iGEM 2025 — Team agora-kor

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

## NOTEBOOK

Date: 2025.08.02

Aim: To quantify the concentrations of three representative ginsenos
ides in the ginseng extracts

Protocols: HPLC (high performance liquid chromatography)

Results: Among the compounds analyzed, ginsenoside Rg1 was the most abundant (6.61 ± 0.99 mg/g), followed by ginsenoside Rb1 (3.52 ± 0.49 mg/g). In contrast, 20(S)-ginsenoside Rg3 was not detected in the extract.

Date: 2025.08.02

Aim: To measure bioaccessibility of MPs by ginseng extracts and analyze the supernatant and the pellet through an in vitro digestion system

Protocols: In vitro digestion biomimick system

Results: The digest obtained through the in vitro digestion using human simulation system at different ginseng concentrations (low, med, high) was the supernatant and pellet portions were well separated and properly used in all experiments.

Date: 2025.08.02

Aim: To measure cytotoxicity assessment of microplastics with ginseng extracts in liver

Protocols: Analysis of cell viability in Hepg2 cell

Results: Co-treatment with low concentration ginseng extracts did not significantly improve viability compared to MPs alone. However, medium and high ginseng extracts concentrations significantly enhanced cell viability, reaching ~120–125% of control values (p < 0.05). The results demonstrate that ginseng extract exerts a protective effect against MP-induced cytotoxicity in HepG2 cells.

Date: 2025.08.02

Aim: To measure oxidative stress assessment of digesta supernatant. In liver

Protocols: Assay of reactive oxygen species (ROS) in Hepg2 cell

Results: The MPs + GS (Med) and MPs + GS (High) groups exhibited significantly higher scavenging activity (~60–65%) compared to the Low group (~30%, p < 0.05), indicating the antioxidant potential of GS at sufficient concentrations. The results suggest that ginseng extract (GS) effectively attenuates microplastic-induced oxidative stress in HepG2 cells.

Date: 2025.08.02

Aim: To measure fluorescence MPs remaining in the digesta supernatant and the bioaccessibility of MPs after in vitro gastrointestinal digestion in the presence of varying concentrations of ginseng extract.

Protocols: Microplate reader

Results: The MPs+GS (low) and MPs+GS (med) groups showed moderate decreases, though not statistically different from each other (p > 0.05). Notably, the MPs+GS (high) group demonstrated a significant reduction in MP bioaccessibility (p < 0.05), decreasing to approximately 60% of the control level, suggesting that high-dose GE co-treatment reduces the solubilized, absorbable fraction of microplastics.

Date: 2025.08.09

Aim: To measure hyperspectral imaging that spectroscopic method of evaluating amine group at 1050-1100 nm in digesta pellet and supernatant

Protocols: Hyperspectral imaging (HSI)

Results: The pellet showed higher reflectance at all concentrations than
supernatant. These results mean that the amount of MPs excreted from the body is more than absorbed. Comparison by concentration, the reflectance was highest in the order of MPs+GS (med), MPs+GS (high), and MPs+GS (low), showing that MPs excretion was best at the medium concentration.

Date: 2025.08.09

Aim: To measure particle size of digesta pellet and compare the MPs size of the aggregated particles by ginseng concentration.

Protocols: Dynamic Light Scattering (DLS)

Results: The particle size of only MPs, MPs+GS (low), MPs+GS (med), and MPs+GS (high) were 2430.45 nm, 5262.6 nm, 8203.45 nm, and 3459.05 nm, respectively. The results mean MPs+GS (med) showed the most aggregation of MPs, followed by low concentration and then high concentration.

Date: 2025.08.09

Aim: To measure dispersion of MPs and ginseng aggregates using zeta potential

Protocols: Zeta-potential

Results: Only MPs have a zeta potential of 46.920 mV, indicating stable dispersion with moderate repulsion between particles. MPs + GS (Low) show a decreased zeta potential of 40.965 mV, suggesting reduced repulsive force and shorter distance between particles, which may lead to mild aggregation. MPs + GS (Med) slightly increase to 44.675 mV, meaning repulsion is partially restored, allowing particles to remain more evenly dispersed. MPs + GS (High) show a significantly higher zeta potential of 58.205 mV.

Date: 2025.08.08

Aim: To visualize fluorescence MPs and identify particles of MPs and ginseng aggregates.

Protocols: Fluorescence microscope

Results: Only MPs showed the most fluorescence and MPs+GS (med) showed the greatest aggregation visually. MPs+GS (low) showed high aggregation and dispersion, and MPs+GS (high) showed the
least aggregation. This is consistent with the results of DLS and zeta-potential.

{% endblock %}
Date: 2025.08.02
Aim: To quantify the concentrations of three representative ginsenosides in the ginseng extracts
Protocols: HPLC (high performance liquid chromatography)
Results: Among the compounds analyzed, ginsenoside Rg1 was the most abundant (6.61 ± 0.99 mg/g), followed by ginsenoside Rb1 (3.52 ± 0.49 mg/g). In contrast, 20(S)-ginsenoside Rg3 was not detected in the extract.
Date: 2025.08.02
Aim: To measure bioaccessibility of MPs by ginseng extracts and analyze the supernatant and the pellet through an in vitro digestion system
Protocols: In vitro digestion biomimick system
Results: The digest obtained through the in vitro digestion using human simulation system at different ginseng concentrations (low, med, high) was the supernatant and pellet portions were well separated and properly used in all experiments.
Date: 2025.08.02
Aim: To measure cytotoxicity assessment of microplastics with ginseng extracts in liver
Protocols: Analysis of cell viability in Hepg2 cell
Results: Co-treatment with low concentration ginseng extracts did not significantly improve viability compared to MPs alone. However, medium and high ginseng extracts concentrations significantly enhanced cell viability, reaching ~120–125% of control values (p < 0.05). The results demonstrate that ginseng extract exerts a protective effect against MP-induced cytotoxicity in HepG2 cells.
Date: 2025.08.02
Aim: To measure oxidative stress assessment of digesta supernatant. In liver
Protocols: Assay of reactive oxygen species (ROS) in Hepg2 cell
Results: The MPs + GS (Med) and MPs + GS (High) groups exhibited significantly higher scavenging activity (~60–65%) compared to the Low group (~30%, p < 0.05), indicating the antioxidant potential of GS at sufficient concentrations. The results suggest that ginseng extract (GS) effectively attenuates microplastic-induced oxidative stress in HepG2 cells.
Date: 2025.08.02
Aim: To measure fluorescence MPs remaining in the digesta supernatant and the bioaccessibility of MPs after in vitro gastrointestinal digestion in the presence of varying concentrations of ginseng extract.
Protocols: Microplate reader
Results: The MPs+GS (low) and MPs+GS (med) groups showed moderate decreases, though not statistically different from each other (p > 0.05). Notably, the MPs+GS (high) group demonstrated a significant reduction in MP bioaccessibility (p < 0.05), decreasing to approximately 60% of the control level, suggesting that high-dose GE co-treatment reduces the solubilized, absorbable fraction of microplastics.
Date: 2025.08.09
Aim: To measure hyperspectral imaging that spectroscopic method of evaluating amine group at 1050-1100 nm in digesta pellet and supernatant
Protocols: Hyperspectral imaging (HSI)
Results: The pellet showed higher reflectance at all concentrations than supernatant. These results mean that the amount of MPs excreted from the body is more than absorbed. Comparison by concentration, the reflectance was highest in the order of MPs+GS (med), MPs+GS (high), and MPs+GS (low), showing that MPs excretion was best at the medium concentration.
Date: 2025.08.09
Aim: To measure particle size of digesta pellet and compare the MPs size of the aggregated particles by ginseng concentration.
Protocols: Dynamic Light Scattering (DLS)
Results: The particle size of only MPs, MPs+GS (low), MPs+GS (med), and MPs+GS (high) were 2430.45 nm, 5262.6 nm, 8203.45 nm, and 3459.05 nm, respectively. The results mean MPs+GS (med) showed the most aggregation of MPs, followed by low concentration and then high concentration.
Date: 2025.08.09
Aim: To measure dispersion of MPs and ginseng aggregates using zeta potential
Protocols: Zeta-potential
Results: Only MPs have a zeta potential of 46.920 mV, indicating stable dispersion with moderate repulsion between particles. MPs + GS (Low) show a decreased zeta potential of 40.965 mV, suggesting reduced repulsive force and shorter distance between particles, which may lead to mild aggregation. MPs + GS (Med) slightly increase to 44.675 mV, meaning repulsion is partially restored, allowing particles to remain more evenly dispersed. MPs + GS (High) show a significantly higher zeta potential of 58.205 mV.
Date: 2025.08.08
Aim: To visualize fluorescence MPs and identify particles of MPs and ginseng aggregates.
Protocols: Fluorescence microscope
Results: Only MPs showed the most fluorescence and MPs+GS (med) showed the greatest aggregation visually. MPs+GS (low) showed high aggregation and dispersion, and MPs+GS (high) showed the least aggregation. This is consistent with the results of DLS and zeta-potential.

