GYNOPHYTO

About the Team

Lahye Yun

Korea International School, Jeju Campus (KISJ) G11

Interests:

Biomedical research
Environmental studies

Research Projects:

Development of a Natural Ointment Using Active Ingredient Content and Antibacterial Activity of Extracts from Ten Plants Including Artemisia capillaris
A Systematic Analysis of the Anticancer Activities of Medicinal Plant Extracts in Triple-Negative Breast Cancer (MDA-MB-231) cells
Analysis of antibacterial activity of acne bacteria found in Sasa quelpaertensis extract and efficacy as cosmetics

Future Goals

I want to pursue biology and continue developing innovative solutions to real-world health and environmental problems.

YeJin Ahn

St. Johnsbury Academy Jeju (SJA), Grade 11

Interests:

Cosmetics
Watching scientific videos (mostly about nature)

Research Projects:

Sasa Quelpaertensis-Based Feminine Hygiene Wash
Jeju Jori Tea for Diabetes Management

Future Goals

Later, I hope to attend medical school and become a doctor, where I can continue exploring my interest in health, especially women's health.

Who are We?

We are a female-led research team from Jeju. We are 11th graders, Lahye Yun and Yejin Ahn, and we conduct research using a variety of plant resources that grow on Jeju Island.

Background

While we were continuously conducting research using Jeju plant resources, we witnessed a close friend's mother struggle with the side effects of radiation therapy. That experience led us to ask, "Is there a safer therapeutic candidate?"

Goal

Using MDA-MB-231 cells, we aimed to explore biocompatible, plant-based therapeutic candidates from domestic plants that could complement the limitations of radiation therapy.

Research Background

Triple-Negative Breast Cancer

Triple-negative breast cancer lacks estrogen, progesterone, and HER2 receptors. Because these key receptors are absent, this does not respond to targeted or hormone-based treatments and is considered the most difficult form of breast cancer to treat.

In this study, we used the MDA-MB-231 cell line, which represents triple-negative breast cancer, an aggressive form that accounts for about 15-20% of cases.

Limitations of Current Therapies

Treatment for triple-negative breast cancer mainly relies on chemotherapy and radiation therapy due to the limited effectiveness of targeted therapy. However, commonly used chemotherapeutic drugs cause serious side effects.

Cisplatin

Induces nephrotoxicity, along with damaging both cancer cells and normal cells.

Radiation Therapy

Significant side effects affecting overall patient quality of life and immune function.

Plant-Derived Therapeutic Potential

Selected Plant Species

Hypochaeris radicata

Artemisia capillaris

Viscum coloratum

Euphorbia maculata

Tetragonia tetragonioides

Current applications of plant-derived compounds in cancer treatment highlight their significant therapeutic potential. Their lower toxicity and established safety profiles highlight the need for effective plant-derived treatments for triple-negative breast cancer.

Research Process

Plant Extraction Method

Collection

Collect plants from Jeju Island

Grinding

Grind into fine powder

Extraction

Ultrasound-assisted extraction

Freeze-Drying

Filtration, concentration, freeze-drying

Experimental Methods

1. MTT Cell Viability Assay

Confirm cytotoxicity and cell proliferation inhibitory effects

2. Wound Healing Assay

Evaluate cell migration inhibition

3. Colony Formation Assay

Long-term proliferation assessment

4. Apoptosis & Cell Cycle Analysis

Flow cytometry-based analysis

5. qPCR & Western Blot

mRNA and protein expression analysis

Experimental Methods

MTT Assay

Cell seeding: 1x10^4 cells/well
96-well plates, 24h incubation
Extract treatment: 0-1000 ug/mL
MTT: 0.5 mg/mL, 2-4 hours
Measurement at 570 nm

Wound Healing

6-well plates, confluent monolayer
Scratch with 200 uL pipette tip
0.5% FBS assay medium
Monitoring: 0, 6, 12, 16 hours

Colony Formation

6-well plates, 1,000 cells/well
10-14 days culture
Acetic acid:methanol (3:1) fixation
Crystal violet (0.5%) staining

