells that had grown to exponential phase and reached 70% confluence density were used in all experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Ginger Extract\u003c/h2\u003e\u003cp\u003eGinger rhizomes weighing 250 g, purchased from Zagazig market in Egypt, were used in all experiments of this research. To prepare the ethanolic ginger extract (GEE), rhizomes were dried and ground, then extracted using 70% aqueous ethanol for 72 h. With the help of a rotary evaporator (Buchi R-210, Switzerland) at 45\u0026deg;C and 175 mbar pressure, ginger ethanol extract was filtered and concentrated. The resulting 4 g of brown extract was stored in the refrigerator at 4\u0026deg;C until use. To prepare GEE stock solution, dimethyl sulfoxide (DMSO) was used, then the solution was diluted to obtain different concentrations (Osman et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Cell Treatments\u003c/h2\u003e\u003cp\u003eTo study the effect of ethanolic ginger extract on cell viability using MTT assay, MCF-7 cells were seeded and allowed to adhere for 24 h. The cells were then incubated for 24, 48 and 72 h with ginger extract at the following concentrations: 0, 7.8, 15.6, 31.25, 62.5, 125, 250, 500 and 1000 \u0026micro;g/mL. Morphological changes were studied using inverted microscopy and crystal violet-stained cells. Cell cycle progression and apoptosis were analyzed using flow cytometry to investigate the effects of GEE on cell lineage.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. MTT Assay\u003c/h2\u003e\u003cp\u003eUsing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Merck) MTT assay, the cytotoxic effect of ethanolic ginger extract was investigated. This assay is based on the conversion of MTT to formazan crystals by living cells, which determines mitochondrial activity. Cancer cell lines were cultured in medium in Corning\u0026reg; 96-well tissue culture plates at a concentration of 5\u0026times;10⁴ cells/well in 100 \u0026micro;L medium, incubated for 24 h. After cell adhesion to the plates, the culture medium was removed and ethanolic ginger extract was added at different concentrations of 7.8, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 \u0026micro;g/mL in three replicates for each concentration. As a control, 0.5% DMSO was used in each of the 96-well plates. All were incubated for 24, 48, and 72 h. After the incubation period, the media were removed and replaced with 100 \u0026micro;L of RPMI-1640 medium and 10 \u0026micro;L of MTT stock solution (5 mg MTT in 1 mL PBS). They were incubated at 37\u0026deg;C and 5% CO₂ for 4 h. The blue formazan crystals were dissolved in 50 \u0026micro;L of DMSO, mixed well, and incubated at 37\u0026deg;C for 10 min. The optical density of the dissolved formazan was then measured at 570 nm with a reference wavelength of 630 nm using a microplate reader (SunRise, TECAN, Inc., USA) to determine the number of viable cells, and the percentage of viability was calculated as follows:\u003c/p\u003e\u003cp\u003e(ODt/ODc) \u0026times; 100%\u003c/p\u003e\u003cp\u003eWhere: ODt is the average optical density of the treated wells, and ODc is the average optical density of the controls.\u003c/p\u003e\u003cp\u003eThe 50% half-maximal inhibitory concentration (IC₅₀), the concentration required to cause toxic effects in 50% of healthy cells, was estimated by graphing the dose-response curve for each concentration using GraphPad Prism software (version 8.0, GraphPad Software Inc., USA). The relationship between surviving cells and drug concentration was plotted to obtain the survival curve for each tumor cell line after treatment (Mossman, 1983).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Morphological Changes\u003c/h2\u003e\u003cp\u003eTo study the morphological changes in the treated cells compared with the control cells, ethanolic ginger extract at concentrations of 100, 200 and 500 \u0026micro;g/mL was added to the MCF-7 cell line, and each concentration was incubated for 24, 48 and 72 h. After that, the plates were inverted to remove the medium, and the wells were washed three times with 300 \u0026micro;L of phosphate-buffered saline pH 7.2. 10% formalin was used for 15 min at room temperature to fix the cells, then the cells were stained with 100 \u0026micro;L of 0.25% crystal violet for 20 min. Finally, the plates were rinsed with deionized water to remove excess dye and allowed to dry for observing the pathological cellular effects microscopically at 200\u0026times; using an inverted microscope (CKX41; Olympus, Japan) equipped with a digital microscope camera for image capture (Rezano et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3. Apoptosis and Cell Cycle Analysis\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section4\"\u003e\u003ch2\u003e2.3.3.1. Apoptosis Analysis\u003c/h2\u003e\u003cp\u003eDead cells were analyzed using BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) and an Annexin V-FITC detection kit. MCF-7 and control cells were cultured in a confluent monolayer and then treated with ethanolic ginger extract at concentrations of 100, 200, and 500 \u0026micro;g/mL. After incubation for 24, 48, and 72 h, cells were harvested and washed twice in phosphate-buffered saline (PBS) for 20 min each time. Cells were centrifuged for 5 min and resuspended in 500 \u0026micro;L of binding buffer (BD, Franklin Lakes, NJ). They were then resuspended in 100 \u0026micro;L of binding buffer with 1 \u0026micro;L of FITC-Annexin V (Becton Dickinson BD PharmingenTM, Heidelberg, Germany) and incubated for 40 min at 4\u0026deg;C. The cells were then washed and resuspended in 150 \u0026micro;L of binding buffer supplemented with 1 \u0026micro;L of DAPI (1 \u0026micro;g/mL