CONTRIBUTION

Overview

In our project, we aimed to address the urgent issue of microplastic toxicity in the human body. Inspired by prior iGEM contributions on bioremediation and biosafety, we sought to extend this work by exploring whether Korean ginseng extract (GS), rich in bioactive ginsenosides, could mitigate the harmful effects of ingested microplastics. Our contribution combines experimental validation and mechanistic insight: we show how ginsenosides interact with microplastics during digestion, reduce their bioaccessibility, induce aggregation, and protect liver cells from oxidative damage. We hope these results broaden the scope of natural product applications in synthetic biology, providing a foundation for future therapeutic and functional food strategies.

Contribution from Our Team Agora 2025

We contributed by:

  • Demonstrating that ginseng extract reduces the gastrointestinal bioaccessibility of microplastics in vitro.
  • Showing that ginsenosides promote aggregation and sedimentation of microplastics through electrostatic and amphiphilic interactions.
  • Providing evidence that GS protects HepG2 liver cells from oxidative stress and cytotoxicity induced by microplastics.
  • Sharing quantitative data, imaging results, and mechanistic interpretations that can guide other teams studying toxin neutralization, food safety, or microplastic degradation.

We believe our findings offer a novel perspective on using natural products to combat microplastic pollution in the human body, and we hope future iGEM teams can build upon our work to develop effective bioremediation strategies or functional foods.

1. Identification of Ginsenosides in Extract with Gradient Elution

We first quantified the main ginsenoside components in our ginseng extract using HPLC.

NO. Compound Contents (mg/g)
1 Ginsenoside Rb1 3.52 +/- 0.49
2 Ginsenoside Rg1 6.61 +/- 0.99
1 20(S)-Ginsenoside Rg3 ND*

*ND: not detected.

Result: Rg1 and Rb1 were the dominant ginsenosides, while Rg3 was not detected. This profile is important since Rg1 and Rb1 is known for surfactant-like properties that facilitate particle interactions.

Gradient Elution to speed up HPLC-UV

In a reversed-phase HPLC, the mobile phase is polar and stationary phase nonpolar.

If the mobile phase is "weak", i.e. more polar like water, nonpolar (hydrophobic) analyte molecules have a very low affinity for the mobile phase. To minimize their contact with the polar solvent, they strongly adsorb onto the nonpolar stationary phase. This results in high retention and the analyte moves very slowly.

On the other hand, a "strong" mobile phase like acetonitrile(ACN) is less polar and has more affinity to the nonpolar analytes, meaning the molecules would move faster.

Either way, using a single type of mobile phase creates a problem: if using a polar phase, it takes a very long time to get the nonpolar analytes to come out, and the signal will be wide and short due to diffusion and is hard to distinguish.

Conversely, using a strong more nonpolar phase means different analytes will arrive with little time difference, making it hard to distinguish between compounds.

Gradient Elution is a technique to use and mix two different phases, but with different concentrations for each at different time.

The schedule first starts with the majority being weaker phase (~95%) to get the more polar analytes out.

Then, it gradually increases the concentration of the stronger phase to speed up the process therefore reduce the likelihood of getting a diffused, wide and short signal from strongly nonpolar molecules.

This way, we can get a sharper signal and faster analysis time.

2. Effect on Bioaccessibility of Microplastics

We used an in vitro digestion model to simulate gastrointestinal conditions and measured the proportion of microplastics in the soluble fraction (bioaccessible).

Figure 1. Bioaccessibility of microplastics (MPs) following in vitro digestion in the absence or presence of ginseng extract (GE) at low, medium, and high concentrations. Data are expressed as mean ± SD (n = 3). Bars with different lowercase letters indicate significant differences at p < 0.05. The GE-H group showed a significantly lower MP bioaccessibility compared to control, indicating reduced intestinal availability of MPs upon high-concentration GE co-treatment

Result: GS reduced MP bioaccessibility in a dose-dependent manner, with the high-concentration GS group showing the most significant decrease.

3. Aggregation and Zeta Potential Analysis

Figure 2. Fuorescence microscope images (scale bar= 400 μm) (A) and particle size distribution profiles including diffential intensity (B), differential volume (C), differential number (D) of pellets from MPs and MPs with GS obtained after in vitro digestion.

