Exploring the Therapeutic Potential of Ruta graveolens: Balancing Cytotoxicity and Wound Closure in Skin Cells.

Description

This research project investigates the concentration-dependent biological effects of Ruta graveolens extract on two skin-related cell models: HaCaT human keratinocytes and NIH/3T3 mouse fibroblasts. The project aims to evaluate whether extract concentrations that preserve cell viability can influence cellular proliferation and in-vitro wound-closure responses. Students will participate in plant extraction, cell culture, cytotoxicity assessment using the MTT assay, cell-growth measurements, scratch assays, image analysis, and statistical interpretation. The project will emphasize the relationship between extract concentration, cellular viability, proliferation, and wound closure. Students will learn to distinguish potentially useful cellular responses from harmful cytotoxic effects and understand the limitations of interpreting scratch closure as evidence of wound healing. Through supervised laboratory work, literature review, data analysis, and scientific reporting, students will develop practical skills in experimental design, laboratory techniques, quantitative analysis, teamwork, and scientific communication. The findings are expected to provide preliminary in-vitro evidence to guide further research into the biological activity and safety considerations of Ruta graveolens extract. The study will not establish clinical wound-healing efficacy or therapeutic safety in humans. More details: Research Problem Although Ruta graveolens L. has been investigated for various cytotoxic and biological activities, further investigation is needed to clarify the relationship between extract concentration, cell viability, proliferation, and scratch-closure responses in skin-related cell models. Gandlevskiy et al. (2025) examined R. graveolens extract and extract-mediated silver nanoparticles in HaCaT keratinocytes and L929 fibroblasts. However, their study primarily focused on the synthesis and biological evaluation of silver nanoparticles. Therefore, further investigation is warranted to clarify whether the plant extract itself is associated with changes in scratch closure at concentrations that preserve cell viability, and whether these changes may be influenced by alterations in cell proliferation. This question is important because scratch closure is influenced by multiple cellular processes, including cell migration and proliferation. If extract concentrations reduce cell viability or inhibit proliferation, changes in scratch closure may not reflect a specific effect on cell migration. Moreover, R. graveolens contains bioactive compounds, including furanocoumarins, that raise relevant safety considerations because of their association with phototoxic reactions. Establishing how different extract concentrations affect cellular viability and scratch closure is therefore an important preliminary step in evaluating the plant’s potential skin-related applications. A further consideration is whether cellular responses differ between keratinocytes and fibroblasts. HaCaT cells provide a human-derived model of epidermal keratinocytes, whereas NIH/3T3 cells are mouse-derived fibroblasts. Although both models are relevant to studying cellular processes associated with skin repair, their responses cannot be assumed to be equivalent because they differ in both cell type and species origin. Evaluating both models may help clarify whether the effects of the extract vary across these cellular contexts. Accordingly, the central research problem is the limited understanding of how R. graveolens extract concentration relates to cell viability, proliferation, and scratch closure in HaCaT keratinocytes and NIH/3T3 fibroblasts. This study will investigate these outcomes together to determine whether concentrations that preserve cell viability are associated with measurable changes in proliferation and scratch closure. The findings will provide preliminary in-vitro evidence to support further investigation of the plant’s potential skin-related biological effects, without assuming that changes in scratch closure alone demonstrate improved wound healing. Aim To determine whether non-cytotoxic concentrations of Ruta graveolens extract alter cell proliferation and scratch closure differently in HaCaT keratinocytes and NIH/3T3 fibroblasts. 4. Specific Objectives 1. To prepare a hydroethanolic extract of Ruta graveolens and determine its extraction yield. 2. To evaluate the concentration-dependent effects of the extract on cell viability in HaCaT keratinocytes and NIH/3T3 fibroblasts. 3. To investigate the effects of non-cytotoxic extract concentrations on cellular proliferation and scratch closure in both cell models. 