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

## ENGINEERING

* [Introduction](#introduction)
* [Ginsenoside Type Identification](#ginesenoside)
* [In Vitro Digestion Model](#digestion)
* [HepG2 Cellular Assay](#hepg2)

#### INTRODUCTION

We build an in vitro digestion system of ginsenosides and microplastics to measure its bioaccessibility and structural aggregation, and a HepG2 cellular culture to measure its hepatoprotection and oxidative stress.
The engineering decisions were based on previous research, and our own empirical laboratory investigations. Here we explain the thought processes behind our decisions to build an in vitro digestion system and the measurement of
various pertinent characteristics.

---

#### GINSENOSIDE TYPE IDENTIFICATION

![HPLC Chromatogram of Ginsenoside Extract](https://static.igem.wiki/teams/5993/images/hplc.webp)

*HPLC Chromatogram of Ginsenoside Extract*

##### Design

Not all ginsengs are the same. The type and concentration of ginsenosides can vary based on the species of ginseng, its age, the part of the plant used, and how it was processed.

For example, Raw (white) ginseng contains different types of ginsenosides than steamed (red) ginseng.

In a white ginseng, the most common types are Rb1, Rb2, Rg1, Rc, Rf, Re. When ginseng is steamed or heated in some other way, those ginsenosides chemically convert to minor ginsenosides, such as 20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5.

We measure only Rb1, Rg1, 20(S)-Rg3 as those have shown relevant ROS-scavenging property and protection against oxidative stress in hepatocytes. In a typical ginseng extract, ginsenosides type Rg1 and Rb1 would be predominant whereas Rg3 concentration would be small.

For analysis, high-performance liquid chromatography-ultraviolet (HPLC-UV) procedure was designed with gradient elution, since it works with non-volatile, high molecular weight compounds like ginsenosides.

##### Build

An extraction protocol was made to isolate the active compounds from raw ginseng powder. First, accurately weigh 10 g of the sample, then add 100 mL of distilled water, and use a sonicator for 20 minutes to lyse the plant cells and release the ginsenosides.
The resulting mixture was then centrifuged at 1000 rpm for 15 minutes to separate the solid debris, and the liquid extract was filtered through a 0.45 µm syringe filter to prepare it for analysis.

To get a proper comparison, analytical standards for Rg1, Rg2, 20(S)-Rg3 were purchased. An analytical workflow was established using a Nanospace SI-2 HPLC system with a photodiode array (PDA) detector and a Cadenza CD-C18 column.
Then, a precise gradient elution method was developed using water and acetonitrile as the mobile phase, gradually increasing the concentration of ACN, then repeating the cycles of washing with each phase separately two times, allowing for the separation and quantification of key ginsenosides (Rg1, Rb1, Rg3) by monitoring UV absorbance at 203 nm.

This provided a quantitative baseline of the active compounds in the extract.

##### Test & Learn

It was found that the peaks of Ginsenoside Rg1, Ginsenoside Rb1, and 20(S)-Ginsenoside Rg3 were not well separated despite the procedure, and the reproducibility was poor.
To address this, the centrifuge condition in the sample extraction step was modified from 1000 rpm to 4000 rpm, which enabled clear separation of the supernatant and pellet.

After this modification, the HPLC chromatograms showed distinct peaks for Rg1, and Rb1 with good reproducibility.
As expected, Rg1 and Rb1 were the predominant ginsenosides in the extract, while 20(S)-Rg3 was not detected, likely due to its low concentration in white ginseng.

---

#### IN VITRO DIGESTION MODEL

![In Vitro Digestion Model Ingredients](https://static.igem.wiki/teams/5993/images/digestion-ingredients.webp)

*In Vitro Digestion Model Ingredients*

##### Design

We simulate the ingestion of microplastics with ginsenosides by an in vitro digestion model.
As nutrient absorption mostly happens in the small intestine, we skip the gastric phase and only model the small intestine.

Test Samples:

* Fluorescent microplastics (MP): amine-modified polystyrene particles with a diameter of 1 μm were used. The fluorescent tag is crucial for later quantification.
* Ginseng Extract (GS): ginsenosides extracted from ginseng powder from the previous cycle.
* Treatment Groups: The MPs were co-treated with GS at three different volume-to-weight ratios: 1:1 (Low), 1:2 (Medium), and 1:4 (High).
* Control Groups: A control group containing only MPs was prepared to establish a baseline for bioaccessibility and toxicity, and another with only GS as a negative control.

Experiments:

* Bioaccessibility Test: we test the bioaccessibility of the MP-GS mixture against the control by measuring the fluorescence of the MP in the supernatant after centrifugation.
* Microstructural Measurements: The centrifuged samples are diluted in distilled water, and are measured for their microstructural properties like particle size distribution or zeta potential.
* Hyperspectral Imaging: The centrifuged samples are imaged with hyperspectral camera, with the pellet and supernatant imaged separately to measure their reflectivity.

##### Build

To simulate the chemical and physical conditions of the human small intestine, we take the MP+GS samples, and two MP only controls.
and for each one we add a phosphate buffer, and a precise cocktail of digestive enzymes (porcine pancreatin, lipase) and bile extract.
The pH was adjusted to 7.0, and the mixture was incubated at 37 ∘C at 200 rpm to mimic digestion.
Lastly, after digestion, the samples are placed in ice bath to inhibit the enzymes.

##### Test & Learn

![pellet supernatant](https://static.igem.wiki/teams/5993/images/pellet-supernatant.webp)
![absorbance analyzer](https://static.igem.wiki/teams/5993/images/absorbance-analyzer.webp)

*The microplate reader measures the fluorescence of centrifuged samples*

Bioaccessibility Test:

First, digested samples are measured for their fluorescence before centrifugation to get a baseline.
Then, the samples are centrifuged at 4500 rpm for 30 minutes at 4 ∘C.
The samples are separated into a liquid supernatant (representing the bioaccessible fraction available for absorption) and a solid pellet.
For each sample, the amount of MPs in the supernatant was quantified by taking out 3 small samples from the supernatant,
then measuring each fluorescence, calculating the mean and the standard deviation, with a microplate reader with excitation/emission wavelengths of 470/505 nm.

Metric: Bioaccessibility (%)

Bioaccessibility (%) = (Fluorescence of supernatant / Baseline Fluorescence) × 100%

Bioaccessibility was lower in all GS treated samples compared to the control, and the bioaccessibility decreased as the concentration of GS increased.

Microstructural Measurements

Microscopy:

We wanted to qualitatively observe the aggregation of MPs, so we took the pellets from the centrifuged samples, diluted to 0.1% in distilled water, and took the fluorescence microscope images.
As expected, the control sample showed a bright and uniform distribution of individual MPs, while the GS treated samples showed larger, irregularly shaped aggregates with a lower overall fluorescence intensity.