in PBS) (Invitrogen, Life Technologies, Darmstadt, Germany) to make the cells ready for analysis and detection of early and late apoptosis and necrosis (Jamalzadeh et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section4\"\u003e\u003ch2\u003e2.3.3.2. Cell Cycle Analysis\u003c/h2\u003e\u003cp\u003eCell cycle analysis was performed using the CycleTESTTM PLUS DNA Detection Kit (Becton Dickinson Immune Cytometry Systems, San Jose, CA) to determine the effect of active cultures on the cell cycle distribution of the MCF-7 cell line. Treated and control cells were stained with propidium iodide after RNase treatment, following treatment with ethanolic ginger extract at concentrations of 100, 200, and 500 \u0026micro;g/mL and incubating them for 24, 48, and 72 h. Cell cycle distribution was calculated using Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA) (Eldenha et al., 2017; Wagdy et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis of the experiments was performed using the SPSS statistical analysis program (version 25.0, IBM Corp., USA), and the data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation for experiments conducted in triplicate. The independent t-test was used to compare the treated and control cells, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThis section presents the experimental findings on the anticancer and cytotoxic effects of ethanolic ginger extract (GEE) on MCF-7 breast cancer cells. The results are organized into three main categories: (1) cell viability and inhibition effects, (2) morphological changes, and (3) apoptosis and cell cycle analysis.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Effect of Ethanolic Ginger Extract on MCF-7 Cell Viability and Inhibition\u003c/h2\u003e\u003cp\u003eThe MTT assay demonstrated that GEE exerted significant dose- and time-dependent cytotoxic effects on MCF-7 breast cancer cells. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, cell viability decreased progressively with increasing GEE concentration and incubation time. After 24 hours of treatment, significant inhibition (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) began at 62.5 \u0026micro;g/mL (1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65%), reaching maximum inhibition at 1000 \u0026micro;g/mL (87.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92%). For 48-hour treatment, inhibition started at a lower concentration of 31.25 \u0026micro;g/mL (1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64%) and reached 94.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68% at 1000 \u0026micro;g/mL. The 72-hour treatment showed the most potent effect, with inhibition beginning at the lowest tested concentration of 7.8 \u0026micro;g/mL (4.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43%) and achieving near-complete inhibition at 1000 \u0026micro;g/mL (97.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42%).\u003c/p\u003e\u003cp\u003eThe half-maximal inhibitory concentration (IC₅₀) values, representing the concentration required to inhibit 50% of cell growth, decreased significantly with longer incubation periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The IC₅₀ values were 420.04\u0026thinsp;\u0026plusmn;\u0026thinsp;16.68, 124.23\u0026thinsp;\u0026plusmn;\u0026thinsp;8.91, and 60.74\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 \u0026micro;g/mL for 24, 48, and 72 hours of treatment, respectively. This inverse relationship between incubation time and IC₅₀ values indicates that longer exposure to GEE substantially enhances its cytotoxic efficacy against MCF-7 cells.\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\u003eEffect of ethanolic ginger extract on MCF-7 cell viability and inhibition at different concentrations and incubation periods.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\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\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eIncubation Time\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.8 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.6 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31.25 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e62.5 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e125 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e250 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e500 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1000 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eViability %\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\u003e98.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e94.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e75.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e38.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e12.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInhibition %\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e5.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e24.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e61.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e87.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e48 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eViability %\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\u003e98.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e88.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e49.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e30.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e17.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e5.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInhibition %\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e11.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e50.