| 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 |
| 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 |
|
||||||||||||||||||||||||||||||||||||||||||
| Injection Volume | 10 ul | ||||||||||||||||||||||||||||||||||||||||||
| Flow Rate | 0.4 mL/min | ||||||||||||||||||||||||||||||||||||||||||
| Temperature | 40℃ | ||||||||||||||||||||||||||||||||||||||||||
| Run Time | 93 min |
| 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) |
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.
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
| Instrument | Thermo Scientific. Varioskan Flash Microplate Reader |
| Mode | Fluorescence |
| Excitation Wavelength | 470 nm |
| Emission Wavelength | 505 nm |
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.
| 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 |
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 suspension5. 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 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 method
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
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).
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.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.
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.
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:
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.
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.
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
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:
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)