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. 2026 Jul 16;13:101502. doi: 10.1016/j.crfs.2026.101502

Mechanistic insights into the lipid-lowering effects of phycocyanin through human intestinal and liver organoids

Sonoko Tokunaga a,⁎, Yu Takahashi b,⁎⁎, Shinya Takeno a, Yumika Taniguchi a, Takashi Sasaki e, Shintaro Sato c,d, Toshiaki Matsuda a, Ryuichiro Sato e
PMCID: PMC13393639  PMID: 42494838

Abstract

Dyslipidemia is a major risk factor for cardiovascular and cerebrovascular diseases. Phycocyanin (PC), a pigment derived from Arthrospira platensis, improves hyperlipidemia in vivo; however, its mechanisms remain elusive. This study investigated whether PC influences lipid metabolism, particularly lipoprotein secretion dynamics, using human small intestine and liver organoids, which are physiologically relevant in vitro models. In small intestinal organoids, PC significantly suppressed chylomicron marker apolipoprotein (Apo) B-48 secretion without affecting its expression and intracellular triglyceride levels. Peptidomic analysis using mass spectrometry identified MKTPL as a key peptide among digestion-derived peptides of PC that exhibited an inhibitory effect on chylomicron secretion similar to that of intact PC. In liver organoids, digested PC (dPC) and MKTPL reduced the secretion of very-low-density lipoprotein marker ApoB-100. These results revealed the novel action of PC and dPC in preventing lipoprotein secretion in both intestinal and liver cells, providing mechanistic insights into their lipid-lowering effects.

Keywords: Phycocyanin, Human intestinal organoids, Human liver organoids, Chylomicron, Very-low-density lipoprotein

Graphical abstract

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Highlights

  • •

    Phycocyanin suppresses chylomicron secretion in intestinal organoids.

  • •

    Peptidomics revealed a novel phycocyanin peptide profile after simulated digestion.

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    One of the digested phycocyanin peptides, MKTPL, inhibits chylomicron secretion.

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    Digested phycocyanin and MKTPL reduce VLDL secretion in liver organoids.

1. Introduction

Obesity is a major public health concern worldwide. Since 1990, obesity prevalence has increased by 155.1% in males and 104.9% in females, and more than 50% global adult population (approximately 3.8 billion people) may become overweight or obese by 2050 (Ng et al., 2025). Multiple reports have indicated that obesity is a significant risk factor for various chronic and potentially fatal diseases including cardiovascular disorders, hyperlipidemia, and type 2 diabetes (Nikolopoulou and Kadoglou, 2012; Vucenik and Stains, 2012). Once these conditions manifest, they not only reduce the quality of life, but also contribute to rising healthcare costs worldwide (Malik et al., 2013). Therefore, preventive strategies against obesity are urgently required on a global scale.

Phycocyanin (PC), a blue protein pigment derived from the edible cyanobacterium Arthrospira platensis (commonly known as Spirulina), has long been used as a food source for humans and has garnered interest because of its diverse physiological functions (Lafarga et al., 2020). It exhibits antitumor (Hao et al., 2018), hepatoprotective (Vadiraja et al., 1998), and neuroprotective effects (Li et al., 2020). Its potential to improve insulin resistance and ameliorate dyslipidemia has been demonstrated in various animal models(Gao et al., 2016; Hao et al., 2022; Ou et al., 2012, 2013). Specifically, PC exerts beneficial effects on serum lipid profiles, including reduced total cholesterol, triglycerides, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) levels (Husain et al., 2022; Nagaoka et al., 2005). However, it remains unclear in which tissues PC exerts its effects and by what mechanisms it improves lipid metabolism. Oral administration of PC has improved liver lipid profiles and functions in the same diabetic models in which dyslipidemia develops (Hao et al., 2022; Husain et al., 2022), implying that PC and/or its metabolites move from the small intestine to the liver, thereby affecting systemic lipid metabolism. However, functional studies on PC in these tissues have not been conducted.

Established cell lines and animal models have been widely used to study intestinal and hepatic functions and metabolism. However, these models do not always accurately reproduce human physiological responses. Unlike humans, mice and rats lack cholesteryl ester transfer proteins, resulting in low plasma LDL and high plasma high-density lipoprotein (HDL) levels, which activate reverse cholesterol transport and prevent atherosclerosis development (Hogarth et al., 2003). Furthermore, cell lines, such as colon carcinoma-derived Caco-2 and hepatocellular carcinoma-derived HepG2 cells, exhibit chromosomal abnormalities and altered lipid transport capacity, limiting their ability to fully reflect normal cellular physiology (Ghadimi et al., 2000; Levy et al., 1999; Sviridov et al., 2003).

Organoids are physiologically relevant organ-like models that can be generated from tissue-derived stem cells or induced pluripotent stem cells (iPSC) (Kretzschmar and Clevers, 2016). Previously, we developed a simple method to create intestinal epithelial cell (IEC) monolayers from human small intestinal organoids (HIOs) (Takahashi et al., 2017). This approach exposes the luminal surface to nutritional stimuli and enables the measurement of epithelial barrier function as transepithelial electrical resistance (TEER) values. IECs derived from human iPSC-derived small intestinal organoids (hiPSOs) mainly consist of enterocytes, as well as Paneth and goblet cells, and exhibit physiologically relevant TEER values compared to Caco-2 cells. Moreover, these IECs show sodium/glucose cotransporter 1 (SGLT1)-mediated glucose transport, which cannot be replicated in Caco-2 cells, highlighting their physiological validity and usefulness (Takahashi et al., 2022).

In enterocytes, dietary lipids are converted into triglycerides and packed with apolipoprotein (Apo) B-48 by microsomal triglyceride transfer protein (MTP) to form chylomicrons. ApoB-48, produced by RNA editing of ApoB-100 mRNA (Chan et al., 1997), serves as the structural protein of chylomicrons, while ApoB-100 is specific to VLDL. Each chylomicron or VLDL particle contains a single molecule of ApoB-48 or ApoB-100, respectively. We previously showed that HIOs and human liver organoids (HLOs) secrete physiologically relevant chylomicron and VLDL, respectively, unlike conventional cell lines (Kuboyama-Sasaki et al., 2024; Takahashi et al., 2022). We therefore considered these organoid models to possess physiologically relevant metabolic functions and hypothesized that they would enable us to investigate the mechanisms by which PC improves lipid metabolism, which are difficult to analyze in complex in vivo systems. In this study, we revealed that PC suppressed chylomicron secretion in HIO-derived IEC monolayers. Furthermore, by using simulated human digestive fluids, we obtained digested PC and identified MKTPL, a digested peptide of PC, which also inhibited chylomicron and VLDL secretions in HIO-derived IEC monolayers and HLOs, respectively. It should be noted that while MTP inhibitors elevate intracellular triglyceride levels, which can lead to fatty liver, PC suppressed chylomicron and VLDL secretion without altering intracellular triglyceride levels. Therefore, from the standpoint of safety, PC appears to have a distinct advantage. Although pharmacological interventions for prevention remain limited, increasing attention has been directed toward the role of diet and bioactive compounds in food (Lafarga et al., 2020). This study supports the notion that PC may serve as a functional food that can be routinely consumed to help improve lipid metabolism.