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e69.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e82.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e94.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e72 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eViability %\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\u003e95.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e91.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e79.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e48.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e36.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e21.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e8.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInhibition %\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e51.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e63.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e78.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e91.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e97.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Morphological Changes in MCF-7 Cells Induced by Ethanolic Ginger Extract\u003c/h2\u003e\u003cp\u003eInverted microscopy revealed significant morphological alterations in MCF-7 cells treated with GEE at concentrations of 100, 200, and 500 \u0026micro;g/mL for 24, 48, and 72 hours. The observed changes were both dose- and time-dependent, with the most pronounced effects at 500 \u0026micro;g/mL after 72 hours of treatment. Compared to the control cells, which displayed typical epithelial morphology with regular shapes and intact cell membranes, the treated cells exhibited characteristic apoptotic features, including:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eCell shrinkage and rounding\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eMembrane blebbing and formation of apoptotic bodies\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eDetachment from the cultural surface\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eDecreased cell density and increased intercellular spaces\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eNuclear condensation and fragmentation\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThese morphological changes are consistent with the induction of apoptosis and correlate with the cytotoxicity results from the MTT assay. The progressive nature of these changes with increasing concentration and incubation time further supports the dose- and time-dependent cytotoxic effects of GEE on MCF-7 cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Effects of Ethanolic Ginger Extract on Apoptosis and Cell Cycle Progression\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1. Apoptosis Analysis\u003c/h2\u003e\u003cp\u003eFlow cytometry analysis using Annexin V-FITC/DAPI staining revealed that GEE induced significant apoptosis in MCF-7 cells in a concentration- and time-dependent manner (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After 24 hours of treatment, early apoptosis was most prominent at 100 \u0026micro;g/mL (18.73%), while late apoptosis and necrosis were highest at 100 \u0026micro;g/mL (6.18%) and 500 \u0026micro;g/mL (9.91%), respectively. At 48 hours, early apoptosis peaked at 200 \u0026micro;g/mL (19.61%), late apoptosis at 100 \u0026micro;g/mL (16.02%), and necrosis at 500 \u0026micro;g/mL (21.62%). After 72 hours, early apoptosis increased with concentration, reaching 15.05% at 500 \u0026micro;g/mL, while late apoptosis was highest at 200 \u0026micro;g/mL (7.69%) and necrosis at 500 \u0026micro;g/mL (4.41%). All treated groups showed significantly higher rates of apoptosis compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which exhibited minimal apoptosis (early: 0.53%, late: 0.21%) and necrosis (1.09%).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eApoptosis assessment of MCF-7 cells treated with different concentrations of ethanolic ginger extract for 24, 48, and 72 hours.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIncubation Time\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e48 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e15.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e21.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e72 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e15.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2. Cell Cycle Analysis\u003c/h2\u003e\u003cp\u003eCell cycle analysis by flow cytometry revealed that GEE treatment induced significant alterations in cell cycle distribution of MCF-7 cells (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The most notable effect was the accumulation of cells in the G2/M phase, particularly at higher concentrations and longer incubation periods. After 48 hours of treatment with 500 \u0026micro;g/mL GEE, the percentage of cells in G2/M phase reached 31.09%, compared to 20.43% in the control group. Similarly, after 72 hours of treatment with 500 \u0026micro;g/mL, G2/M phase accumulation was 27.51%. This G2/M phase arrest was accompanied by a corresponding decrease in the G0/G1 phase population, particularly at 500 \u0026micro;g/mL after 48 hours (44.93% vs. 55.41% in control).\u003c/p\u003e\u003cp\u003eThe S phase population also showed concentration-dependent changes, with the highest percentage (31.55%) observed at 200 \u0026micro;g/mL after 48 hours of treatment. These results indicate that GEE induces cell cycle arrest primarily at the G2/M phase, preventing the completion of mitosis and contributing to the observed cytotoxic effects. The cell cycle arrest was most pronounced at higher concentrations (500 \u0026micro;g/mL) and longer incubation periods (48 and 72 hours), correlating with the cytotoxicity and apoptosis data.