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.

---

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.

---

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).

---

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.

---

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.

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.

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.

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.