Sample Zeta Potential (mV)
MPs 46.92
MPS + GS (Low) 40.965
MPS + GS (Med) 44.675
MPS + GS (High) 58.205

Fluorescence microscopy and particle size distribution showed that GS induced MP aggregation, particularly at medium concentrations. Zeta potential analysis confirmed changes in electrostatic stability.

  • Control MPs remained dispersed.
  • With GS, particles formed larger clusters and showed altered surface charge, consistent with amphiphilic ginsenoside interactions.
4. Hyperspectral Imaging of MP Distribution

Hyperspectral imaging provided further evidence that GS promoted MP sedimentation into the pellet fraction. Pellet reflectance was higher than supernatant reflectance, especially at 1050–1100 nm, confirming aggregation.

Figure 3. Hyperspectral imaging of digesta pellet and supernatant of MPs and GS.

Near-infrared hyperspectral reflectance spectra (1000–1700 nm) of digesta pellet and supernatant fractions obtained after co-treatment of microplastics (MPs) with ginseng extract (GS) at low, medium, and high concentrations. The inset magnifies the 1000–1200 nm range, highlighting concentration-dependent spectral changes. The pellet fractions exhibited higher reflectance compared to supernatants, especially at 1050–1100 nm, suggesting increased MP aggregation and sedimentation at higher GS concentrations.

5. Protective Effects in HepG2 Cells

We evaluated the cytotoxicity and oxidative stress induced by MPs in HepG2 liver carcinoma cells, and whether GS could mitigate these effects.

  • Cell Viability Assay: We used the MTT assay to measure cell viability after exposure to MPs and GS. This assay measures the metabolic activity of mitochondria, which is a reliable indicator of cell viability and health.
  • Reactive Oxygen Species (ROS) Assay: We used the DCFH-DA assay to measure intracellular ROS levels as an indicator of oxidative stress.

Figure 4. Cell viability (%) of HepG2 cell after digestion of MPs, GS, and MPs with GS. It was normalized by control according to various concentrations. The vertical bars represent the standard error of the mean of six replications. Different letters indicate a significant difference among groups (p<0.05). Cell viability (%) of HepG2 cells after exposure to digested samples containing microplastics (MPs), ginseng extract (GS), or their combinations at low, medium, and high concentrations. Data are expressed as mean ± standard error (n = 6), normalized to control. Different letters (a, b) indicate significant differences among groups (p < 0.05). Asterisks (*) indicate significant difference from the MPs-only group, and number signs (#) indicate significant difference from the GS-only group.

Figure 5. Effect of GS on scavenging activity of reactive oxygen species (ROS) in HepG2 cell. Effect of ginseng extract (GS) on reactive oxygen species (ROS) production and scavenging activity in HepG2 cells. (A) Intracellular ROS production (%) in cells treated with MPs or GS alone, normalized to untreated control.(B) ROS scavenging activity (%) of GS co-treated groups (MPs + GS at low, medium, and high concentrations), normalized to negative control (NC). Bars represent mean ± SD (n = 3). Different letters indicate significant differences among groups (p < 0.05).

Results:

  • MP exposure significantly reduced HepG2 cell viability compared to controls.
  • Co-treatment with GS improved cell viability in a dose-dependent manner, with the highest GS concentration showing the most pronounced protective effect.
  • MPs increased intracellular ROS levels compared to control, indicating oxidative stress.
  • GS co-treatment reduced ROS levels, demonstrating its antioxidant properties and protective effect against MP-induced oxidative damage.
Conclusion

Our findings establish ginseng extract as a natural agent that reduces the bioaccessibility and toxicity of ingested microplastics by:

  1. Measuring the concentraton of ginsenosides quickly and efficiently.
  2. Promoting aggregation and sedimentation of MPs during digestion.
  3. Lowering gastrointestinal absorption potential.
  4. Protecting liver cells against oxidative stress and cytotoxicity.

This contribution offers both mechanistic insight and practical potential for future iGEM teams exploring biosafety, natural-product interactions, or environmental health protection.