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCell cycle distribution of MCF-7 cells treated with different concentrations of ethanolic ginger extract for 24, 48, and 72 hours.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIncubation Time\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e500 \u0026micro;g/mL\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e24 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e55.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e52.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e47.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e51.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e28.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e22.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e26.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e48 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e55.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e51.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e49.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e44.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e23.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e31.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e72 hours\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e55.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e57.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e55.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e48.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e21.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e24.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e23.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e27.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn summary, our results demonstrate that ethanolic ginger extract exerts potent anticancer effects on MCF-7 breast cancer cells through multiple mechanisms: (1) dose- and time-dependent reduction in cell viability, (2) induction of characteristic apoptotic morphological changes, (3) activation of both early and late apoptotic pathways, and (4) cell cycle arrest primarily at the G2/M phase. These effects were most pronounced at higher concentrations (200\u0026ndash;500 \u0026micro;g/mL) and longer incubation periods (48\u0026ndash;72 hours), suggesting that both dose and duration of exposure are critical factors in the anticancer activity of ginger extract against breast cancer cells.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eRecently, many anticancer drugs have been derived from natural sources, such as plants and herbs, as well as their compounds (Abdallah et al., 2010; Chunarkar-Patil et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Numerous studies have demonstrated that ginger exhibits antioxidant and anti-inflammatory properties (Nile and Park, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Research has also revealed the anticancer effect of ginger on various human cancer cells (Habib et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Afshin et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In vitro studies have also shown that the flavonoid and polyphenol components present in ginger rhizome are capable of suppressing cancerous tumors (Mao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Asuzu et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Anusha et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, the MCF-7 human breast cancer cell line served as a model to evaluate ethanolic ginger extract's effects on cytotoxicity, viability, morphology, apoptosis, and cell cycle progression (Al-Kishawi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Abdallah et al., 2010).\u003c/p\u003e\u003cp\u003eOur MTT assay demonstrated that ginger extract significantly reduced MCF-7 cell viability in a dose- and time-dependent manner (Elkady et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Inhibition increased with higher concentrations (ranging from 0 to 1000 \u0026micro;g/mL) and longer incubation periods (24, 48, and 72 h), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. There was a significant difference in cell viability between treated cells and the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The total activity values of ginger extract IC₅₀ (the concentration causing toxic effects in 50% of healthy cells) for 24, 48, and 72 h of treatment were 420.04\u0026thinsp;\u0026plusmn;\u0026thinsp;16.68, 124.23\u0026thinsp;\u0026plusmn;\u0026thinsp;8.91, and 60.74\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 \u0026micro;g/mL, respectively (Churiyah et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In one study also using MTT assay, Afshina et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) observed that the viability of MCF-7 cells decreased significantly with increasing concentration of ginger extract (2.5, 5, and 10 \u0026micro;g/mL) for a 48 h incubation period compared to the control group.