Exosome-like nanoparticles from the Okinawan medicinal plant Talinum fruticosum exhibit subset-specific bioactivities in vitro: Suppression of IL-6 production and reduction of intracellular reactive oxygen species

Introduction

Growing evidence indicates that many animal cell types secrete nano-sized membranous vesicles, known as exosomes, that mediate intercellular communication (1,2). Exosomes deliver bioactive cargo molecules, including proteins, nucleic acids, and small-molecule metabolites, via paracrine and endocrine mechanisms, thereby modulating the metabolism and biological functions of recipient cells (1,2). In recent years, it has become evident that plant cells also produce extracellular vesicles known as exosome-like nanoparticles (ELNs), which share morphological and functional characteristics with animal exosomes (3). These plant ELNs are enriched with natural compounds such as flavonoids, terpenoids, and alkaloids, and exhibit cross-kingdom biological activities in mammalian cells, including anti-inflammatory, antioxidant, anti-tumor, anti-senescent, and neuroprotective effects (4,5).

Talinum fruticosum (L.) Juss., an edible medicinal plant belonging to the family Talinaceae, is distributed across tropical and subtropical regions, particularly in West Africa, South America, and South/Southeast Asia (6-9). Notably, major botanical authorities such as Plants of the World Online and the Flora of North America treat T. fruticosum as a synonym or conspecific of T. triangulare (10,11). Studies on T. triangulare have reported its anti-inflammatory and immunomodulatory effects in animal models, which we cite here as supportive background for the pharmacological relevance of T. fruticosum (12,13). Similarly, T. paniculatum is treated as closely related and/or occasionally listed under the same taxonomic treatment as T. fruticosum in certain plant classification databases, including the Global Biodiversity Information Facility (6,14). In ethnobotanical contexts, T. paniculatum is often confused with T. fruticosum due to their morphological similarity, and both species are traditionally used for comparable medicinal purposes. Although scientific studies on T. paniculatum are limited, emerging evidence indicates its cardioprotective potential in rat models through antioxidant activity and angiotensin-converting enzyme inhibition (15).

Okinawa, Japan, a region globally recognized as a ‘blue zone’ for its high longevity, has a unique culture in which the boundary between food and medicine has traditionally been blurred in daily life (16-19). T. fruticosum (locally known as ‘Shibiran’) is both cultivated and naturalized in Okinawa (6), and has long been recognized as a traditional food-medicine plant. It has been commonly consumed in the Okinawan diet as a leafy vegetable for maintaining general health and traditionally used as a folk remedy for managing ailments such as gastric discomfort, diarrhea, cough, asthma, and inflammatory swelling (20). These uses are consistent with Japanese-language ethnobotanical records that document its role in the food-medicine continuum deeply rooted in Okinawan culture. The Okinawan use of T. fruticosum highlights a form of empirical knowledge that has long informed local health practices, yet remains underrepresented in English-language scientific literature.

While T. fruticosum has long been valued as an edible medicinal plant, the biological basis of its health benefits remains largely unexplored. In this study, as part of our broader ethnopharmacological project focusing on Okinawan medicinal plants, we identified ELNs from T. fruticosum leaves (TfELNs). We hypothesized that TfELNs may contribute to the health-promoting effects of T. fruticosum and aimed to evaluate their potential immunomodulatory and antioxidant activities using a macrophage-based in vitro assay.

Materials and methods

Plant material identification and source information

Fresh aerial parts of Talinum fruticosum (L.) Juss. were obtained from Atarasu market, a local farmers' market located in Hirara-Nishizato, Miyakojima-shi, Okinawa, Japan, on July 1, 2024. This market is known for direct sales of freshly harvested local produce by regional farmers. The plants were harvested on the same day at the Uchima farm, located in Hirara-Shimozato, Miyakojima-shi, Okinawa, Japan.

Plant identification was initially carried out by the authors based on morphological characteristics described in the published literature and authoritative botanical databases (e.g., Plants of the World Online). Key features included leaf shape, flower bud morphology, and the characteristic triple-ridged structure of the rachis. Detailed photographic documentation of the plant is provided in Figs. S1 and S2.