2. Results

2.1. PC does not affect the epithelial integrity of hiPSO-derived IEC monolayers

PC is a phycobiliprotein found in cyanobacteria and a major component of the phycobilisome rod structure (Fig. 1A). It consists of two subunits (α and β) and is classified into C-phycocyanin (C-PC) and allophycocyanin (Allo-PC), which contain three or two phycocyanobilin chromophores (Fig. 1B). In this study, we examined the effects of PC in monolayer IECs established from three-dimensional hiPSOs (Fig. S1A). We first determined the non-toxic concentration range of PC based on cell viability assays and selected up to 20 μg/mL PC for further evaluation (Fig. S1B). IECs were treated with various PC concentrations and the expression levels of the epithelial marker gene VIL1, Paneth cell marker LYZ, goblet cell marker MUC2, and stem cell marker LGR5 were analyzed. No significant changes in gene expression were observed following PC treatment (Fig. 1C), suggesting that PC did not influence the constituent cell proportion in IECs.

Fig. 1.

Fig. 1

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PC does not affect the fundamental properties of HIOs. (A) Schematic structure of the phycobilisome in Arthrospira platensis. The phycobilisome contains three allo-PCs as a central core from which six C-PC rods extend outward. Phycoerythrin (PE) hexamers overlap with each other to form rod-like elongated sections. (B) Cartoon representation of C-PC-αβ monomer (left panel) and Allo-PC-αβ monomer (right panel) of A. platensis, based on X-ray crystallographic data (PDB ID: 1gh0). The α- and β-subunits are represented in cyan and green, respectively. PC chromophores are presented in gray stick representation. (C) Expression of organoid marker genes in TkDN4-M organoid-derived IECs after PC treatment. (D and E) The transepithelial electrical resistance (TEER) values of TkDN4-M organoid-derived IECs cultured in (D) growth medium (GM) or (E) intestinal epithelial differentiation medium (IDM) were measured on the indicated days. Data are presented as mean ± SEM (n = 3). No significant difference versus the no-treatment group (Dunnett's test, p > 0.05).

To further assess the impact of PC on epithelial barrier function, TEER values were measured, which indicated tight junction formation. IECs cultured in intestinal organoid growth or epithelial differentiation media were treated with various PC concentrations. The TEER values were not significantly different between the PC-treated and untreated groups (Fig. 1D and E). Therefore, PC may not detrimentally affect the epithelial condition of IEC properties.

2.2. PC suppresses chylomicron secretion in IECs

To evaluate the cellular uptake of PC, we harnessed its intrinsic autofluorescence to estimate the absorption rate in IECs. Fluorescence intensity measurements revealed a concentration-dependent PC uptake by the cells (Fig. 2A). Quantitative analysis detected 0.16% and 0.28% PC in cell lysates after adding 5 and 20 μg/mL PC, respectively, demonstrating that IECs are capable of taking up PC, although the proportion was small. Western blotting confirmed dose-dependent intracellular PC accumulation (Fig. 2B).

Fig. 2.

Fig. 2

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PC suppresses chylomicron secretion but does not affect glucose uptake in HIOs. (A and B) TkDN4-M organoid-derived IECs were treated with PC for 24 h. After harvesting, the cells were lysed with RIPA buffer. (A) The fluorescence values of PC were determined (excitation/emission wavelengths of 540/640 nm). (B) The whole cell lysate was subjected to SDS-PAGE, followed by western blotting using an anti-PC antibody. (C) TkDN4-M organoid-derived IECs were treated with 10 μM phlorizin (Pz.) or PC for 2 h before treatment with 100 μM AMG for 1 h. LC–MS determined intracellular AMG levels. (D and E) ApoB-48 secretion from TkDN4-M organoid-derived IECs (D) and hPIOs organoid-derived IECs (E) treated with 500 μM OA for 48 h in the presence or absence of 1 μM CP-346086 (CP) or PC in the basal medium containing 10% charcoal stripped FBS. SDS-PAGE and western blotting determined ApoB-48. (F) Intracellular triglyceride levels in TkDN4-M organoid-derived IECs after treatment with 500 μM OA and 1 μM CP or 20 μg/mL PC for 48 h. Data are presented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01 versus non-treatment group (Dunnett's test).

To evaluate the effect of PC on nutrient absorption in the intestinal epithelium, we first assessed glucose uptake via key transporters. Carbohydrates ingested from the diet are hydrolyzed by amylase and further broken down into monosaccharides by α-glucosidase in the small intestine, followed by absorption through glucose transporters (Elferink et al., 2020). SGLT1 is a major glucose transporter in the small intestine (Röder et al., 2014). Traditionally, SGLT1 activity has been assessed using radiolabeled methyl α-D-glucopyranoside (AMG), an SGLT1-specific substrate. In this study, we developed a quantitative LC–MS-based approach that does not require a radiolabeled substrate or specialized facilities. When AMG was added to IECs, its intracellular accumulation increased. Furthermore, treatment with the SGLT inhibitors phlorizin (Pz.) and sotagliflozin (St.) suppressed SGLT1-regulated glucose uptake (Fig. S2), indicating the assay validity. However, PC did not alter glucose uptake at the tested concentrations (Fig. 2C). Moreover, glucose uptake via GLUT2, another key transporter involved in glucose absorption and efflux (Röder et al., 2014), was measured using 2-deoxyglucose (2DG) as a substrate in an enzymatic colorimetric assay; however, no significant changes were observed (Fig. S3). These results suggested that PC does not affect glucose absorption in IECs.