4. To compare concentration-dependent responses between HaCaT keratinocytes and NIH/3T3 fibroblasts and determine whether changes in scratch closure occur at concentrations that preserve cell viability. Hypothesis It is hypothesized that Ruta graveolens extract will exhibit concentration-dependent effects on cell viability, proliferation, and scratch closure in HaCaT keratinocytes and NIH/3T3 fibroblasts. At least one concentration range is expected to preserve cell viability while producing measurable changes in cellular proliferation or scratch closure, with responses potentially differing between the two cell models. This hypothesis will not be supported if all concentrations that preserve cell viability produce no measurable changes in proliferation or scratch closure, or if the observed changes occur only at concentrations associated with substantial loss of cell viability. Cell Culture Models Two cell models will be used in this study: HaCaT human keratinocytes and NIH/3T3 mouse fibroblasts. HaCaT cells are available in the cell culture laboratories at Arab American University, whereas NIH/3T3 fibroblasts will be obtained from a laboratory in Nablus. HaCaT cells will be used to investigate the effects of Ruta graveolens extract on keratinocyte viability, proliferation, and scratch closure, while NIH/3T3 fibroblasts will be used to evaluate these responses in fibroblasts. Using both models will allow the investigation of extract responses in two cell types relevant to skin-related cellular processes. However, because the models differ in both cell type and species origin, differences in their responses cannot be attributed exclusively to either factor . This limitation will be considered when interpreting the results. Both cell lines will be maintained according to established laboratory procedures. Information regarding cell source, authentication status, passage range, and mycoplasma-testing procedures will be documented based on available laboratory records. Assessment of Cytotoxicity The cytotoxic effects of Ruta graveolens extract will be evaluated in HaCaT human keratinocytes and NIH/3T3 mouse fibroblasts using the MTT assay. Cells will be seeded into 96-well plates at an appropriate density and allowed to attach before treatment. The cells will then be exposed to a preliminary range of extract concentrations, initially extending up to 500 µg/mL, provided the extract remains suitably soluble. The final concentration levels and exposure duration will be selected based on relevant published literature and preliminary pilot experiments. Cell viability will be assessed by comparing the results with untreated and vehicle-treated controls. The vehicle concentration will be kept at a level compatible with cell viability, and appropriate biological and technical replicates will be included according to the established laboratory protocol. Since colored plant extracts may interfere with colorimetric measurements, extract-only blanks will be included where appropriate to assess potential interference with the MTT signal. If feasible, an independent cell viability or cell-death assay will be used to support the MTT findings. The concentration–response results will guide the selection of concentrations for subsequent proliferation and scratch-closure experiments. Concentrations associated with substantial loss of viability will not be interpreted as evidence of a beneficial skin-related effect. Assessment of Cytostatic Effects The effects of Ruta graveolens extract on cell proliferation will be assessed at selected non-cytotoxic concentrations using direct cell counting. Cell growth will be evaluated at 24 and 48 hours and compared with untreated and vehicle-treated controls. Cell proliferation results will be interpreted alongside MTT viability results to distinguish reduced cell growth from loss of cell viability. Reduced cell growth accompanied by relatively preserved viability will be considered consistent with a potential cytostatic effect, whereas a substantial reduction in viability may indicate cytotoxicity. Since MTT measures metabolic activity rather than cell proliferation directly, the two methods will be interpreted together. To investigate whether the cytostatic effect is reversible, a washout and recovery experiment will be performed after removal of the extract. Reversibility will be concluded only if recovery of cell growth is experimentally demonstrated. In-Vitro Wound-Closure Assay A scratch assay will be used to evaluate wound-closure responses in HaCaT and NIH/3T3 cells treated with selected concentrations of Ruta graveolens extract based on the cytotoxicity results. A consistent scratch will be created in the cell monolayer using the same procedure across all treatment and control groups. Detached cells will be removed, and the selected treatments will be applied. Images will be captured immediately after scratching (0 hours) and at defined follow-up time points, including 12 and 24 hours, using consistent imaging conditions. Scratch areas will be quantified using ImageJ, and percentage wound closure will be calculated relative to the initial scratch area at 0 hours. Measurements will be performed using a consistent analysis procedure across all groups. Independent experimental repeats will be conducted to assess the reproducibility of the findings, and image analysis will be performed using predefined criteria to minimize observer bias. Untreated and vehicle-treated cells will serve as controls. Extract concentrations will be selected based on the cytotoxicity results to minimize the influence of substantial cell death on wound-closure measurements. Because scratch closure can be influenced by both cell migration and proliferation, the findings will be interpreted alongside the cell viability and proliferation results. The assay will be described as measuring wound-closure responses and will not, by itself, be considered definitive evidence of cell migration or wound healing. Statistical Analysis ● The primary endpoint will be cell viability measured by the MTT assay following treatment with Ruta graveolens extract. Secondary endpoints will include cell growth and