Particle Size Distribution:

Dynamic Light Scattering (DLS) measured the average particle size, while Zeta Potential analysis measured the electrical potential of the slipping plane, which indicates how stable a colloidal dispersion is.
As the concentration of GS increased, the average particle size increased significantly, indicating that GS promotes aggregation of MPs.

Hyperspectral Imaging:

Hyperspectral Imaging (HSI) scanned the pellet and supernatant fractions to generate a spectral profile, allowing for a highly sensitive analysis of where the MPs were accumulating.

We discovered that the halogen illumination of HSI caused changes in the coloration of fluorescent microplastics.
To minimize this effect, HSI was performed only after completing all other experiments.
Compared to initial attempts, this adjustment markedly reduced the influence of illumination on the results.

Metric: Average Particle Size (nm), Zeta Potential (mV), Reflectance (%).

We created a static in vitro digestion model. However, in the future we can create a dynamic digestion model that more closely mimics the peristaltic movements and changing conditions of the human gastrointestinal tract.
We can also test different types of microplastics, as well as real-world samples extracted from food or water.

---

#### HEPG2 CELLULAR ASSAY

##### Design

We use HepG2 cells, a human liver cancer cell line, to model the hepatotoxicity induced by microplastics and the protective effects of ginsenosides.
HepG2 cells are widely used in toxicology studies due to their human origin and retention of many liver-specific functions.
We expose HepG2 cells to microplastics with and without ginsenosides, then measure cell viability and oxidative stress markers.

Test Samples:

* Fluorescent microplastics (MP): amine-modified polystyrene particles with a diameter of 1 μm were used.
* Ginseng Extract (GS): ginsenosides extracted from ginseng powder from the previous cycle.
* Treatment Groups: The MPs were co-treated with GS at three different volume-to-weight ratios: 1:1 (Low), 1:2 (Medium), and 1:4 (High).
* Control Groups: A control group containing only MPs was prepared to establish a baseline for toxicity, and another with only GS as a negative control.

![MTT reaction](https://static.igem.wiki/teams/5993/images/mtt-reaction.webp)

*MTT assay reaction: tetrazolium is converted to purple formazan inside mitrochodria.*

Experiments:

* Cell Viability Assay: We use 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 use the DCFH-DA assay to measure intracellular ROS levels as an indicator of oxidative stress. DCFH-DA turns into DCFA by intracellular enzymes, and reactive oxygen species can oxidize it into fluorescent DCF. The fluorescence would then reflect the concentration of ROS in the cells.

##### Build

HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
Cells were maintained at 37 ∘C in a humidified atmosphere with 5% CO2.

For experiments, cells were seeded in 96-well plates at a density of 1 × 10^4 cells/well and allowed to adhere overnight.
After 24 hours, cells were treated with MPs and GS at the specified ratios for 24 hours.

Following treatment, cell viability was assessed using MTT solution, and incubated for 2 hours. As MTT only dyes living cells purple, measuring the absorbance gives the viability relative to control. A microplate reader with excitation/emission wavelengths of 570/630 nm was used to measure the absorbance.

Absorbance was measured at 450 nm using a microplate reader.

For ROS measurement, cells were incubated with DCFH-DA (10 µM) for 30 minutes at 37 ∘C in the dark. After washing with PBS, fluorescence intensity was measured using a microplate reader with excitation/emission wavelengths of 485/535 nm.

DCFH-DA

![dcfh-da](https://static.igem.wiki/teams/5993/images/dcfh-da.webp)

DCFH

![dcfh](https://static.igem.wiki/teams/5993/images/dcfh.webp)

DCF

![dcf](https://static.igem.wiki/teams/5993/images/dcf.webp)

*ROS assay: DCFH-DA enters a cell, and is first deacetylized into DCFH by esterase enzyme. If any reactive oxygen species exist in the cell, DCFH reacts with them to form fluorescent DCF, whose fluorescence can then be measured.*

##### Test & Learn

Cell Viability Assay:

The MTT assay results showed that exposure to MPs significantly reduced HepG2 cell viability compared to the control group.
However, co-treatment with GS at all tested ratios improved cell viability in a dose-dependent manner, with the highest GS concentration (1:4 ratio) showing the most pronounced protective effect.

Reactive Oxygen Species (ROS) Assay:

The DCFH-DA assay revealed that MP exposure led to a significant increase in intracellular ROS levels, indicating oxidative stress.
Co-treatment with GS effectively reduced ROS levels, again in a dose-dependent manner, suggesting that ginsenosides can mitigate MP-induced oxidative stress in HepG2 cells.

Metric: Cell Viability (% of control), ROS Levels (fluorescence intensity).

Future studies could explore the molecular mechanisms underlying the protective effects of ginsenosides, such as their impact on antioxidant enzyme expression or signaling pathways involved in oxidative stress response.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

## EXPERIMENTS

* [Materials](#materials)
* [Protocols](#protocols)
  + [Analysis of Ginsenosides](#analysis)
  + [In Vitro Digestion Model](#digestion)
  + [Fluorescence Detection via Microplate Reader](#fluorescence)
  + [Spectroscopic analysis of amino group (-NH2)](#hyperspectral)
  + [Cell Thawing](#thawing)
  + [Media Change](#media-change)
  + [Subculture](#subculture)
  + [Seeding](#seeding)
  + [Cell Viability Assay](#cell-viability)
  + [Intracellular ROS Measurement](#ros)
* [Methods](#methods)
  + [Analysis of ginsenoside from GS using HPLC-UV](#methods-1)
  + [In vitro digestion model](#methods-2)
  + [Characterization of MP structure](#methods-3)
  + [Hyperspectral analysis](#methods-4)
  + [Measurement of cell viability by MTT assay](#methods-5)
  + [GS on the intracellular ROS level](#methods-6)
  + [Statistical Analysis](#methods-7)

#### MATERIALS

* Ginseng powder was purchased from the commercial market in South Korea and then extracted.
* Amine-modified fluorescent polystyrene (MPS) particles, with diameters of 1 µm (FluoSpheres® Fluorescent Microspheres), were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
* DMEM with phenol red was purchased from Biowest (Riverside, MO, USA).
* α-amylase from porcine pancreas, pepsin from human gastric mucosa, pancreatin, lipase from porcine pancreas, and bile extract from porcine pancreas were supplied by Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
* Fetal bovine serum (FBS) was supplied by Cytiva (Marlborough, MA, USA).
* Dulbecco’s phosphate-buffered saline (DPBS) and penicillin/streptomycin (P/S) were supplied by Corning Inc. (Corning, NY, USA).
* All chemicals were analytical grade and stored according to their storage conditions.
* Analytical standards of Ginsenoside Rg1, Ginsenoside Rb1, and 20(S)-Ginsenoside Rg3 were obtained from TCI Chemical (Tokyo, Japan).
* Methanol, water, and acetonitrile (ACN) of high-performance liquid chromatography (HPLC) grade were provided by J.T. Baker (Phillipsburg, NJ, USA).
* The ginseng concentrate used as samples was obtained from BTC Corporation (Gwacheon-si, Gyeonggi-do, Republic of Korea).

#### PROTOCOLS

##### Analysis of Ginsenosides

###### Analytical Method

Standard: 5, 10, 20, 50 ug/mL(mg/kg)

| Ginsenoside Name | CAS No. | Molecular Weight | Retention Time (min) |
| --- | --- | --- | --- |
| Ginsenoside Rg1 | 112246-15-8 | 800.49 | 21.540 |
| Ginsenoside Rb1 | 41753-43-9 | 1108.6 | 36.722 |
| 20(S)-Ginsenoside Rg3 | 14197-60-5 | 784.49 | 52.360 |

###### Sample

1. Ginseng

###### Sample Preparation

1. Accurately weigh 10 g of the sample and add 100 mL of distilled water.
2. Extract the sample using a sonicator for 20 minutes.
3. Centrifuge at 4,000 rpm, 4 °C for 10 minutes.
4. Filter using a Whatman 0.45 um Syringe Filter.
5. Analyze using HPLC.