Next, we investigated the effects of PC on lipid metabolism. When hiPSO-derived IECs loaded with oleic acid (OA) were treated with a MTP inhibitor, CP-346086, secretion of ApoB-48 into the basolateral side was confirmed to be suppressed. Interestingly, PC significantly reduced ApoB-48 secretion in a dose-dependent manner (Fig. 2D), indicating that the improved in vivo lipid metabolism could be attributed to chylomicron secretion suppression. Similar results were obtained in IECs derived from hiPSOs differentiated from a different iPSC line (Fig. S4) as well as from human primary ileum organoids (hPIOs) (Fig. 2E). Importantly, intracellular triglyceride levels remained unchanged following PC treatment (Fig. 2F), in clear contrast to the increase observed with CP-346086. These results suggest that PC significantly inhibits chylomicron secretion.

2.3. PC does not affect ApoB-48 synthesis in IECs

Based on the above results, we investigated whether PC influenced the expression of Apo-B. Quantitative PCR (qPCR) analysis revealed no significant changes in APOB mRNA levels upon PC treatment compared with that in the control (Fig. 3A). In addition, western blotting showed that PC treatment did not reduce the intracellular ApoB-48 levels (Fig. 3B). These results indicated that PC did not alter ApoB-48 expression.

Fig. 3.

Fig. 3

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PC does not affect ApoB-48 synthesis in HIOs. (A) APOB mRNA expression in 500 μM OA-treated TkDN4-M organoid-derived IECs for 48 h. (B) Intracellular ApoB-48 protein levels in TkDN4 M organoid-derived IECs treated with 500 μM OA for 48 h in the presence or absence of 1 μM CP or 20 μg/mL PC in the basal medium with 10% charcoal stripped FBS. (C) ApoB-48 secretion from TkDN4-M organoid-derived IECs treated with 500 μM OA for 48 h in the presence of 1 μM CP, 20 μg/mL PC (left panel), and co-treatment with 10 μM MG132 (right panel) in the basal medium containing 10% charcoal stripped FBS. (D) Effects of 1 μM CP, 20 μg/mL PC, and co-treatment with 10 μM Triacsin C (TC) on ApoB-48 secretion from 500 μM OA-treated TkDN4-M organoid-derived IECs for 48 h. SDS-PAGE and western blotting determined ApoB-48. Data are presented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01 versus non-treatment group (Dunnett's test).

Lipid-free ApoB-48 is rapidly degraded by the ubiquitin–proteasome pathway (Ginsberg and Fisher, 2009; Olofsson and Borén, 2012). To assess whether PC affects ApoB-48 degradation that can lead to its secretion inhibition, IECs were co-treated with PC and the proteasome inhibitor MG132. Although PC alone reduced extracellular ApoB-48 secretion (Fig. 3C, left panel), co-treatment with MG132 did not significantly decrease ApoB-48 levels (Fig. 3C, right panel). These results suggested that PC may, at least in part, participate in ApoB-48 degradation.

Next, we examined whether PC affects triglyceride synthesis, a process essential for chylomicron production. Treating IECs with Triacsin C (TC), an inhibitor of long-chain acyl-CoA synthetase that suppresses triglyceride synthesis (Igal et al., 1997) significantly reduced ApoB-48 secretion, and the combined treatment with TC and PC caused an even greater reduction (Fig. 3D), suggesting that the mechanism of action of PC differs from that of TC. Taken together with the data in Fig. 2F, these results suggest that PC inhibits chylomicron production by promoting intracellular ApoB-48 degradation, rather than by suppressing ApoB-48 or triglyceride biosynthesis.

2.4. Simulated PC digestion reveals peptide fragmentation and characteristic sequences

Oral PC administration improves lipid metabolism in mice and rats by reducing blood triglyceride levels (Husain et al., 2022; Nagaoka et al., 2005). Since PC may undergo digestion before reaching the small intestine and undigested PC may exert physiological effects, we next performed an in vitro simulated human digestion assay using digestive fluids, including simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF), which mimic the human oral, gastric, and intestinal environments (Fig. 4A) (Brodkorb et al., 2019). After stopping the digestive enzymatic reaction with pefablock, PC was subjected to polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue (CBB) staining and zinc staining to determine its molecular weight. Zinc staining exploits the properties of the chromophore phycocyanobilin, which binds to zinc and becomes excitable under UV light, thereby enabling PC visualization (Berkelman and Lagarias, 1986). After CBB and zinc staining, digested PC (dPC) revealed degradation of its α- and β-subunits, which typically appear around 15 kDa (Fig. 4B). This suggests that the simulated digestion was performed appropriately. To obtain a detailed profile of the molecular weight distribution of PC following digestion, gel filtration chromatography was performed. The results indicated that most peptides in the dPC samples were <1000 Da (Fig. 4C). The digestion products (<3 kDa) were subsequently subjected to peptidomic analysis using LC–MS/MS. In this analysis, peptide sequences corresponding to PC consisting of ≥5 residues were identified through both de novo sequencing and database searching, whereas peptides of 2–5 residues were identified by deep de novo analysis (Fig. 4D). The identified peptides are summarized in Table 1, Table 2. Among the short peptides (2–5 residues), the dipeptide YA accounted for the highest relative abundance (6.1%), followed by the pentapeptide MKTPL (3.5%; Table 1). Among peptides with ≥6 residues, IAGGTGPM was the most abundant (11.3%; Table 2). Notably, sequences containing “GGTGPM” and “IAGGTGP” were frequently observed (cumulative relative abundances of 17.9% and 17.7%, respectively), indicating recurrent fragmentation patterns.

Fig. 4.

Fig. 4

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Simulated PC digestion. (A) Simulated digestion conditions and flow. SSF, simulated saliva fluid; SGF, simulated gastric fluid; SIF, simulated intestinal fluid. (B) PC was digested using pepsin or pepsin pancreatin. Equal PC-equivalent amounts of the digests were subjected to SDS-PAGE, followed by zinc (left panel) and CBB (right panel) staining. (C) The PC samples after digestion treatment were subjected to GPC analysis. (D) The distribution numbers by peptide length are presented in two groups: peptides with 2–5 (left panel) and ≥6 (right panel) residues.

Table 1.

2-5 a.a. peptides relative amount: Top 25.