To further authenticate the taxonomic identity of the plant material used for TfELN preparation, DNA barcoding was conducted using two plastid loci, rbcL and matK. DNA extraction, PCR amplification, and Sanger sequencing were outsourced to Fasmac Co., Ltd.. For rbcL, PCR was performed using primers rbcLa-F/rbcLa-R, and a 553-bp sequence excluding primer regions was obtained. For matK, PCR was performed using primers 390F/1326R, and an 852-bp sequence excluding primer regions and adjacent low-quality ends was obtained from a 942-bp amplicon. Primer sequences were obtained from the Barcode of Life Data (BOLD) Systems Primer Database.rbcLa-F: 5'-ATGTCACCACAAACAGAGACTAAAGC-3'; rbcLa-R: 5'-GTAAAATCAAGTCCACCRCG-3' (R=A/G); 390F: 5'-CGATCTATTCATTCAATATTTC-3'; 1326R: 5'-TCTAGCACACGAAAGTCGAAGT-3'.

Sequence similarity searches were performed using BLASTN against the NCBI nucleotide database. The rbcL and matK sequences obtained in this study showed high similarity to publicly available T. fruticosum reference sequences (Figs. S3 and S4). Because plastid barcode loci may show limited interspecific divergence in closely related taxa, the final species identification was made by integrating the DNA barcoding results with the diagnostic morphological characteristics shown in Figs. S1 and S2 (including the three longitudinal ridges on the rachis). Accordingly, the plant material used in this study was treated as T. fruticosum in subsequent experiments.

Preparation of TfELNs

Fresh T. fruticosum plant (Fig. 1A) was obtained from a local farm in Miyako Island, Okinawa Prefecture, Japan. TfELNs were prepared as previously described with slight modifications (21). Leaves were washed, briefly sterilized with 70% ethanol, and homogenized using a kitchen blender (Acasas A-1200; Eco Setsubi Inc.). The homogenate was centrifuged at 16,000 x g for 40 min at 4˚C (Avanti J-26S XP; Beckman Coulter). The supernatant was then centrifuged at 170,000 x g for 90 min at 4˚C (Optima XE-90 Ultracentrifuge; Beckman Coulter), and the pellet was resuspended in phosphate-buffered saline (PBS). Subsequently, 60/45/30/8% sucrose density gradient ultracentrifugation was performed at 155,000 x g for 90 min at 4˚C. Two visible bands at the 8/30% (B1) and 30/45% (B2) interfaces were collected using a 20G syringe needle (TERUMO). The B1 and B2 fractions were diluted with distilled water and centrifuged at 170,000 x g for 90 min at 4˚C. The pellet was resuspended in 1 ml of 30% sucrose solution for cryoprotection. The purified sample was filtered through a 0.22-µm polyethersulfone membrane filter (Merck Millipore) and stored at -80˚C.

Figure 1 Isolation and characterization of TfELNs. (A) Fresh Talinum fruticosum leaves. (B) Purification of TfELNs using sucrose density gradient ultracentrifugation: Upper and lower arrowheads indicate TfELN B1 and B2 bands, respectively. Particle size distributions of purified TfELN (C) B1 and (D) B2, determined by NTA. Representative TEM images at 50,000x magnification are shown as insets in each panel. Scale bar, 100 nm. (E) Cytotoxicity of TfELN B1 and B2 in THP-1 macrophages. Box plots: horizontal line, median; red cross, mean. Brackets with ‘ns’ (not significant) indicate specific comparisons between the control group and each treatment group (P>0.05). Additionally, no statistically significant differences were found between any other pairs in the post hoc analysis (all P>0.05). TfELN, Talinum fruticosum-derived exosome-like nanoparticle; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy.

Characterization of TfELNs

Size distribution and particle concentration of TfELNs were measured using nanoparticle tracking analysis (NTA) (NanoSight NS300; Malvern Panalytical). Protein concentration was determined with the Qubit Protein Assay Kit (Thermo Fisher Scientific, Inc.). The yield was calculated as the number of particles and proteins in TfELNs per weight of fresh T. fruticosum leaf. TfELN morphology was analyzed using transmission electron microscopy (TEM) (JEM-2000EX; JEOL) by the Hanaichi UltraStructure Research Institute (Okazaki).