###### Analytical conditions for HPLC

|  |  |
| --- | --- |
| Instrument | Josaka Soda HPLC |
| Detector | UV (203 nm) |
| Column | Almtakt 3.0 um Cadenza CD-C18 250 x 3 mm (CD036) A: Water B: ACN |
| Gradient | | Time (min) | A (%) | B (%) | | --- | --- | --- | | 0.5 | 85 | 15 | | 28.5 | 70 | 30 | | 30.5 | 68 | 32 | | 36.5 | 62 | 38 | | 47.5 | 57 | 43 | | 54.0 | 45 | 55 | | 62.0 | 45 | 55 | | 70.0 | 30 | 70 | | 76.0 | 10 | 90 | | 76.1 | 5 | 95 | | 86.1 | 5 | 95 | | 86.1 | 85 | 15 | | 93.0 | 85 | 15 | |
| Injection Volume | 10 ul |
| Flow Rate | 0.4 mL/min |
| Temperature | 40℃ |
| Run Time | 93 min |

---

##### In Vitro Digestion Model

###### Samples

| Sample No | Sample Name |
| --- | --- |
| 1 | Negative Control |
| 2 | Control (MPs only) |
| 3 | Sample (GS only) |
| 4 | GS-L (2.5mg(125uL) MPs + 125ug(6.25uL) GS) |
| 5 | GS-M (2.5mg(125uL) MPs + 250ug(12.5uL) GS) |
| 6 | GS-H (2.5mg(125uL) MPs + 500ug(25uL) GS) |

###### Intestinal Phase

1.Prepare the sample solutions.

2.Add 2.5 mL of Phosphate-Buffered Saline (PBS) to the sample.

3.Add 0.4 mL of pancreatin solution (800 U/mL based on trypsin activity).

4.Add 0.4 mL of bile acid (60 mg/mL 20 mM PB).

5.Add 0.4 mL of lipase (5 mg/mL 20 mM PB).

6.Adjust the pH of the mixture to 7.0 using 1 M NaOH.

7.Add PBS to bring the total volume to 5 mL.

8.After incubation, place the mixture in an ice bath for 10 minutes to inactivate enzyme
activity.

9.Incubate the mixture at 37 °C for 2 hours.

10. Centrifuge (4000 RPM, 30 min, 4°C), and then store them at −80 °C until analysis.

##### Fluorescence Detection via Microplate Reader

###### Analytical Method

Purpose: This study aims to evaluate the potential amount of microplastics absorbed by the human
body by measuring the fluorescence intensity of the supernatant after in vitro digestion. (Fluorescent,
amine-modified polystyrene microspheres (FluoSpheres™) are used as model microplastics.)

Sample Preparation

1.Separate the supernatant after centrifugation

2.Inject the supernatant of each sample into three wells of a 96-well plate

3.Measure the fluorescence using the microplate reader

###### Analytical conditions for Microplate Reader

|  |  |
| --- | --- |
| Instrument | Thermo Scientific. Varioskan Flash Microplate Reader |
| Mode | Fluorescence |
| Excitation Wavelength | 470 nm |
| Emission Wavelength | 505 nm |

---

##### Spectroscopic analysis of amino group (-NH2)

###### Hyperspectral imaging method

1. Hyperspectral imaging

Sample: Supernatant / pellet of digested 3 mL in conical tube

Set camera parameters based on the spatial orientation of the sample, including its height and distance, before hyperspectral imaging.

Hyperspectral imaging using scanner software.

2. Normalization

Normalize by subtracting the dark and white values from the raw hyperspectral image.

\*Dark value: Reflectance measured under closed-shutter condition

\*White value: Reflectance value that indicates the reference point for the location

3. Reflectance value data

Obtain reflectance data using region of interest (ROI) and spectral profile from normalized hyperspectral image.

Compare reflectance values in range from 1050 to 1100 nm.

###### Hyperspectral camera and software conditions for imaging

| Setting (unit) | Value |
| --- | --- |
| Red band (nm) | 1004.83 |
| Blue band (nm) | 1301.56 |
| Green band (nm) | 1605.92 |
| Positioning speed (mm/s) | 40 |
| Frame rate (Hz) | 3 |
| Exposure time (ms) | 3 |
| Spectral binning | 2 |
| Spatial binning | 1 |
| Trigger mode | Internal |
| Working distance (mm) | 300 |
| Dark subtraction | Shutter |
| White reference | Scanner |

##### Thawing the cell

1. Take out frozen cells from liquid nitrogen storage.

2. Thawing: Immediately immerse the bottom part of the cryovial (below the cap) into a 37 °C water bath.

\*Melts quickly but does not melt all the ice
\*Do not submerge the cap to prevent contamination.

3. Sterilization: Wipe the outside of the cryovial with 70% ethanol to sterilize it.

4. Dilution: Transfer the contents into a centrifuge tube containing pre-warmed growth medium (at least 10× the volume of the thawed cell suspension) to dilute DMSO.

\*Dilute DMSO rapidly by adding culture medium at least 10 times the volume of the cell suspension

5. Centrifugation: Centrifuge the cell suspension at 1300 RPM for 3–5 minutes to pellet the cells.

6. Resuspension and Seeding: Discard the supernatant carefully and resuspend the pellet in fresh growth medium (10% FBS, 1% PS in DMEM). Transfer the cell suspension to an appropriate culture vessel and incubate under standard culture conditions (37 °C, 5% CO₂).

##### Media Change

Media change method

1. Observation: Observe the cells under a microscope to check for confluency and contamination. Proceed with medium change if cells are healthy and growing well.

\*Do not allow the culture medium to turn deep yellow, which indicates over-confluency and nutrient depletion.
\*Avoid reaching 100% confluency, as this can lead to undesirable cell differentiation, nutrient depletion, and reduced cell viability.

2. Medium Removal: Carefully remove the old medium using a pipette or vacuum aspirator, avoiding contact with the cell layer.

3. Washing: Gently wash the cells with pre-warmed PBS (phosphate-buffered saline) to remove residual serum and waste. Repeat the wash twice.

4. Addition of Fresh Medium: Add fresh, pre-warmed complete growth medium (e.g., DMEM with 10% FBS and 1% penicillin-streptomycin) to each well or flask.

5. Incubation: Return the culture vessel to the incubator and maintain at 37 °C, 5% CO₂. Replace the medium every 2–3 days or as needed based on pH (color change), nutrient depletion, or cell growth.

##### Seeding

Seeding method

1. Removal of Spent Medium: Carefully aspirate the old medium without disturbing the attached cells.
2. Washing with PBS: Gently wash the cells with pre-warmed PBS (pH 7.4) to remove serum proteins that can inhibit Trypsin-EDTA activity.
3. Addition of Trypsin-EDTA: Add sufficient Trypsin-EDTA solution (0.25%) to cover the cell surface. This enzyme detaches adherent cells by cleaving proteins anchoring them to the plate.
4. Incubation for Detachment: Place the flask or plate in a 37 °C incubator for 2–5 minutes. Tapping the plate gently may help if detachment is incomplete.
5. Neutralization of Trypsin: Add an equal or greater volume of complete culture medium (containing FBS) to stop trypsin activity.
\*FBS contains inhibitors that neutralize trypsin.6. Centrifugation: Transfer the cell suspension to a 15 mL conical tube and centrifuge at 1300 RPM for 3–5 minutes. This step pellets the cells at the bottom.
7. Removal of Supernatant and Resuspension: Aspirate the supernatant and gently resuspend the cell pellet in 1 mL fresh complete medium.
8. Count Cells: Harvest the cell suspension and count the viable cells using a hemocytometer (e.g., Pellet medium 10 µL + Trypan Blue 10 µL).
\*Use Trypan Blue to distinguish live (unstained) and dead (blue-stained) cells.9. Dilute to Desired Concentration: Based on the calculated viable cell count, dilute the cells in fresh pre-warmed complete growth medium to reach the desired seeding density (e.g., 2 × 10⁴ cells/well for 96-well plate).
10. Plate Cells Evenly: Using a multichannel or single pipette, dispense the calculated volume into each well of the culture plate.
\*Ensure even distribution of cells to avoid clustering or edge effects.11. Incubate: Place the plate into a humidified incubator (37 °C, 5% CO₂) for 24 hours.
12. Proceed with Experiment: Once attachment and morphology are confirmed, proceed with subsequent treatment or assay setup as planned.