Peptide Tag length m/z RT Mass PTM Relative abundant (%) origin
YA 2 253.1167 12.9642 252.111 6.1 APCβ, CPCα, CPCβ, APCβ18
MKTPL 5 295.17081 20.5226 588.3305 3.5 CPCα
KNFP 4 253.14055 18.6591 504.2696 3.4 -
FDA 3 352.14819 19.7465 351.143 3.2 CPCβ
LLL 3 358.26776 23.0497 357.2627 3.1 -
PAAGY 5 239.6188 12.7292 477.2223 2.9 -
KM(+15.99)TPL 5 303.16809 18.666 604.3254 Oxidation (M) 2.8 -
ANHG 4 199.59151 15.3609 397.171 2.3 CPCα
ANGH 4 199.59151 15.3609 397.171 2.3 -
FD 2 281.11157 18.1888 280.1059 2.1 APCα, APCβ, CPCβ, APCβ18
VRE 3 202.11752 16.0966 402.2227 1.9 APCα
LL 2 245.18454 20.7246 244.1787 1.7 APCα, APCβ, APCβ18
YNKFP 5 334.67163 20.2489 667.3329 1.6 CPCα
KFNYP 5 334.67145 20.1812 667.3329 1.4 -
M(+15.99)EKA 4 247.61604 13.3909 493.2206 Oxidation (M) 1.3 -
M(+15.99)EAK 4 247.61604 13.3909 493.2206 Oxidation (M) 1.3 -
LRE 3 209.12529 13.5729 416.2383 1.2 CPCβ, APCβ18
YT 2 283.12616 12.2668 282.1216 1.2 APCβ, CPCα, CPCβ, APCβ18
VSLA 4 389.23709 20.4425 388.2322 1.1 -
LAGL 4 373.24237 21.5553 372.2372 1.1 -
TPL 3 330.20023 19.9199 329.1951 1.1 CPCα
TY 2 283.12616 12.2668 282.1216 1.1 CPCα, CPCβ, APCα, APCβ, APCβ18
DY 2 297.10629 18.265 296.1008 1.0 APCα, APCβ, CPCα, APCβ18
VREM(+15.99) 4 275.63477 12.8188 549.2581 Oxidation (M) 1.0 APCα
M(+15.99)EVR 4 275.63477 12.8188 549.2581 Oxidation (M) 1.0 -
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APCα: Allophycocyanin alpha subunit, APCβ: Allophycocyanin beta subunit, CPCα: C-phycocyanin alpha subunit, CPCβ: C-phycocyanin beta subunit.

Table 2.

Relative amount of peptides of 6 amino acids or more attributed to PC: Top 25.

Peptide (808 peptides) Description Mass Length m/z RT Relative abundant (%) PTM
IAGGTGPM C-phycocyanin alpha subunit 702.337 8 703.3338 36.66 11.3
IAGIDE C-phycocyanin alpha subunit 616.3068 6 617.3118 36.75 5.1
NSLGVPI Allophycocyanin beta subunit, Allophycocyanin beta-18 subunit 698.3962 7 699.3994 60.18 4.3
MQDAIT Allophycocyanin beta subunit 677.3054 6 678.3132 29.77 3.8
FAEQPQ C-phycocyanin beta subunit 718.3286 6 719.3317 25.95 3.4
IAPGGNA(+14.02) C-phycocyanin beta subunit, Allophycocyanin-B alpha subunit 612.3231 7 613.3267 19.7 3.3 Methylation(C-term)
SLGTPI Allophycocyanin alpha subunit 586.3326 6 587.3363 44.17 3.3
FATGEL Allophycocyanin beta subunit 636.3119 6 637.3157 56.33 3.2
ALGTPGSS C-phycocyanin beta subunit 688.3392 8 689.345 25.46 3.0
RPDVVSPGGNA Allophycocyanin alpha subunit 1067.5359 11 1068.5364 27.37 2.9
IAGGTGPM(+15.99) C-phycocyanin alpha subunit 718.332 8 360.1708 19.61 2.9 Oxidation (M)
YSDITRPGGNM(+14.02) Allophycocyanin beta subunit 1223.5604 11 1224.5621 48.91 2.4 Methylation(C-term)
YSDITRPGGN(+14.02) Allophycocyanin beta subunit 1092.52 10 547.2648 29.22 2.2 Methylation(C-term)
GIVAGDVTPI Allophycocyanin alpha subunit 940.5229 10 941.5303 80.13 1.7
TFELSPS C-phycocyanin alpha subunit 779.3701 7 780.3724 58.69 1.5
IVNDPA C-phycocyanin beta subunit 627.3228 6 628.3264 24.41 1.4
LSSTEIQ C-phycocyanin alpha subunit 776.3916 7 777.4001 30.05 1.4
SLGTPIE Allophycocyanin alpha subunit 715.3752 7 716.3775 42.18 1.4
YLSPGEL Allophycocyanin alpha subunit 777.3909 7 778.3957 70.51 1.3
LIAGGTGPM C-phycocyanin alpha subunit 815.4211 9 816.4233 55.67 1.3
YSDITRPGGN(+30.01)M Allophycocyanin beta subunit 1239.5554 11 620.7823 30.46 1.2 Hydroxymethyl
AIVNDPA C-phycocyanin beta subunit 698.3599 7 699.3641 33.49 1.1
QKRPDVVSPGGNA Allophycocyanin alpha subunit 1323.6895 13 662.8486 29.84 1.1
VSIADSQG C-phycocyanin alpha subunit 775.3712 8 776.3741 30.76 1.1
GDVTPI Allophycocyanin alpha subunit 600.3119 6 601.3175 34.84 1.0
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2.5. dPC suppresses chylomicron secretion in IECs

To investigate whether dPC exhibited biological activity, we treated hiPSO-derived monolayer IECs with PC digestion products in the presence of fatty acids. Similar to undigested PC, dPC significantly reduced ApoB-48 secretion and did not affect intracellular triglyceride levels (Fig. 5A and S5) and SGLT1-mediated glucose uptake (Fig. S6).

Fig. 5.

Fig. 5

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Inhibitory effect of dPC on chylomicron secretion in HIOs. (A) ApoB-48 secretion from TkDN4-M organoid-derived IECs treated with 500 μM OA for 48 h in the presence or absense of 1 μM CP or dPC in the basal medium with 10% charcoal stripped FBS. (B) Effects of 1 μM CP, 20 μg/mL PC, 20 μg/mL dPC, or 50 μM MKTPL peptide on ApoB-48 secretion from 500 μM OA-treated TkDN4-M organoid-derived IECs for 48 h. SDS-PAGE and western blotting determined ApoB-48. Data are presented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01 versus non-treatment group (Dunnett's test).