Preparation of THP-1 macrophages

The THP-1 human monocytic cell line was purchased from KAC Co., Ltd. (EC88081201-FF) and cultured in RPMI-1640 medium (FUJIFILM Wako Pure Chemical) supplemented with 10% fetal bovine serum (MP Biomedicals), 1% GlutaMAX (Thermo Fisher Scientific, Inc.), and 1X Antibiotic-Antimycotic (Thermo Fisher Scientific, Inc.). To induce macrophage differentiation, THP-1 cells were suspended in RPMI-1640 medium containing 160 nM phorbol 12-myristate 13-acetate (Abcam) and seeded at 2x104 cells/well in a 96-well plate (SANPLATEC) (22,23). After 48 h incubation at 37˚C, adherent THP-1 macrophages were washed with PBS and incubated for an additional 24 h before lipopolysaccharide (LPS) stimulation.

Cytotoxicity of TfELNs

THP-1 macrophages were treated with TfELNs (109-1012 particles/ml) in RPMI-1640 medium for 48 h. Assays with 1012 particles/ml of TfELN B1 could not be performed due to its extremely low yield. Cell viability was assessed using the Cell Counting Kit-8 (Dojindo Laboratories).

In vitro cytokine release assay

THP-1 macrophages were pretreated with TfELNs (1011 particles/ml) in RPMI-1640 medium for 24 h and then stimulated with 0.5 mg/ml Salmonella typhimurium-derived LPS (Fujifilm Wako Pure Chemical) for 24 h. For dose-dependency experiments, TfELNs (109-1012 particles/ml) were used. Dexamethasone, a steroidal anti-inflammatory drug, was used as a positive control. Cytokine levels in the culture supernatant were measured using the V-PLEX Pro-inflammatory Panel 1 Human Kit (Meso Scale Discovery) and the MESO QuickPlex SQ 120MM (Meso Scale Discovery).

Intracellular reactive oxygen species (ROS) assay

THP-1 macrophages were pretreated with TfELNs (108-1011 particles/ml) in RPMI-1640 medium for 24 h and then stimulated with 1 mM hydrogen peroxide (H2O2) for 4 h. The intracellular ROS levels were measured using a ROS Assay Kit (Photo-oxidation Resistant DCFH-DA; Dojindo Laboratories) according to the manufacturer's instructions, with a Spark multimode microplate reader (Tecan Japan Co., Ltd.).

Statistical analyses

All statistical analyses were performed using XLSTAT (Lumivero). Outlier detection was not performed; all collected data were included. Normality was tested using the Shapiro-Wilk test. Homogeneity of variances was assessed using either Bartlett's or Brown-Forsythe test, depending on the distribution of the data.

Based on these results, appropriate parametric or non-parametric methods were applied as follows. Data presented in Fig. 1E were analyzed using Welch's ANOVA, followed by the Games-Howell post hoc test. Fig. 2A data were analyzed with the Kruskal-Wallis test, followed by the Conover-Iman test with Bonferroni correction. For Fig. 2B (no LPS stimulation), the Kruskal-Wallis test was used, followed by Dunn-Bonferroni post hoc test. For Fig. 2B (LPS stimulation), Welch's ANOVA was performed. While the Games-Howell test served as the primary analysis for robust, exploratory all-pairs comparisons, Dunnett's test was specifically employed for comparisons against the control group to provide optimized statistical power for these primary reference points. Group comparisons for Fig. 3A were conducted using the Kruskal-Wallis test, with Dunn-Bonferroni post hoc test. For Fig. 3B, Welch's ANOVA was applied, followed by the Games-Howell post hoc test. Statistical significance was set at P<0.05.

Figure 2 Selective inhibition of IL-6 production by TfELNs. (A) Levels of representative pro-inflammatory cytokines in LPS-stimulated THP-1 macrophages, with or without pretreatment (1011 particles/ml) of TfELN B1 or B2. Only IL-6 was significantly suppressed. (B) Dose-response of IL-6 inhibition by TfELN B2. Box plots: horizontal line, median; red cross, mean. To avoid visual clutter, brackets are only shown for significant differences or comparisons with the control group; all other pairwise comparisons were non-significant (P>0.05). Statistical significance is indicated as follows: *P<0.05; **P<0.01; ***P<0.001; ns, not significant. TfELN, Talinum fruticosum-derived exosome-like nanoparticle; IL, interleukin; LPS, lipopolysaccharide.