###### Formula

1. Average cell count × Dilution factor × 104 = Cells per mL
2. Desired total number of cells / Cells per mL = Required Volume (mL)
3. Average cell count × Dilution factor × 104 (correction factor) : 1 = Desired number of cells : Required volume (X)

What is the 104 factor?

A Hemocytometer chamber has a defined volume: 1mm×1mm×0.1mm = 0.1mm3 = 0.0001mL

Therefore, to convert the cell number in 0.0001 mL to cells/mL, we multiply by:

1/0.0001 = 104

##### Subculture

1. Observation: Subculture should be performed when the cells reach approximately 70–80% confluency.

\*Do not allow cells to reach full (100%) confluency, as this may cause growth arrest or unwanted differentiation.

2. Removal of Spent Medium: Carefully aspirate the old medium without disturbing the attached cells.

3. Washing with PBS: Gently wash the cells with pre-warmed PBS (pH 7.4) to remove serum proteins that can inhibit Trypsin-EDTA activity.

4. Addition of Trypsin-EDTA: Add sufficient Trypsin-EDTA solution (0.25%) to cover the cell surface. This enzyme detaches adherent cells by cleaving proteins anchoring them to the plate.

5. Incubation for Detachment: Place the flask or plate in a 37 °C incubator for 2–5 minutes. Tapping the plate gently may help if detachment is incomplete.

6. Neutralization of Trypsin: Add an equal or greater volume of complete culture medium (containing FBS) to stop trypsin activity.

\*FBS contains inhibitors that neutralize trypsin.

7. Centrifugation: Transfer the cell suspension to a 15 mL conical tube and centrifuge at 1300 RPM for 3–5 minutes. This step pellets the cells at the bottom.

8. Removal of Supernatant and Resuspension: Aspirate the supernatant and gently resuspend the cell pellet in fresh complete medium.

9. Transfer to New Culture Plate: Dispense the resuspended cells into a new culture plate or flask at the desired seeding density. And return the plate to a humidified incubator (37 °C, 5% CO₂).

##### Cell Viability Assay

MTT assay method

1. Seeding: HepG2 cells (2 × 10⁴ cells/well) were seeded in a 96-well plate with 200 µL of DMEM supplemented with 10% FBS and 1% penicillin-streptomycin.

2. Incubation: The cells were incubated for 24 hours at 37 °C in a 5% CO₂ atmosphere.

3. Sample treatment: After washing with DPBS, 200 µL of DMEM (containing 10% FBS and 1% penicillin-streptomycin) with serially diluted digested supernatant (10, 50, and 100 mg/g) was added to each well.

4. Incubation: The cells were incubated for another 24 hours at 37 °C in a 5% CO₂ incubator.

5. MTT solution treatment: After washing with DPBS, MTT solution (0.5 mg/mL in DMEM) was added to each well.

\*MTT solution, Avoid exposure to direct light. Handle in the dark.

6. Incubation: The plate was incubated for 2 hours at 37 °C in a 5% CO₂ atmosphere.

7. DMSO treatment: After incubation, 200 µL of DMSO was added to each well to dissolve the formazan crystals, followed by a 10-minute incubation at 37 °C.

8. Absorbance measurement: The absorbance was measured at 570 nm and 630 nm using a microplate reader.

\*The 630 nm value was subtracted as a background correction for DMEM.

\*The absorbance value should be within the range of 0.1 to 1.0 for accurate measurements.

##### Reactive Oxygen Species (ROS) Assay

ROS assay method

1. Seeding: HepG2 cells (2 × 10⁴ cells/well) were seeded in a 96-well plate with 200 µL of DMEM supplemented with 10% FBS and 1% penicillin-streptomycin.

2. Incubation: The cells were incubated for 24 hours at 37 °C in a 5% CO₂ atmosphere.

3. Sample treatment: After washing with DPBS, 200 µL of DMEM (containing 10% FBS and 1% penicillin-streptomycin) with serially diluted digested supernatant (10, 50, and 100 mg/g) was added to each well.

4. Incubation: The cells were incubated for another 24 hours at 37 °C in a 5% CO₂ incubator.

5. 2’7’-DCFH-DA solution treatment: Removing the medium, 2’7’-DCFH-DA solution (10 μM in DMEM) was added to each well.

\*2’7’-DCFH-DA solution, Avoid exposure to direct light. Handle in the dark.

6. Incubation: The plate was incubated for 30-minute at 37 °C in a 5% CO₂ atmosphere.

7. Fluorescence measurement: The fluorescence was measured at 488 nm for excitation and 525 nm for emission using a microplate reader.

#### METHODS

##### Analysis of ginsenoside from GS using HPLC-UV

The analysis of standards of ginsenosides, and ginsenoside components in GS were
conducted using the following method. HPLC analysis was conducted using the Nanospace
SI-2 system with a photodiode array (PDA) detector (Nanospace SI-2, Osaka SODA Co.,
Ltd., Osaka, Japan) and the Cadenza CD-C18 column (250 mm × 3 mm, 3.0 μm, Imtakt,
Kyoto, Japan). The mobile phase consisted of distilled water (A) and acetonitrile (B), with
the gradient elution programmed as follows: 15% B at 0.5 min, increasing to 30% at 28.5
min, 32% at 30.5 min, 38% at 36.5 min, and 43% at 47.5 min. The proportion of B was
further increased to 55% at 54.0 min and maintained at this level until 62.0 min. The flow
rate was set to 0.4 mL/min. The injection volume was set to 10 µL, and the column oven
temperature was maintained at 40°C. The wavelength of the UV detector was set to 203
nm.

---

##### Bioaccessibility of MPs with RGS by in vitro digestion model system

To define the bioaccessibility of MPs or MPs with RGSs was estimated by using an in
vitro digestion model system, which slightly imitates the method conducted in the previous
studies (Choi et al., 2017; Choi et al., 2019). 125 µL of 1 µm MPs (20000 µg/mL) was cotreated with GS (20000 µg/mL) in a ratio of 1:1, 1:2, and 1:4 (volume: weight), and then
mixed with 2.5 mL of 20 mM phosphate buffer for the sample. Simulates the small intestine
phase of human digestion. 400 µL bile acid, 400 µL lipase, and 400 µL pancreatin were
added to the mixture, and the pH value was set at 7.0 ± 0.1 by 1 M NaOH or 1M HCl. The
mixtures were incubated for 120 min at bath at 37 ℃ at 200 rpm. After digestion, all
samples were adjusted to the same volume by adding 20 mM phosphate buffer.
Subsequently, the mixtures were centrifuged for 30 min at 4000 rpm in a refrigerated
centrifuge (4 ℃). The separated supernatant and pellet were applied for further analyses.
The supernatants of the sample were measured through a microplate reader (Varioskan
Flash, Thermo Scientific, CA) at an excitation wavelength of 470 nm and an emission
wavelength of 505 to confirm bioaccessibility, and the value of bioaccessibility of MPs
were calculated as follows:

##### Measurement of strucutral characterization MPs

The pellets obtained from digest of 1 µm fluorescent MPs, as well as those derived from
MPs co-treated with GS at ratios of 1:1, 1:2, and 1:4, were diluted to 0.1% in distilled water.
Among the dispersed pellets, structural changes were visualized for fluorescent MPs
through fluorescence microscopy (Invitrogen™ EVOS™ FL Digital Inverted Fluorescence
Microscope, Thermo Scientific, CA), and the change in particle size and zeta potential of
pellet were measured using both a Particle Size Analyzer (Delsa™ Nano Series, Beckman
Coulter, Brea, USA), and a Zeta Potential Analyzer (ELSZneo, Otsuka Electronics, Osaka,
Japan), respectively.