We further focused on the biological function of the specific peptides identified in dPC. Treatment with the pentapeptide MKTPL, the second most abundant short peptide detected, markedly reduced ApoB-48 secretion (Fig. 5B). Conversely, treatment with the mixed peptides, IAGGTGPM, IAGGTGP, and GGTGPM, which had the highest relative abundance among the longer peptides, did not suppress ApoB-48 secretion (Fig. S7). These results suggest that dPC inhibited chylomicron secretion similar to undigested PC and MKTPL generated from PC digestion suppressed chylomicron secretion in IECs in cooperation with undigested PC.

2.6. dPC suppresses VLDL secretion in liver organoids

Chylomicrons and VLDL share a common secretion mechanism, characterized by MTP-dependent lipid transfer and ApoB-mediated particle assembly in the endoplasmic reticulum (Hussain et al., 2003). Since the liver receives molecules absorbed from the intestine directly via the portal vein, the absorbed PC, dPC, and derived peptides may act on hepatocytes to modulate lipid metabolism. Considering the suppressive effect of PC on chylomicron secretion in IECs, we investigated its impact on VLDL secretion in hepatocytes using iPSC-derived HLOs (Fig. S8A), which secrete VLDL at physiologically appropriate densities, unlike conventional cell lines such as HepG2 cells (Kuboyama-Sasaki et al., 2024).

Treating dPC with HLOs in the presence of fatty acids significantly reduced ApoB-100 secretion (Fig. 6A). A similar suppressive effect was observed for the undigested PC (Fig. S8B). To further examine this effect, OptiPrep density gradient ultracentrifugation was performed to isolate the VLDL fraction according to previous reports (Kuboyama-Sasaki et al., 2024), and ApoB-100 levels were quantified. When HLOs were treated with OA, the proportion of VLDL fraction among the total lipoproteins increased (Fig. S9), whereas co-treatment with dPC reduced ApoB-100 levels in the VLDL fraction by approximately 30% (right panel of Fig. 6B), indicating that dPC suppressed VLDL secretion. Furthermore, MKTPL treatment reduced ApoB-100 secretion (Fig. 6C), which was consistent with the chylomicron secretion suppression by MKTPL in IECs. These results demonstrate that dPC inhibits VLDL secretion and MKTPL may contribute to this activity. Taken together, PC and dPC would co-operate to suppress chylomicron secretion in IECs and VLDL secretion in hepatocytes.

Fig. 6.

Fig. 6

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Inhibitory effect of dPC on VLDL secretion in HLOs. (A) ApoB-100 secretion from TkDN4-M-derived HLOs treated with 500 μM OA for 48 h in the presence or absence of 1 μM CP or dPC in the basal medium with 10% charcoal stripped FBS. SDS-PAGE and western blotting determined ApoB-100. (B) After lipoprotein fractionation of TkDN4-M-derived HLO culture supernatants treated with 500 μM OA for 48 h in the basal medium, the same volume of each fraction was subjected to SDS-PAGE, followed by western blotting. Proportion of lipoprotein among lipoprotein fractions (left, lower panel) and ApoB-100 levels in the VLDL fraction (right panel) were determined by densitometric analysis. (C) Effects of 1 μM CP or MKTPL peptide on ApoB-100 secretion from 500 μM OA-treated TkDN4-M-derived HLOs for 48 h. SDS-PAGE and western blotting determined ApoB-100. Data are presented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01 versus non-treatment group (Dunnett's test).

3. Discussion

The small intestine plays a critical role in systemically providing lipids by re-esterifying free fatty acids into triglycerides and transporting them to peripheral tissues as chylomicrons. Similarly, hepatocytes, which account for approximately 80% liver mass, are essential for maintaining whole-body lipid homeostasis through synthesis and secretion of VLDL (Ben-Moshe and Itzkovitz, 2019). Therefore, to elucidate how PC improves blood glucose and lipid profiles in vivo, this study focused on nutrient metabolism in the small intestine and liver. Compared to established cell lines and animal models, HIOs and HLOs recapitulate physiological processes, including SGLT1-mediated glucose transport and secretion of chylomicron and VLDL (Kuboyama-Sasaki et al., 2024; Takahashi et al., 2022) making them suitable models for elucidating the molecular mechanisms of metabolism.

Using HIOs, we evaluated SGLT1-and GLUT2-dependent glucose uptake and found that PC did not affect glucose absorption by IECs. In contrast, we found that PC markedly suppressed chylomicron secretion in IECs. This effect is considered one of the mechanisms contributing to the improved lipid metabolism observed in vivo following PC administration, including changes in plasma triglyceride, VLDL, and LDL levels (Hussain et al., 2003; Nagaoka et al., 2005; Ou et al., 2013). Mechanistically, PC did not alter the intracellular triglyceride levels in IECs, suggesting that PC may not affect fatty acid uptake or de novo triglyceride synthesis. In contrast to the MTP inhibitor CP-346086, which reduced intracellular ApoB-48 levels likely by increasing lipid-free ApoB-48 that is rapidly degraded through the ubiquitin–proteasome pathway (Ginsberg and Fisher, 2009; Olofsson and Borén, 2012), PC did not alter intracellular ApoB-48 protein levels. Moreover, inhibition of proteasome using MG132 abolished the suppressive effect of PC on ApoB-48 secretion. These results suggest that PC would inhibit the secretion of chylomicrons after their assembly within IECs, thereby indirectly increasing the intracellular pool of lipid-free ApoB-48. Chylomicrons are secreted from IECs through Golgi-mediated exocytosis (Kumar and Mansbach, 1999), although the underlying molecular mechanisms remain unclear. Future studies using HIOs will clarify the detailed mechanism on how PC interferes with the chylomicron secretory pathway.

In this study, we simulated PC digestion in humans and performed peptidomic analysis to provide important insights into PC fragmentation patterns. Previous studies using enzymatic digestion and LC–MS/MS have identified PC-derived peptides, including IAGGTGPM and MKTPL (Sommella et al., 2019), which were also detected in this study. Unlike in previous studies, we successfully identified peptides with ≤5 residues. By combining conventional database searches with de novo and deep learning-based approaches, we improved the accuracy of unknown peptide identification and increased the number of PC-derived peptides identified by more than three-fold, offering a more comprehensive profile of PC fragments. Both dPC and the MKTPL peptide, identified by peptidomics, suppressed chylomicron secretion, suggesting that MKTPL is proposed to be an active component of dPC. Notably, MKTPL corresponds to residues 1–5 of C-PC α-subunit and constitutes a region exposed while associated with the β-subunit. This structural feature raises the possibility that the MKTPL segment in intact PC also directly interacts with IECs, contributing to its inhibitory effect. In addition to PC-derived bioactive peptides that exhibit various biological activities, such as inhibition of melanin formation, antioxidant properties, and DPPⅣ inhibitory activity (Kose and Oncel, 2022; Minic et al., 2016; Pan et al., 2020; Xu et al., 2022), this study is the first to elucidate the lipid-lowering effects of PC in IECs and hepatocytes.