Figure 3 Differential antioxidant activity of TfELN subsets. Intracellular ROS levels in H2O2-treated THP-1 macrophages were measured using DCFH-DA fluorescence intensity, and values are expressed as RFU. (A) Pretreatment with B1 significantly reduced ROS levels, (B) whereas pretreatment with B2 had no detectable antioxidant effect. Box plots: horizontal line, median; red cross, mean. To avoid visual clutter, brackets are only shown for significant differences or comparisons with the control group; all other pairwise comparisons were non-significant (P>0.05). Statistical significance is indicated as follows: *P<0.05; **P<0.01; ***P<0.001; ns, not significant. TfELN, Talinum fruticosum-derived exosome-like nanoparticle; ROS, reactive oxygen species; RFU, relative fluorescence units; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate.

Results

Isolation and characterization of TfELNs

We successfully isolated two distinct fractions of TfELNs, referred to as TfELN B1 and B2, from T. fruticosum leaves (Fig. 1B). The average yields per gram of fresh T. fruticosum leaves were 2.94x109 particles (1.36 µg of protein) for TfELN B1 and 5.26x1010 particles (12.4 µg of protein) for TfELN B2. NTA revealed that the size distribution of TfELN B1 ranged from 117.8 to 212.6 nm (D10-D90), with mean and mode particle sizes of 162.9 nm and 131.5 nm, respectively (Fig. 1C). TfELN B2 had a slightly smaller particle size distribution, ranging from 78.5 to 185.1 nm (D10-D90), with a mean of 128.8 nm and a mode of 96.1 nm (Fig. 1D). TEM analysis confirmed that both TfELN B1 and B2 exhibited spherical vesicle morphology typical of exosomes, with diameters ranging from approximately 50 to 110 nm (insets in Fig. 1C and D). No notable morphological differences were observed between the two fractions.

TfELNs exhibit subset-specific anti-inflammatory effects

We first evaluated the cytotoxicity of TfELNs on THP-1-derived macrophages. Treatment with various concentrations of TfELN B1 (109-1011 particles/ml) and B2 (109-1012 particles/ml) did not significantly affect cell viability (Fig. 1E), indicating the high biocompatibility of TfELNs. Due to the low yield of B1, 1011 particles/ml was used as the maximum feasible dose for subsequent assays.

Next, the anti-inflammatory potential of TfELNs was assessed using LPS-stimulated THP-1 macrophages. LPS stimulation markedly increased the production of inflammatory cytokines compared with the unstimulated control (Fig. 2A). Among the tested cytokines, interleukin-6 (IL-6) production was significantly reduced by pretreatment with TfELN B2 (53.5% inhibition), whereas TfELN B1 induced a modest but non-significant reduction (34.6% inhibition). Neither fraction significantly affected the production of other pro-inflammatory cytokines. Dose-dependent suppression of IL-6 production was observed upon treatment with increasing concentrations of TfELN B2 (109-1012 particles/ml) (Fig. 2B). IL-6 inhibition was statistically significant only at 1011 and 1012 particles/ml. These findings suggest that TfELNs, particularly the B2 subset, exert anti-inflammatory effects through selective suppression of IL-6 production in activated macrophages.

TfELNs exhibit subset-specific antioxidant effects

We next examined the antioxidant effects of TfELNs. THP-1 macrophages were pretreated with TfELNs (108-1011 particles/ml) and then exposed to H2O2 to induce oxidative stress, with intracellular ROS levels quantified using DCFH-DA fluorescence. Pretreatment with B1 resulted in a significant reduction in ROS levels compared with the control at particle concentrations of 109-1011 (Fig. 3A). In contrast, B2 did not show any significant effect on ROS levels across the tested particle concentrations (Fig. 3B), demonstrating the subset-specific antioxidant effects of TfELNs.

Discussion

In this study, we identified ELNs derived from T. fruticosum leaves and demonstrated their selective inhibitory effect on IL-6 production in an LPS-induced in vitro inflammation model. TfELNs suppressed IL-6 secretion in a dose-dependent manner without affecting other cytokines or inducing cytotoxicity, indicating a specific immunomodulatory effect rather than a general anti-inflammatory or cytotoxic response. IL-6 is a key mediator of inflammation and contributes to the pathogenesis of several autoimmune diseases (24), including rheumatoid arthritis (25), systemic lupus erythematosus (26), and Castleman disease (27). Its excessive production is also associated with severe systemic inflammation, such as cytokine storms in COVID-19 and sepsis (28,29), and supports the growth and survival of multiple myeloma and ovarian endometriosis cells (30,31). Although pharmacological inhibitors for IL-6 signaling are clinically available (32), their use is limited by high cost and the potential for adverse effects with long-term treatment (33). In contrast, TfELNs are derived from edible plant material using simple preparation methods, offering a potentially safer and more accessible approach for modulating IL-6-driven inflammation.