##### Measurement of structural changes of MPs by hyperspectral imaging (HSI)

A hyperspectral camera (Specim FX17, Geostory, Finland), with an invisible
wavelength range of 900 to 1700 nm, was used for taking images of the supernatant and
pellet in the digest of MPs, and then each image was divided into 112 bands. Spectral
imaging software (Lumo scanner, Middleton Spectral Vision, USA) was used to capture
the image, and after capturing, normalization was carried out to subtract the reflections
value that affect the sample measurements. Reflection value data and graphs obtained from
normalized hyperspectral imaging (HSI) of the samples were analyzed using a software
program (ENVI, NV5, USA). Reflection value data was saved in pixel units and plotted as
a graph against wavelength.

##### Measurement of cell viability by MTT assay

The cell viability of HepG2 cell was measured using a modified version of the 3-(4,5
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as outlined in a
previous study (Kumar et al., 2018). HepG2 cells were seeded with 2 x 104 cells/well in a
96-well cell culture plate. After seeding, the cell was incubated at 5% CO2, 37 ℃ for 24
hours. The cells were treated with in vitro digests that had been diluted 1:9 with DMEM.
Each treatment was incubated separately at 37 ℃ with 5% CO2 for 24 hours. After
removing the sample solution, 0.5 mg/mL MTT in DMEM was added, and the cells were
cultured for 2 hours under the same conditions as above. Then, 200 µL dimethyl sulfoxide
(DMSO) was added to dissolve the formazan derived from MTT immediately after
removing the MTT solution. It was incubated at 37 ℃ for 10 minutes. The optical density
(O.D.) was measured at 570 nm for the MTT signal and 630 nm for the background by
using a microplate reader (Varioskan Flash, Thermo Scientific, CA). The cell viability (%)
was calculated by using the following equation

##### Preventive effect of GS on the intracellular ROS level induced by MPs in HepG2 cell

A slightly modified version of the method described in a previous study was used to
measure the preventive effect of RGS on ROS levels induced by MPs (Yu et al., 2021).
HepG2 cell was seeded into 96-well plates (2 x 104 cells/well) and treated with in vitro
digests that had been diluted 1:9 with DMEM. After incubation for 24 hours (5% CO2,
37 ℃), a 10 μM 2’7’-DCFH-DA solution was added to each well for 30 minutes.
Fluorescence was measured at 488 nm for excitation and 525 nm for emission using a
microplate reader (Varioskan Flash, Thermo Scientific, CA). The protective effect of RGS
on MPs-induced ROS was calculated as follows:

##### Statistical analysis

All the experiments were performed in triplicate, and results were expressed as the
means ± standard deviation (SD). A one-way analysis of variance (ANOVA) was
performed to measure the significant differences between the groups at the significant
value of p < 0.05 by using Graphed Prism 6.0 software (Graphpad, CA, USA)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

## RESULTS

* [Identification and quantification of ginsenosides from GS](#one)
* [Effect of GS on bioaccessibility of MPs during in vitro digestion](#two)
* [Effect of ginseng extract on microplastic aggregation in simulated digestion](#three)
* [Hyperspectral analysis of pellet and supernatant fractions](#four)
* [Protective effects of ginseng extract on microplastic-Induced cytotoxicity](#five)
* [Antioxidant activity of ginseng Extract in microplastic-treated HepG2 Cells](#six)
* [Conclusion and Future Directions](#seven)

##### Identification and quantification of ginsenosides from GS

| 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.*

*Table 1: Concentration of ginsenosides Rb1, Rg1, 20(S)-Rg3 in the extract*

The concentrations of three representative ginsenosides in the GS were quantified using
HPLC and are summarized in Table 1. Among the compounds analyzed, ginsenoside Rg1
was the most abundant (6.61 ± 0.99 mg/g), followed by ginsenoside Rb1 (3.52 ± 0.49 mg/g).
In contrast, 20(S)-ginsenoside Rg3 was not detected in the extract. These results indicate
that the GE used in this study is enriched in protopanaxatriol-type ginsenosides (e.g., Rg1),
which are known for their amphiphilic and biointeractive properties. The ginsenoside
profile of the GE used in this study may underline its observed ability to modulate
microplastic behavior during digestion. Notably, ginsenoside Rg1—the most abundant
component—has been previously reported to possess surfactant-like properties, attributed
to its hydrophobic aglycone backbone and hydrophilic sugar moieties (Christensen, 2009).
This amphiphilic nature facilitates interfacial interactions with hydrophobic surfaces, such
as plastic polymers, potentially contributing to the aggregation or encapsulation of MPs
during gastrointestinal simulation (Liu et al., 2019). The absence of Rg3, a less polar
ginsenoside often associated with high-temperature processed ginseng, suggests that the
extract retains a more native saponin profile, favoring surface binding rather than deep
partitioning into hydrophobic domains.

---

##### Effect of GS on bioaccessibility of MPs during in vitro digestion

![](https://static.igem.wiki/teams/5993/images/bioaccessiblity.webp)

*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*

Figure 1 shows the bioaccessibility of MPs after in vitro gastrointestinal digestion
in the presence of varying concentrations of GS. The control group, containing MPs alone,
exhibited the highest bioaccessibility value (approximately 28.90 ± 0.24%), indicating that
most of the particles remained in the solubilized fraction. In contrast, GS-treated groups
exhibited a concentration-dependent reduction in MP bioaccessibility. The GS-L and GS
M groups showed moderate decreases, though not statistically different from each other (p
> 0.05). Notably, the GS-H group demonstrated a significant reduction in MP
bioaccessibility (p < 0.05), decreasing to approximately 60% of the control level,
suggesting that high-dose GS co-treatment reduces the solubilized, absorbable fraction of
microplastics. The significant reduction in MPs bioaccessibility in the GS-H group
suggests that high concentrations of ginseng extract can effectively limit the
gastrointestinal availability of microplastics. This is likely due to the aggregation or
entrapment of MPs induced by amphiphilic compounds such as ginsenosides, which can
simultaneously interact with hydrophobic plastic surfaces and aqueous digestive media
(Christensen, 2009); Liu et al., 2019. The formation of larger aggregates or their
incorporation into semi-solid digestion residues may cause these particles to sediment or
become physically inaccessible, thereby reducing their presence in the supernatant fraction
used to assess bioaccessibility. These results are in agreement with previous reports that
describe how dietary fibers or polyphenol-rich plant extracts can entrap or flocculate
nanoparticles and microplastics, reducing their diffusion and cellular uptake (Ojo et al.,
2020; Stock et al., 2019).

Given that bioaccessibility is a prerequisite for intestinal absorption, the observed
decrease implies that GE may serve as a natural mitigation agent, lowering the potential
systemic exposure to microplastics following oral ingestion. This effect may be especially
valuable in functional food applications or dietary interventions aimed at reducing
microplastic absorption in humans.