This study has several limitations. First, HIOs do not fully recapitulate in vivo conditions because they lack immune cells, gut microbiota, and other components of the intestinal microenvironment. Nevertheless, this simplified model enables us to assess the direct effects of PC-derived peptides on IECs, suggesting that the small intestinal epithelium may be a site of action. Second, although several major PC-derived peptides were evaluated, many other peptides identified in this study were not investigated. Further studies are needed to clarify their contributions to the biological effects of PC.

In conclusion, the use of human intestinal and liver organoids revealed that PC modulates lipid metabolism by suppressing chylomicron and VLDL secretion, respectively, which could not be detected using animal models or conventional cell lines. To the best of our knowledge, this is the first study to investigate the lipid-modulating effects of PC using human organoids. Importantly, unlike MTP inhibitors, PC had minimal effects on intracellular triglyceride levels, suggesting its potential as a safe daily supplement. Our findings also suggest that PC may have therapeutic potential not only for dyslipidemia but also for chronic diseases in which dysregulated lipid metabolism contributes to disease progression, including inflammatory bowel disease, metabolic dysfunction-associated steatotic liver disease, and cardiovascular disorders such as atherosclerosis. This study provides mechanistic insights based on an in vitro HIO model and is not intended to establish the safety of PC. However, PC has a long history of use as a food ingredient, and its safety has been evaluated in both human and animal studies (Imai et al., 2019; Naidu et al., 1999). In addition, PC has been used in the Japanese Foods with Function Claims system (FFC; notification No. G1419), supporting its practical use as a functional food ingredient. Nevertheless, the safety and efficacy of PC at doses relevant to lipid regulation and chylomicron suppression should be confirmed in future in vivo and clinical studies. Future clinical trials are essential to validate these effects and establish organoid models as promising platforms for evaluating the metabolic effects of dietary bioactive compounds.

4. Methods

4.1. Materials

Human RSPO1 and Oncostatin M were purchased from R&D Systems. Human HGF, gastrin I, transforming growth factor-α, and mouse epidermal growth factor (EGF) were purchased from PeproTech. Defined fetal bovine serum (dFBS) was purchased from GE Healthcare. Penicillin–streptomycin–glutamine (PSG), Advanced Dulbecco's modified Eagle's medium (DMEM)/F12, FBS, GlutaMAX Supplement (100×), HEPES (1 M), TrypLE Express, and human vitronectin were purchased from Thermo Fisher Scientific. High-glucose DMEM (high glucose), 30% bovine serum albumin, CultureSure A83-01, Y-27632, 2-Deoxy-D-glucose (2DG) and zinc acetate dihydrate were purchased from FUJIFILM Wako. HCM Hepatocyte Culture Medium BulletKit was purchased from Lonza. SB202190, CP-346086 dihydrate, porcine pepsin, porcine pancreatin (8xUSP specifications), and Pefabloc were purchased from Sigma-Aldrich. Gentamicin sulfate solution (50 mg/mL) and OA were purchased from Nacalai Tesque. AMG, Pz., phloretin and MG132 were purchased from Tokyo Chemical Industry. St. was purchased from Selleck Chemicals. Matrigel was purchased from Corning, Inc. TC was purchased from Cayman Chemical. Antibodies against β-actin (A5441) and ApoB (ab7616) were purchased from Sigma-Aldrich and Abcam, respectively. Spirulina is a dried A. platensis product obtained from DIC Co., Ltd. (Tokyo, Japan). PC was purified from aqueous Spirulina extract by filtration and ultrafiltration. The PC content in the sample was 66.93% (C-PC: 54.04%, Allo-PC: 12.89%).

4.2. Cell culture

L-WRNH, L-RN, and L-RHF2 cell lines have been previously established (Kuboyama-Sasaki et al., 2024; Takahashi et al., 2018). All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Conditioned medium (CM) from L-WRNH and L-RN cells was prepared from culture supernatants collected after seeding 1.4 × 106 cells per 35 mm dish and cultured for 72 h. CM from L-RHF2 cells was obtained from supernatants harvested after seeding 5.0 × 106 cells per 100 mm dish and culturing for 72 h. All collected supernatants were filtered through 0.2 μm membrane filters and stored at −80°C until use.

Human iPSC lines TkDN4-M (Stem Cell Bank at the University of Tokyo) and iPS11 (Alstem Cell Advancements) were maintained as colonies under feeder-free conditions on vitronectin (Gibco)-coated plates with essential 8 medium (Gibco) and mTeSRTM Plus-cGMP (STEMCELL Technologies), respectively. The cells were passaged every three days before reaching confluence.

4.3. Differentiation of human iPSCs into intestinal and liver organoids

Human iPSCs (TkDN4-M or iPS11) were differentiated into intestinal or liver organoids as described previously (Kuboyama-Sasaki et al., 2024; Takahashi et al., 2018). Briefly, 80–90% confluent iPSCs were sequentially treated with definitive endoderm (DE)1, DE2, and DE3 media (Table S1) for 24 h to induce DE differentiation. For primitive gut formation, the DE cells were cultured in foregut spheroid differentiation medium (Table S1) for 3 days. Floating spheroids were collected, embedded in Matrigel in Nunc Multidish 4 well plates (Thermo Fisher Scientific), and cultured in IEC growth medium (Table S1) for 14 days to generate HIOs. For differentiation into HLOs, foregut spheroids were first cultured for 3 days in posterior foregut differentiation medium (Table S1), followed by hepatocyte differentiation medium (HDM) for additional 10 days. The culture medium was replaced every 3 days.

