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.

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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.

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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

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.

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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 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.

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.