---

##### Effect of ginseng extract on microplastic aggregation in simulated digestion

![](https://static.igem.wiki/teams/5993/images/microstructural.webp)

*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.*

The influence of GS on the aggregation behavior of MPs was visualized using
fluorescence microscopy and quantified through particle size distribution analysis (Figure
2). In the absence of GS (control), MPs were well-dispersed with minimal aggregation,
showing multiple size populations from submicron to tens of micrometers. Upon treatment
with low concentrations of GS, fluorescence images revealed the emergence of localized
clustering, with corresponding particle size histograms indicating a modest rightward shift
in volume-weighted distribution. Notably, medium-concentration treatment resulted in
pronounced aggregate formation, with the fluorescence image displaying a single large
fluorescent mass and the size distribution peaking sharply above 10 μm. Interestingly, the
high-concentration group displayed fewer visible particles and a relatively narrow particle
size distribution dominated by large aggregates, possibly due to sedimentation or
encapsulation of particles into larger, denser complexes. Across all treatments, the number
weighted particle size plots confirmed the shift in population toward fewer but larger
particles, indicating that GS induced MPs agglomeration in a concentration-dependent
manner.

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

*Table 2: Zeta potential of microplastics (MPs) in the absence or presence of ginseng extract (GE) at low, medium, and high concentrations*

Plain MPs have a zeta potential of 46.92 mV, indicating stable dispersion with
moderate repulsion between particles (Table 2). MPs + GS (Low) show a decreased zeta
potential of 40.965 mV, suggesting reduced repulsive force and shorter distance between
particles, which may lead to mild aggregation (Table 2). MPs + GS (Med) slightly increase
to 44.675 mV, meaning repulsion is partially restored, allowing particles to remain more
evenly dispersed (Table 2). MPs + GS (High) show a significantly higher zeta potential of
58.205 mV, indicating stronger electrostatic repulsion, resulting in greater distance
between particles and increased colloidal stability (Table 2). It can also suggest that
particles are getting larger due to increased surface interactions or molecular layering.

GS is rich in amphiphilic phytochemicals such as ginsenosides, known for their
surfactant-like properties due to their triterpenoid backbone and sugar moieties
(Christensen, 2009; Sun et al., 2017). These molecules can interact with hydrophobic
plastic surfaces via Van der Waals forces or hydrophobic interactions, while
simultaneously forming hydrogen bonds with water or other polar groups in the medium
(Dąbrowski, 2001; Wang et al., 2021). This dual affinity may facilitate bridging
flocculation, wherein ginsenosides act as molecular linkers between microplastic particles,
leading to aggregation (Bolto & Gregory, 2007); Liu et al., 2019. At higher concentrations,
such bridging may be enhanced, forming denser clusters that sediment out of solution or
become entrapped within gelatinous pellets, as seen in the fluorescence and particle size
data. The observed aggregation of microplastics induced by ginseng extract has important
implications for their gastrointestinal fate. In the simulated digestion model, ginsenoside
mediated flocculation led to the formation of large, visible microplastic aggregates,
especially at medium and high concentrations. These larger agglomerates are less likely to
permeate the intestinal barrier, such as the Caco-2 monolayer or mucus layer, compared to
their nanoscale or submicron counterparts.

Previous studies have shown that particle size is a critical determinant of
microplastic uptake, with particles smaller than 10 μm being more readily internalized by
intestinal epithelial cells via endocytosis or transcellular transport (Stock et al., 2019;
Walczak et al., 2015). The formation of larger aggregates exceeding tens of microns, as
seen in our fluorescence images and size distribution data, may thus significantly reduce
the probability of microplastic translocation across the gut epithelium. Moreover, the
potential encapsulation of microplastics into gelatinous pellets, possibly consisting of
ginseng polysaccharides or glycoprotein-like matrices, may further impair their mobility
and bioavailability in the digestive tract. Such matrix entrapment could physically prevent
interaction between microplastics and intestinal cells, acting as a natural barrier similar to
dietary fibers or mucin-like complexes (Ojo et al., 2020).

---

##### Hyperspectral analysis of pellet and supernatant fractions

![](https://static.igem.wiki/teams/5993/images/hyperspectral.webp)

*Figure 3. Hyperspectral imaging of digested pellet and supernatant of MPs and GS.*

Figure 3 presents the hyperspectral reflectance profiles of the digesta pellet and
supernatant fractions obtained after co-incubation of microplastics with varying
concentrations of GS. Across all samples, pellet fractions exhibited significantly higher
reflectance values than the corresponding supernatants, particularly in the 1000–1200 nm
region. Within this range, distinct concentration-dependent patterns emerged. The medium
concentration GS group (pellet\_MPs+GS\_Med) showed the highest reflectance intensity,
followed by the high and low GS concentrations. Conversely, the supernatant fractions
showed a corresponding decline in reflectance with increasing GS concentration,
indicating progressive sedimentation or removal of MPs from the soluble phase. Key
inflection points near 1050 nm, 1075 nm, and 1100 nm, highlighted in the magnified region
suggest specific wavelength zones where differences in MP accumulation or matrix
interactions are most pronounced. The hyperspectral reflectance data corroborates the
earlier findings of MP aggregation and bioaccessibility reduction in the presence of ginseng
extract. Higher reflectance in pellet fractions—particularly in the near-infrared region
suggests increased accumulation of particulate matter, likely due to ginsenoside-mediated
flocculation or entrapment of MPs (Chu et al., 2019).

Notably, the medium GS concentration produced the highest reflectance intensity
in pellets, possibly reflecting an optimal concentration range for bridging flocculation,
where sufficient amphiphilic molecules bridge MPs without excessive self-aggregation.
The declining reflectance in supernatants implies fewer free MPs remain in the soluble
phase, further supporting the notion that GE promotes MP removal from the bioaccessible
fraction. This spectral behavior aligns with previous studies employing hyperspectral
imaging to track nanoparticle aggregation and dispersion in biological or simulated
environments (Lu et al., 2020). Reflectance peaks in the 1050–1100 nm range have been
associated with organic matter absorption, which may also reflect GS-polysaccharide–MP
complex formation. Collectively, these results demonstrate that hyperspectral imaging
provides a non-destructive, quantitative tool to monitor MPs aggregation and sequestration
dynamics during digestion.

---

##### Protective effects of ginseng extract on microplastic-Induced cytotoxicity

![](https://static.igem.wiki/teams/5993/images/cell-viability.webp)

*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 4 illustrates the protective effects of ginseng extract (GS) against
microplastic (MP)-induced cytotoxicity in HepG2 liver cells. Panel A shows that exposure to
MPs alone significantly reduced cell viability to approximately 65% compared to the
untreated control group, indicating notable cytotoxic effects. However, co-treatment with
GS at increasing ratios (1:1, 1:2, and 1:4 MP:GS) progressively restored cell viability in a
dose-dependent manner. The highest GS concentration (1:4 ratio) nearly normalized viability
to control levels (~95%), demonstrating strong cytoprotective effects. Panel B shows that
MP exposure markedly elevated intracellular reactive oxygen species (ROS) levels to about
250% of control, indicating oxidative stress as a key mechanism of toxicity. Co-treatment
with GS significantly attenuated ROS generation in a concentration-dependent fashion, with
the highest GS dose reducing ROS levels back to near baseline (~110% of control). These
findings suggest that GS mitigates MP-induced oxidative damage, likely through its known
antioxidant properties.

##### Antioxidant activity of ginseng Extract in microplastic-treated HepG2 Cells

![](https://static.igem.wiki/teams/5993/images/cell-viability.webp)

*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).*

Figure 5A shows that treatment with MPs significantly increased intracellular ROS
production in HepG2 cells to approximately 113% of the control level. In contrast, GS
alone maintained ROS levels comparable to or slightly below control. As shown in Figure
5B, co-treatment with GS significantly enhanced ROS scavenging activity in a
concentration-dependent manner. The MPs + GS (Med) and MPs + GS (High) groups
exhibited significantly higher scavenging activity (~60–65%) compared to the Low group
(~30%, p < 0.05), indicating the antioxidant potential of GS at sufficient concentrations.
The results suggest that GS effectively attenuates microplastic-induced oxidative stress in
HepG2 cells. The elevation of ROS production by MPs (Figure 5A) is consistent with
known mechanisms of cellular stress and membrane disruption triggered by micro- and
nanoplastics (Schirinzi et al., 2017). However, the addition of GS markedly enhanced ROS
scavenging capacity, particularly at medium and high concentrations, as shown in Figure
5B.