4.4. Organoid culture and passage

Organoids embedded in Matrigel were washed with phosphate-buffered saline (PBS) (−), collected in TrypLE Express solution (Thermo Fisher Scientific), and incubated at 37°C for 15 min. The recovered organoids were mechanically dissociated by pipetting 30 times using a P1000 micropipette (Gilson) fitted with a P1000 pipette tip (WATSON 706C), followed by centrifugation at 440 × g for 3 min. The specified pipette and tip are representative examples, and equivalent products from other manufacturers may also be used. After removing the supernatant, the organoids were washed with 10 mL basal medium (Table S1). Following centrifugation at 440 × g for 3 min, the organoids were resuspended on ice in Matrigel supplemented with 20% intestinal organoid growth medium (GM) or liver organoid expansion medium (EM) (Table S1). Suspension aliquots were dispensed into the wells of a Nunc Multidish 4-well plate (Thermo Fisher Scientific), avoiding contact with the edges of each well, and allowed to solidify for 15 min at 37°C in a 5% CO2 incubator. Subsequently, 500 μL human organoid culture medium (GM or EM) was added to each well. The culture medium was replaced every 3 days. The organoids were passaged every 6–7 d at a split ratio of 1:8 to 1:20. All cells were maintained at 37°C in a 5% CO2 incubator. hPIOs, established from an 82-year-old Japanese female surgical specimen, were cultured in the same manner as iPSOs, in accordance with the method described by (Takahashi et al., 2023). Experiments using hPIOs were conducted in accordance with the Declaration of Helsinki and approved by the institutional review boards of the University of Tokyo (No. 18-341). The tissue samples were collected after obtaining informed consent from the patients.

4.5. Monolayer culture of intestinal organoid-derived cells

After recovery from Matrigel, the organoids were digested by incubation in TrypLE Express solution at 37°C for 15 min, followed by mechanical dissociation through vigorous 30 times pipetting, as described for passaging. The organoids were collected by centrifugation at 440× g for 3 min. After centrifugation, the organoids were washed with the basal medium, recovered by centrifugation, resuspended in human organoid culture medium, passed through a 40 μm nylon mesh (Corning), and seeded on type I collagen-coated 6-, 12-, 24-, or 96-well plates or transwell inserts. The culture medium was replaced every 2–3 days. Cell confluence was validated by microscopic observation or measuring the TEER values.

To evaluate ApoB-48 secretion and SGLT1 activity in the intestinal organoids, the medium was replaced with intestinal epithelial differentiation medium (ID; Table S1). To determine ApoB-100 secretion activity in liver organoids, the medium was replaced with HDM (Table S1).

4.6. Cell viability determination

After harvesting from Matrigel, the organoids were disrupted. The cells were seeded at 1.0 × 104 cells/well in a 96-well plate containing GM and cultured until they reached confluence. After the cells became confluent, the culture medium was replaced with IDM and the cells were maintained at 37°C in a 5% CO2 incubator for 2–3 days. Confluent cells were incubated with each test sample for 24 h. Subsequently, CellTiter Glo® 3D reagent (Promega) was added to each well, followed by incubation at room temperature for 30 min. Luminescence signals were measured using Varioskan LUX (Thermo Fisher Scientific) according to the manufacturer's instructions.

4.7. Western blotting

After independently collecting 600 μL culture supernatant from the basolateral sides of the Transwell, a six-fold concentrated Laemmli sample buffer (1 mM Tris–HCl, pH 6.8; 30% glycerol; 10% sodium dodecyl sulfate; 600 mM dithiothreitol; 0.03% bromophenol blue) was added to the basolateral medium and the mixture was boiled for 5 min. Equal volumes of each sample were subjected to SDS-PAGE and transferred on polyvinylidene difluoride membranes. The membrane was blocked with EzBlock Chemi (Atto, Japan) at room temperature for 30 min, incubated overnight at 4°C with the primary antibody (anti-ApoB, 1:1000), and then incubated with the secondary antibody for 1 h at room temperature. Chemiluminescent signals were detected using the Fusion Solo S imaging system (Vilber).

4.8. Quantitative reverse transcription–polymerase chain reaction (RT–qPCR)

Total cellular RNA was isolated using a RNeasy Mini Kit (Qiagen, Hilden, Germany). Complementary DNA was synthesized with the ReverTra Ace® qPCR RT Master Mix (TOYOBO, Japan). Quantitative analysis of mRNA expression was performed by fluorescence real time PCR using THUNDERBIRD® SYBR™ qPCR Mix (TOYOBO, Japan) on a StepOnePlus system (Thermo Fisher Scientific). Primer sequences are listed in Table S2.

4.9. SGLT activity measurement

After culturing on collagen I-coated 12-well plates with IDM for two days, hiPSO-derived IECs were incubated in choline assay buffer (Table S1) at 37°C for 20 min. The buffer was replaced with sodium assay buffer (Table S1) with or without each concentration of PC, 10 μM Pz., and 1 μM St. After incubation at 37°C for 2 h, the cells were treated with 100 μM AMG at 37°C for another 1 h, washed twice with cold choline assay buffer, and lysed in 150 μL EasyPep™ MS Sample Prep Kits Lysis Buffer (Thermo Fisher Scientific). The cell lysates (100 μL) were mixed with 100 μL 4 ppm xylitol solution (internal standard) and diluted with 5 mL distilled water. The diluted samples were applied to a Sep Pak® C18 Plus short cartridge (Waters) pre-conditioned with 10 mL 100% acetonitrile and 10 mL distilled water. After loading 5 mL diluted sample, the eluate was collected using 5 mL distilled water. The C18-processed samples were evaporated to dryness under reduced pressure, reconstituted in 80% acetonitrile, and further purified using a MonoSpin HILIC column (NH2, GL Sciences) pre-conditioned with 500 μL 50% acetonitrile and 500 μL 90% acetonitrile. The samples (500 μL) were applied and fractions were eluted with 500 μL 90% acetonitrile. After filtration through a 0.2 μm PTFE membrane, 2 μL samples were injected to analyze. AMG was quantified using LC–MS (LCMS-8050, Shimadzu). Chromatographic separation was performed on a HILICpak VG-50 2D column (Shodex) maintained at 40°C. The mobile phase consisted of 0.5% aqueous ammonia (solvent A) and acetonitrile (solvent B), delivered at 0.2 mL/min. The gradient program for solvent B was as follows: 80% from 0 to 2 min, decreased to 10% from 2 to 12 min, held at 10% from 12 to 15 min, returned to 80% at 15.01 min, and maintained at 80% for 25 min. Mass spectrometric detection was performed using electrospray ionization in the negative-ion mode. The desolvation line and heat block temperatures were 250 and 400°C, respectively. The nebulizing and drying gas flow rates were 3 and 10 L/min, respectively. Multiple reaction monitoring was used for the quantification. AMG was monitored by adjusting the precursor to product ion transition m/z 192.90 → 101.00 with a collision energy (CE) of 11.0 eV, while xylitol (m/z 151.00 → 89.00, CE 12.0 eV) was employed.