Apoptosis Assay

Annexin V/PI dual staining
BD Pharmingen Detection Kit
Flow cytometry analysis
Live, early/late apoptosis, necrotic

Cell Cycle

Cold 70% ethanol fixation
PI + RNase A staining
DNA-content histograms
G0/G1 and S/G2M analysis

qPCR

TRI-reagent RNA extraction
260/280 purity check
SYBR Green, 40 cycles
GAPDH normalization

Western Blot Analysis

RIPA buffer protein extraction
BCA quantification
15 ug protein, 10% SDS-PAGE
PVDF membrane transfer

Primary Targets:

NF-kB p65 p-STAT3 STAT3 c-Myc beclin-1

Results

MTT Cell Viability Assay

Before using the extracts in our experiments, we first needed to confirm cytotoxicity.

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We found that Hypochaeris and Tetragonia showed little cytotoxicity (overall), whereas Viscum, Artemisia, and Euphorbia showed cytotoxic effects at higher concentrations.

Wound Healing Assay

For the wound-healing assay, we cultured the cells in 6-well plates, created a scratch using a pipette tip, and then treated the cells with each extract.

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In the control group, the wound area decreased over time. In contrast, when the extracts were applied, the wound area remained largely unchanged, indicating that cell migration and proliferation were inhibited.

Colony Formation Assay

In the long-term culture (colony formation) assay, we observed that all extracts clearly suppressed the ability of MDA-MB-231 cells to form colonies.

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These results indicate that each extract has a strong antiproliferative effect, inhibiting the cells' ability to divide and maintain colonies in a concentration-dependent manner.

Western Blot Analysis

Western blot analysis supported the qPCR results at the protein level.

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Western blot analysis supported these results. Hypochaeris reduced p65 and pSTAT3 expression, while Artemisia decreased c-Myc levels. Viscum showed a dual cell death-inducing pattern, with reduced p65 expression and activation of the RIPK1, RIPK3, and MLKL pathway, indicating involvement of multiple cell death mechanisms.

Cytotoxicity Evaluation

Evaluation of cytotoxicity across all plant extracts.

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Evaluation of cytotoxicity revealed that Tetragonia tetragonoides and Hypochaeris radicata exhibited minimal toxicity even at high concentrations, while other extracts demonstrated dose-dependent effects, highlighting the importance of optimizing concentration ranges for safe therapeutic use.

Result Summary

Hypochaeris radicata

Inhibits wound healing and long-term colony formation
Significant results from an invasive species
Minimal toxicity even at high concentrations
Reduced p65 and pSTAT3 expression

Artemisia capillaris

Strongly inhibits cell migration and colony formation
Induces G2/M arrest
Decreased c-Myc levels

Tetragonia tetragonioides

Shows little to no toxicity at low concentrations
Reduced NF-kB p65 expression
Increased Beclin-1 cleavage

Euphorbia maculata

Inhibits cell migration and colony formation
Higher necrotic and dead populations
Decreased pSTAT3 while total STAT3 unchanged

Viscum coloratum

Shows effects even at low concentrations
Dual cell death-inducing pattern
Reduced p65 expression and RIPK pathway activation

Key Findings

Because each experiment used different treatment concentrations, we cannot directly compare anticancer potency across plants under a standardized condition. Instead, we focused on whether each extract exhibited anticancer effects in MDA-MB-231 cells and on the mechanisms underlying those effects.

Most Promising Candidates

Tetragonia, Artemisia, and Hypochaeris show the greatest potential, given their bioactive compounds as well as their environmental and economic advantages. However, Viscum and Euphorbia should still be retained as potential candidates for further research.

Future Experiments

HPLC & LC-MS

For next steps, we will identify active compounds using HPLC and LC-MS analyses to pinpoint the specific molecules driving the observed anticancer effects.

MPLC

Isolate and test individual fractions through MPLC to determine which specific components or synergistic combinations are responsible for the results.

Animal Studies

While not feasible at this stage, we plan to pursue in vivo validation in future university research, with the goal of ultimately developing a viable therapeutic product.