Another intriguing finding of this study is the functional divergence between the two TfELN subsets: B1 exhibited significant antioxidant activity without detectable anti-inflammatory activity, whereas B2 displayed anti-inflammatory activity without detectable antioxidant effects. This differential activity indicates that TfELN functions are subset-specific and may reflect differences in their molecular cargo, such as distinct profiles of proteins, lipids, or small RNAs (34,35). Such cargo differences could influence recipient macrophages following cellular uptake, for example by modulating inflammatory signaling and oxidative stress-response pathways. In other edible plant-derived ELNs/EVs, reported cargos commonly include small RNAs (e.g., miRNAs), membrane lipids enriched in phospholipids such as phosphatidic acid and phosphatidylcholine, and a limited set of proteins related to stress responses and metabolism (4,5,34,35). In addition, some plant ELNs have been reported to carry plant-derived bioactive small molecules (e.g., flavonoids or isothiocyanates) depending on the source species, which could in principle contribute to anti-inflammatory and/or antioxidant phenotypes (4,5,34,35). Although cargo composition was not assessed here, fraction-dependent differences may underlie the divergent effects of B1 and B2, with potential implications for tailoring TfELN subsets to specific biological contexts. The selective antioxidant effect of B1 could be particularly relevant for mitigating oxidative stress-related conditions, while the anti-inflammatory properties of B2 may play a key role in modulating IL-6-driven immune responses. This functional specialization suggests that isolating or enriching specific EV subsets could enhance therapeutic efficacy for targeted applications (4,36). Further studies are warranted to identify the molecular determinants underlying these subset-specific activities and to explore their potential in in vivo applications.

Plant-derived ELNs with anti-inflammatory activity have been reported from multiple edible and medicinal plants. For example, ELNs isolated from Solanum nigrum berries were shown to attenuate IL-6-related inflammatory responses in LPS-stimulated RAW264.7 macrophages (37), supporting the feasibility of IL-6 modulation by plant EV preparations. In our study, TfELN B2 selectively reduced LPS-induced IL-6 secretion in THP-1 macrophages, whereas other measured pro-inflammatory cytokines were not significantly altered under the same conditions. This profile is consistent with a targeted immunomodulatory effect rather than broad cytokine suppression. Notably, dose reporting varies across the literature (e.g., protein-based µg/ml vs. particle-based particles/ml), and such differences, together with variation in plant source material and isolation workflows, may contribute to apparent differences in effective dose ranges between studies.

The discovery of TfELNs sheds light on why T. fruticosum has long been valued in Okinawa as both food and medicine. The presence of ELNs with specific biological activity provides a molecular rationale for its traditional use and supports the concept that there are ‘no clear boundaries between food and medicine’ (16-19). Moreover, this finding may have broader relevance, as T. fruticosum and similar edible medicinal plants are traditionally used in diverse cultures worldwide (7-9). By elucidating the functional role of ELNs, our results offer a new perspective for interpreting traditional plant-based therapies within a modern biomedical framework.

This study has several limitations. The specific molecular cargos responsible for IL-6 suppression and ROS reduction remain unidentified, and comprehensive multi-omics profiling will be required to elucidate their mechanisms. We also note that dedicated stability testing of TfELNs under storage or physiological conditions was not performed in this study and will be important for future translational applications. Moreover, although marker-based characterization is commonly applied to mammalian exosomes, universally accepted marker panels for plant-derived ELNs have not yet been established, which currently limits standardized comparisons across studies (38,39). In addition, other potentially relevant biological activities, such as anti-senescent or neuroprotective effects, were not investigated in the present study. The current work was designed as an initial, subset-focused in vitro evaluation of selective IL-6 suppression and ROS reduction; broader profiling across additional endpoints and/or cell types will be an important direction for future studies. In vivo studies will also be critical to establish the therapeutic relevance and safety of TfELNs under physiological conditions. Future research should further explore how TfELNs contribute to the health-promoting properties of T. fruticosum, particularly within the framework of Okinawa's longevity culture and its broader ethnopharmacological significance.