percentage wound closure. The experimental unit will be an independent biological experiment, while technical replicate wells will be used to improve measurement reliability and will not be treated as independent biological samples. ● Each experiment will include three independent biological replicates. This number is proposed as an initial basis for exploratory undergraduate research, with the limitation that it may provide limited statistical power to detect small effects. Technical replicates will be included according to the experimental protocol. ● Results will be summarized as mean ± standard deviation. Treatment groups will be compared with the relevant untreated or vehicle-treated controls. For comparisons involving multiple concentrations, one-way ANOVA followed by Dunnett’s post-hoc test will be used when its assumptions are met. Tukey’s test will be used when all pairwise group comparisons are required. For experiments involving multiple factors, an appropriate factorial ANOVA or other suitable statistical method will be selected according to the design and data distribution. Statistical significance will be set at p < 0.05. ●Results will be presented graphically to show differences between treatment groups and, where relevant, concentration-dependent responses. The sample size will be considered appropriate for an exploratory study, and the findings will be interpreted with due consideration of the number of independent biological replicates and the limitations in statistical power. 8. Expected Outcomes and Significance This project is expected to generate preliminary data on the effects of Ruta graveolens extract on HaCaT human keratinocytes and NIH/3T3 mouse fibroblasts, focusing on cell viability, proliferation, and in-vitro wound-closure responses. The findings may: 1. Identify extract concentrations associated with relatively preserved cell viability and distinguish them from concentrations that cause substantial cellular toxicity. 2. Determine whether selected concentrations affect cell proliferation independently of substantial loss of viability. 3. Establish whether the extract alters wound-closure responses in HaCaT and NIH/3T3 cells. 4. Identify concentration-dependent responses that may guide further mechanistic studies. The significance of this study lies in its evaluation of both potentially useful cellular responses and possible harmful effects of R. graveolens extract. Identifying a concentration range that preserves cell viability while influencing cell growth or wound closure may help guide future investigations. Conversely, evidence of substantial cytotoxicity would help identify concentrations that may be unsuitable for further development. Overall, the results will provide preliminary in-vitro evidence to support the design of future studies. They will not, by themselves, establish clinical wound-healing efficacy, prove that the extract treats inflammatory skin diseases. Feasibility and Work Plan The project will be conducted in coordination with the relevant laboratories at Arab American University, under the supervisor’s guidance. The work will begin with laboratory coordination and preparation to confirm the availability of the required equipment, reagents, and experimental materials. The work plan will include the following stages: 1. Laboratory Coordination and Preparation: Coordinate with laboratory staff to prepare the required equipment, reagents, and materials and ensure that the planned experimental procedures can be carried out. 2. Plant Collection and Preparation: Collect the aerial parts of Ruta graveolens and dry them in the shade until adequately dried for grinding and extraction. 3. Cell Sourcing and Coordination: Confirm the availability of the required cell lines. HaCaT cells are available at Arab American University, while coordination with a laboratory in Nablus will be undertaken to obtain NIH/3T3 fibroblasts. 4. Plant Extract Preparation: Grind the dried plant material and prepare the extract according to the established extraction procedure. 5. Cytotoxicity Assessment: Evaluate the effects of the extract on cell viability using the MTT assay and identify suitable concentrations for subsequent experiments. 6. Cell-Growth and Wound-Closure Experiments: Assess cell growth and in-vitro wound-closure responses using selected extract concentrations in the relevant cell models. 7. Data Analysis and Interpretation: Analyze the experimental results, review the findings with the supervisor, and compare the observations with relevant published studies. A literature review will be conducted throughout the project to support experimental planning, address methodological challenges, and guide the interpretation of unexpected findings. The project timeline will remain flexible and will be adjusted according to the progress of the experimental work, the time required for plant collection and drying, cell availability, and the successful completion of each experimental stage. The planned sequence is expected to proceed without significant technical or logistical obstacles; however, the schedule may be revised in consultation with the supervisor if delays or experimental challenges arise. The main deliverables will include the prepared Ruta graveolens extract, cell-viability data, cell-growth measurements and quantitative wound-closure results. The final findings will be compiled and interpreted in relation to the study objectives and relevant scientific literature.