\u003c/p\u003e"},{"header":"5. Conclusions and Recommendations","content":"\u003cp\u003eGinger is well known for its uses as a spice and medicinal remedy due to the diverse biological activities of its extracts. From our research, we conclude that ethanolic ginger extract induces time-dependent cell cycle arrest in G2/M phase, which leads to decreased or impaired autophagy and promotes apoptosis in breast cancer cells. In addition, ginger extract was able to inhibit the proliferation, metastasis, and invasion of MCF-7 breast cancer cells depending on the dose concentration and incubation period. The anticancer effects of ginger extract may arise from its ability to induce changes in multiple cellular processes, including cell division, cell death, and differentiation. Based on these promising results, we recommend conducting extensive in vivo studies to investigate the therapeutic potential of specific ginger extract components against various cancer types, particularly focusing on the mechanisms of cell cycle arrest and apoptosis induction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe study was conducted according to the guidelines of the Declaration of the Libyan Academy and approved by the Ethics Committee of the School of Life Sciences, Libyan Academy - Misurata (SLS-LAM Protocol No. 2024:12, approved on July 21, 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEA and MA carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. BSMA and ARE carried out the immunoassays. AA, AIA, and RAA participated in the sequence alignment. ES participated in the design of the study and performed the statistical analysis. ABM and NIA conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no informed consent because the study was conducted on MCF-7 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Faculty of Pharmacy, University of Jazan, KSA. The authors would like to thank the technical staff of the Cell Culture Laboratory for their assistance with the experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdullah S, Abidin SAZ, Murad NA, Makpol S, Ngah WZW, Yusof YAM. Ginger extract (Zingiber officinale) triggers apoptosis and G0/G1 cells arrest in HCT 116 and HT 29 colon cancer cell lines. Afr J Biochem Res 2010;4:134-42.\u003c/li\u003e\n\u003cli\u003eAfshin A, Khosravi A, Mansouri K, Hasanvand A, Amiri F, Razzaghi-Asl N, et al. Cytotoxic effect of Zingiber officinale hydroalcoholic extract on MCF-7 breast cancer cell line. Res J Pharmacogn 2022;9:53-61.\u003c/li\u003e\n\u003cli\u003eAfshina, F.; Bohloolia, M.; Maghsoudic, A.; Sheikhnejadd, Reza.; Khajehe, M.; Khatibif, A.; Ghaffari-Moghaddame, M.; Ghamarig F. and Sheibanih, N. Anti-tumor activity of ginger extracts in MCF-7 breast cancer cells. 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Applied Sciences, 2021;11, p. 3898.\u003c/li\u003e\n\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":"Ginger extract, MCF-7 cell line, MTT, Cell cycle, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-6875241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6875241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany factors stimulate breast cancer in women, including genetic, chemical, metabolic, environmental, and physical elements. Currently, a diet rich in antioxidants is considered an effective means of controlling cancer growth and spread. Therefore, cytotoxic and anticancer compounds derived from medicinal plants are promising therapeutic agents, as they induce apoptosis pathways. Ginger is one of the medicinal herbs, and its rhizome extracts are used as an anticancer treatment for many types of malignancies. In this study, the viability of human breast cancer cell (MCF-7) was evaluated after treatment with ethanolic ginger extract (GEE) at concentrations of 0.0, 7.8, 15.62, 31.25, 62.50, 125, 250, 500, and 1000 \u0026micro;g/mL for 24, 48 and 72 h using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. GEE significantly reduced the viability of treated cells and increased their inhibition compared to the control group with increasing dose and incubation duration. GEE had significant toxic activity on treated cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with IC₅₀ values based on incubation periods of 24, 48 and 72 hours (420.04\u0026thinsp;\u0026plusmn;\u0026thinsp;16.68, 124.23\u0026thinsp;\u0026plusmn;\u0026thinsp;8.91 and 60.74\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 \u0026micro;g/mL), respectively. Using inverted microscopy, significant morphological changes were observed in MCF-7 cells compared to the control group. The treated cells became smaller and more rounded with a proportional increase in abnormal and dead cells; the morphological changes were most pronounced at a concentration of 500 \u0026micro;g/mL after 72 h of incubation. Flow cytometry measurements revealed a significant induction of apoptosis and cell cycle arrest in the G2/M phase after exposing MCF-7 cells to ginger extract at doses of 100, 200, and 500 \u0026micro;g/mL for 24, 48, and 72 h, compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Meanwhile, no significant change was observed in the G1 phase of cells.\u003c/p\u003e","manuscriptTitle":"Anticancer Activity and Cytotoxic Effect of Rhizome Extract Against MCF-7 Breast cancer Cell Line – an In Vitro study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 11:55:44","doi":"10.21203/rs.3.rs-6875241/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":"bfc4793f-f3d9-4eef-854e-a6f2a555c6e4","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51294495,"name":"Biological sciences/Cancer/Breast cancer"},{"id":51294496,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":51294497,"name":"Health sciences/Oncology/Cancer"},{"id":51294498,"name":"Biological sciences/Cancer"},{"id":51294499,"name":"Health sciences/Oncology"},{"id":51294500,"name":"Health sciences/Medical research"},{"id":51294501,"name":"Health sciences/Medical research/Biomarkers"},{"id":51294502,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":51294503,"name":"Health sciences/Medical research/Stem cell research"}],"tags":[],"updatedAt":"2025-09-17T19:23:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 11:55:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6875241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6875241","identity":"rs-6875241","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}