In conclusion, this study demonstrates that exosome-like nanoparticles from T. fruticosum leaves exhibit subset-specific activities: B2 selectively inhibits IL-6 production, while B1 exerts significant antioxidant effects in vitro. These findings provide a scientific basis for its ethnopharmacological use in Okinawan practices and highlight the broader potential of similar edible medicinal plants worldwide.

Supplementary Material

Morphological features of the plant material (leaf, stem and bud) used for TfELN preparation. (A) Leaves and flower buds attached to stems. (B) Magnified view of the bud area in panel A. (C) Close-up of a stem. (D) Close-up of a flower bud. (E) Close-up of a leaf (adaxial surface). (F) Close-up of a leaf (abaxial surface). TfELN, Talinum fruticosum-derived exosome-like nanoparticle.

Morphological features of the plant material (flower) used for TfELN preparation. (A) A nearly blooming flower. White arrowheads indicate the characteristic longitudinal ridges of the rachis. (B) Close-up of a nearly blooming flower with separated sepals. (C) Close-up of the inner structure of a nearly blooming flower. TfELN, Talinum fruticosum-derived exosome-like nanoparticle.

DNA barcoding of the rbcL locus (553 bp). (A) Screenshot of the top 10 BLASTN hits (Descriptions table) for the rbcL sequence, shown in the default BLASTN order (sorted by E-value) against the NCBI nucleotide database. (B) Screenshot of a representative pairwise alignment between the query rbcL sequence and a reference Talinum fruticosum sequence (top T. fruticosum hit), illustrating the sequence similarity across the aligned region. Note: The rbcL barcode region can be highly conserved among closely related Talinum species; therefore, rbcL barcoding is presented as supportive molecular evidence together with the matK results (Fig. S4) and diagnostic morphological characteristics (Figs. S1 and S2) for species identification. rbcL, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene; bp, base pairs; BLASTN, Basic Local Alignment Search Tool for nucleotides; NCBI, National Center for Biotechnology Information; matK, maturase K gene.

DNA barcoding of the matK locus (852 bp). (A) Screenshot of the top 10 BLASTN hits (Descriptions table) for the matK sequence, shown in the default BLASTN order (sorted by E-value) against the NCBI nucleotide database. (B) Screenshot of a representative pairwise alignment between the query matK sequence and a reference Talinum fruticosum sequence (top T. fruticosum hit), illustrating the sequence similarity across the aligned region. Note: A hit showing 100% identity with 94% query coverage reflects complete identity within the aligned region, with the remaining region not covered by the query sequence (i.e., no overlapping bases available for comparison). Species identification was determined based on the combined evidence from DNA barcoding (Figs. S3 and S4) and morphological characteristics (Figs. S1 and S2). matK, maturase K gene; bp, base pairs; BLASTN, Basic Local Alignment Search Tool for nucleotides; NCBI, National Center for Biotechnology Information.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

YK conceptualized the study, acquired funding, provided key study materials and resources, supervised the work, and reviewed and edited the manuscript. KN curated and managed the data, performed the formal/statistical analyses, conducted the investigations, developed the methodology, administered the project, validated the results, prepared the figures and tables, drafted the original manuscript, and reviewed and edited the manuscript. YA performed the formal/statistical analyses, conducted the investigations, developed the methodology, validated the results, and reviewed and edited the manuscript. TE conceptualized the study, curated and managed the data, administered the project, and reviewed and edited the manuscript. KA conceptualized the study, acquired funding, supervised the work, and reviewed and edited the manuscript. TN conceptualized the study, performed the formal/statistical analyses, supervised the work, validated the results, prepared the figures and tables, drafted the original manuscript, and reviewed and edited the manuscript. KN and TN confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

Yasunari Kageyama is the chief executive officer of IDO Medical Co., Ltd. (Tokyo, Japan) and owns shares of the company. Kazunori Namba, Yuki Akamatsu, and Tomoka Ebisui are employees of IDO Medical Co., Ltd. Koichi Aida is the representative director of Takanawa Clinic (Tokyo, Japan). Tsutomu Nakamura is the director of IDO Medical Co., Ltd. and the scientific adviser of Takanawa Clinic.

References