4.10. GLUT2 activity measurement

After culturing hiPSO-derived IECs on collagen I-coated 12-well plates with IDM for 2 days, the cells were incubated in choline assay buffer (Table S1) at 37°C for 20 min. The buffer was then replaced with sodium assay buffer (Table S1) containing various concentrations of PC, with or without 10 μM Pz., 1 μM St., and 250 μM phloretin. Following incubation at 37°C for 2 h, the cells were treated with 1 mM 2DG at 37°C for an additional 1 h, washed twice with cold choline assay buffer, and lysed in 150 μL of lysis buffer (10 mM Tris-HCl [pH 8.0], 1% Triton X-100). Intracellular 2DG levels were quantified using a 2-Deoxyglucose Uptake Measurement Kit (COSMO BIO Co., Ltd.) according to the manufacturer's instruction.

4.11. Intracellular triglyceride level measurement

After culturing in collagen I-coated 6-well plates with IDM for two days, hiPSO-derived IECs were washed with PBS (−). Hexane: isopropanol (3:2, v/v) was added directly to the culture vessel to extract lipids. After incubation on ice for 5 min, the extracts were collected in 1.5 mL tubes. This procedure was repeated twice. Intracellular triglyceride levels were quantified using a LabAssay™ Triglyceride kit (FUJIFILM Wako) according to the manufacturer's instructions. The amounts of total cellular proteins collected from the remaining cells using RIPA buffer were measured using the BCA Protein Assay Kit (Pierce) to normalize the triglyceride levels.

4.12. In vitro digestion

The in vitro digestion was performed according to the COST InfoGest protocol (Minekus et al., 2014). PC (1 g) was suspended in 20 mL distilled water subjected to in vitro digestion. SSF, SGF, and SIF solutions were prepared as described in Table S1.

During the oral phase, the suspension was mixed with SSF in a 1:1 (v/v) ratio, adjusted to pH 7.0, and incubated at 37°C for 2 min in a thermostatic water bath with continuous agitation. In the gastric phase, the oral digest was combined with SGF at a 1:1 (v/v) ratio and adjusted to pH 3.0. Porcine pepsin (3.8 mg/mL final concentration) was added to the gastric digestion solution and incubated at 37°C for 2 h under continuous stirring. In the intestinal phase, gastric chyme was mixed with SIF at a 1:1 (v/v) ratio and adjusted to pH 7.0. Porcine pancreatin (16.25 mg/mL final concentration) was added and digestion was continued for 2 h at 37°C with continuous stirring. The pH was adjusted using 1 M HCl or 1 M NaOH. At the end of the intestinal phase, digestion was stopped by adding the protease inhibitor Pefabloc. Undigested and dPC, adjusted to equivalent amounts corresponding to the same amount of PC, were subjected to SDS-PAGE and stained with CBB or zinc. The obtained samples were subsequently subjected to peptidomic analysis. Detailed procedures are described in the Supplementary Methods.

4.13. Lipoprotein fraction separation

The lipoprotein fractions of culture supernatant were separated by density gradient ultracentrifugation using OptiPrep (Axis Shield). The cells were pretreated with each PC concentration for 24 h and then exposed to 500 μM OA in the basal medium containing 10% charcoal-stripped FBS with or without PC for 48 h. A total of 4 mL culture supernatant was concentrated to 100 μL using Amicon Ultra 4 centrifugal filter unit equipped with PLHK Ultracel 100 K membranes (Millipore) by centrifugation at 5000× g for 20 min. The concentrated samples were subjected to density gradient ultracentrifugation using 0–46% OptiPrep gradient in a TLS 55 swinging bucket rotor (Beckman) at 173,000× g for 16 h (Accel: 9; Decel: Coast) at 4°C. After centrifugation, the gradient was carefully collected from the top to the bottom in 200 μL aliquots (11 fractions). The density of each fraction was measured using an electric balance. The fractions corresponding to VLDL, intermediate-density lipoprotein (IDL), LDL, and HDL were 0.950–1.006, 1.006–1.019, 1.019–1.063, and 1.063–1.210 g/mL, respectively.

4.14. Quantification and statistical analysis

Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using Student's t-test for comparisons between two groups and Dunnett's test for comparisons among more than three groups, which were determined using R software. Differences were considered statistically significant at p < 0.05 or p < 0.01, which are indicated with asterisks. All quantitative analyses were performed using at least three independent biological replicates.

Author contributions CRediT

Sonoko Tokunaga: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft.

Yu Takahashi: Conceptualization, Methodology, Investigation, Writing – review & editing, Funding acquisition, Project administration.

Shinya Takeno: Investigation.

Yumika Taniguchi: Investigation.

Takashi Sasaki: Supervision.

Shintaro Sato: Resources.

Toshiaki Matsuda: Funding acquisition, Project administration.

Ryuichiro Sato: Funding acquisition, Supervision.

Funding sources

The work was supported by a joint grant from DIC Corporation and grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan under Grant-in-Aid for Scientific Research C (23K05070 to Y.T.) and Challenging Research (Exploratory, 22K18342 to R.S.).

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Toshiaki Matsuda reports financial support was provided by DIC Corporation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study is supported by a collaborative research agreement with DIC Corporation. We would like to thank Hidenori Yamanaka of the Chemicals Evaluation and Research Institute, Japan (CERI), for conducting the analysis. We also thank Editage (www.editage.jp) for English language editing.

Handling Editor: Dr. Yeonhwa Park

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crfs.2026.101502.

Contributor Information

Sonoko Tokunaga, Email: sonoko.tokunaga@ma.dic.co.jp.

Yu Takahashi, Email: ayutaka@g.ecc.u-tokyo.ac.jp.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Multimedia component 1
mmc1.pdf (317KB, pdf)
Multimedia component 2
mmc2.docx (42.7KB, docx)

Data availability

Any additional information required to reanalyze the data in this study is available from the corresponding authors upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
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Multimedia component 2
mmc2.docx (42.7KB, docx)

Data Availability Statement

Any additional information required to reanalyze the data in this study is available from the corresponding authors upon reasonable request.

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