This antioxidant effect is likely mediated by the presence of ginsenosides Rg1 and Rb1,
which are known to activate the Nrf2 signaling pathway, leading to the induction of
antioxidant enzymes such as HO-1 and SOD (Kim et al., 2013; Lee et al., 2021).
Furthermore, the enhanced RSA may also relate to indirect mechanisms such as inhibition
of MP uptake or aggregation-mediated sequestration, reducing cellular exposure to ROS
inducing particles. Together, these findings indicate that GS can mitigate MP-induced
oxidative stress through both direct ROS scavenging and preventive barrier effects,
reinforcing its potential as a dietary protector against environmental contaminants.

The observed cytoprotective effects of GS against MP-induced toxicity in HepG2
cells are likely multifactorial. Ginsenosides and other bioactive compounds in GS possess
potent antioxidant activity, scavenging free radicals and reducing oxidative stress (Attele
et al., 1999; Kim et al., 2013). By lowering intracellular ROS levels, GS may prevent
oxidative damage to cellular components such as lipids, proteins, and DNA, thereby
preserving cell viability. Additionally, GS has been reported to modulate signaling
pathways involved in cell survival and apoptosis, such as the PI3K/Akt and MAPK pathways
(Chen et al., 2018; Lee et al., 2015). Activation of these pro-survival pathways by GS may
further enhance cellular resilience against MP-induced stress. Furthermore, the ability of
GS to reduce the bioaccessibility of MPs during digestion, as demonstrated earlier, may
also contribute to its protective effects by limiting the amount of MPs that reach and
interact with liver cells.

#### Conclusion and Future Directions

This study demonstrates that ginseng extract (GS) has a protective role against
microplastic (MP)-induced adverse effects during simulated digestion and subsequent
cellular exposure. Co-treatment of MPs with GS in an in vitro digestion model led to
pronounced aggregation and sedimentation of MPs, as confirmed by fluorescence imaging,
particle size distribution, and hyperspectral reflectance analyses. These changes resulted in
a significant decrease in MP bioaccessibility, especially at medium and high concentrations
of GS, indicating a reduction in their gastrointestinal availability. Furthermore, GS
effectively mitigated MP-induced cytotoxicity and oxidative stress in HepG2 cells. Co
digestion with GS restored cell viability beyond control levels and significantly reduced
intracellular ROS production. GS also enhanced ROS scavenging activity in a
concentration-dependent manner, with ginsenosides Rg1 and Rb1 likely contributing to
both physical particle entrapment and activation of endogenous antioxidant pathways such
as Nrf2.

Overall, these findings suggest that GS can reduce the intestinal absorption and cellular
toxicity of ingested microplastics through both physical (aggregation, sedimentation) and
biochemical (antioxidant, cytoprotective) mechanisms. Ginseng extract therefore holds
promise as a functional dietary intervention to mitigate the health risks associated with
microplastic exposure. Future research should focus on in vivo validation of these
protective effects, including pharmacokinetic studies to assess MP bioavailability and
systemic distribution following oral GS administration. Additionally, mechanistic studies
exploring the specific molecular pathways involved in GS-mediated cytoprotection and
antioxidant responses will further elucidate its therapeutic potential. Finally, evaluating
the efficacy of GS in complex food matrices and real-world dietary scenarios will be
critical for translating these findings into practical applications for human health.

#### REFERENCE

Christensen, L. P. (2009). Ginsenosides: Chemistry, biosynthesis, analysis, and potential health effects. Advances in Food and Nutrition Research, 55, 1–99. https://doi.org/10.1016/S1043-4526(08)00401-4

Liu, F., et al. (2019). Sorption behavior and mechanism of ginsenosides on plastic
microbeads. Environmental Pollution, 254, 113026.

Stock, V., et al. (2019). Uptake and effects of orally ingested polystyrene microplastic
particles in vitro and in vivo. Archives of Toxicology, 93(7), 1817–1833.

Ojo, O., Feng, Q.-Q., Ojo, O. O., & Wang, X.-H. (2020). The role of dietary fibre in modulating
gut microbiota dysbiosis in patients with type 2 diabetes: a systematic review and meta-
analysis of randomised controlled trials. Nutrients, 12(11), 3239. 507
https://doi.org/https://doi.org/10.3390/nu12113239

Walczak, A. P., et al. (2015). Bioavailability and biodistribution of differently charged
polystyrene nanoparticles upon oral exposure in rats. Journal of Nanomedicine, 10(4),
1097–1110.

Schirinzi, G. F., et al. (2017). Cytotoxic effects of commonly used plasticizers and plastic
particles on human epithelial intestinal Caco-2 cells. Frontiers in Toxicology, 5, 39.

Attele, A. S., Wu, J. A., & Yuan, C. S. (1999). Ginseng pharmacology: Multiple
constituents and multiple actions. Biochemical Pharmacology, 58(11), 1685–1693.
https://doi.org/10.1016/S0006-2952(99)00212-9

Kim, J. H., et al. (2013). Ginsenoside Rg1 suppresses oxidative stress through the Nrf2
pathway in HepG2 cells. Toxicology Letters, 220(2), 226–234.

Bolto, B., & Gregory, J. (2007). Organic polyelectrolytes in water treatment. Water research,
41(11), 2301-2324. https://doi.org/https://doi.org/10.1016/j.watres.2007.03.012
Christensen, L. P. (2009). Ginsenosides chemistry, biosynthesis, analysis, and potential health
effects. Adv Food Nutr Res, 55, 1-99. https://doi.org/10.1016/s1043-4526(08)00401-4

Chu, S.-f., Zhang, Z., Zhou, X., He, W.-b., Chen, C., Luo, P., Liu, D.-d., Ai, Q.-d., Gong, H.-f., &
Wang, Z.-z. (2019). Ginsenoside Rg1 protects against ischemic/reperfusion-induced
neuronal injury through miR-144/Nrf2/ARE pathway. Acta Pharmacologica Sinica, 40(1),
13-25. https://doi.org/10.1038/s41401-018-0154-z

Dąbrowski, A. (2001). Adsorption—from theory to practice. Advances in colloid and interface
science, 93(1-3), 135-224. https://doi.org/https://doi.org/10.1016/j.scitotenv.2021.147819

Jin, S., Jeon, J.-H., Lee, S., Kang, W. Y., Seong, S. J., Yoon, Y.-R., Choi, M.-K., & Song, I.-S.
(2019). Detection of 13 ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh2, F1,
Compound K, 20 (S)-Protopanaxadiol, and 20 (S)-Protopanaxatriol) in human plasma and
application of the analytical method to human pharmacokinetic studies following two
24 week-repeated administration of red ginseng extract. Molecules, 24(14), 2618.
https://doi.org/https://doi.org/10.3390/molecules24142618

Qiao, R., Sheng, C., Lu, Y., Zhang, Y., Ren, H., & Lemos, B. (2019). Microplastics induce
intestinal inflammation, oxidative stress, and disorders of metabolome and microbiome in
zebrafish. Science of the Total Environment, 662, 246-253.
https://doi.org/https://doi.org/10.1016/j.scitotenv.2019.01.245

Sun, M., Ye, Y., Xiao, L., Duan, X., Zhang, Y., & Zhang, H. (2017). Anticancer effects of
ginsenoside Rg3. International journal of molecular medicine, 39(3), 507-518
https://doi.org/https://doi.org/10.3892/ijmm.2017.2857

Lu, G., et al. (2020). Application of hyperspectral imaging in agriculture and food: A
review. Food Reviews International, 36(1), 1–20.

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