The Research Reports No.30 （issued in 2024）

Abstracts of Research Reports funded by the Foundation's Research Grants in FY2023.

30－01

Safety evaluation of food additives by multilayered omics analysis with Caenorhabditis elegans

Yuko Sakaguchi
College of Pharmaceutical Sciences, Ritsumeikan University

　 The nematode Caenorhabditis elegans (C. elegans) has a high percentage of homologous genes to humans based on its whole genome sequence and is used as a model for toxicity assessment in mammals. We aimed to realize multilayered omics analysis of C. elegans for safety assessment of food additives by integrating RNA-sequencing and non-targeted metabolomics into bioassays using C. elegans. In this study, neonicotinoid pesticides (acetamiprid; ACE and clothianidin; CTD) were selected as target substances; ACE and CTD were exposed to L1 larvae, and their RNA was analyzed by next-generation sequencing. For nontargeted metabolomics, larval metabolites were analyzed by LC-Q-TOF/MS. The results showed that the number of genes and metabolites that varied significantly was higher in ACE compared to CTD, raising concerns about the toxicity of ACE. This was also the case in the bioassay. Furthermore, glutathione-related genes and metabolites were significantly up-regulated, suggesting the development of neonicotinoid toxicity due to oxidative stress. The use of C. elegans was shown to enable comprehensive toxicity evaluation from multiple perspectives.

30－02

New perspective on food additive "lactate" Exploring the preventive effects of oral intake of lactate combined with exercise on cognitive decline

Takanori Tsuda
College of Bioscience and Biotechnology, Chubu University

　Exercise has various benefits for the entire body. For example, exercise is an effective strategy for body weight control because it elevates energy expenditure and improves overall glucose and lipid homeostasis as well as insulin sensitivity. Among them, it is well-known that exercise can prevent cognitive decline. Lactate is not a cause of fatigue, but also is thought to be a signaling molecule. Furthermore, it has been reported that exercise induced elevation of blood lactate concentration results in improvement of cognitive function. Although lactate is daily ingested from various foods, there have been no studies on the physiological function of lactate intake as a food additive. Accordingly, the purpose of this study was to verify the effect of oral intake of lactate on preventing cognitive decline, or the amplification of the beneficial effect by combination with exercise. To achieve this objective, experimental animals and exercise methods were investigated. As a result, the use of voluntary exercise and ICR mouse is appropriate for studies of combination with lactate and exercise. Furthermore, we verified the effect of oral intake of lactate on cognitive function, and found that it significantly improved cognitive function. Based on the results, we were able to set the conditions for the next study.　　　

30－03

Development of semi-automated analytical method for determination of polar pesticide residues in foods

Hiroshi Akiyama
Department of Analytical Chemistry, School of Pharmacy and Pharmaceutical Sciences, Hoshi University

　We developed solid-phase analytical derivatization with LC-MS/MS to determine glyphosate, glufosinate and their related metabolites in soybeans. The sample was extracted with water. An aliquot of the supernatant was deproteinized by addition of acetonitrile. The sample extract was applied to Presh-SPE AXs (3 mg) and dehydrated with acetonitrile. The trapped compounds were derivatized using N-(tert-butyl dimethylsilyl)-N-methyltriuoroacetamide at ambient temperature for 1 min and eluted with acetonitrile. The eluate was then injected to LC-MS/MS. The coefficients of determination of the calibration curve indicated good results. The validation studies showed good recovery (97-108%) and precision (<9%). Glyphosate, glufosinate, N-acetyl glyphosate, N-acetyl glufosinate and MPPA were not detected in food soybeans from domestic sources or other countries. However, analysis of feed soybeans and processed soybean meal for feed use detected glyphosate, glufosinate and N-acetyl glufosinate. This method is rapid, simple and reliable for monitoring distributed soybean.

30－04

Analysis of change in physical forms of silver nanoparticle after oral exposure

Kazuya Nagano
School of Pharmaceutical Sciences, Wakayama Medical University

　It is reported that the physical forms of nanoparticles are changed, such as aggregation, ionization and reconstitution. However, these changes in physical forms of nanoparticles in vivo have not been sufficiently clarified. In this study, we tried to analyze the changes in physical forms of silver nanoparticles by using our single-particle ICP-MS method for biological samples.
　When silver nanoparticles were orally administrated, most of the silver detected in the blood was silver ion, suggesting that silver nanoparticles were ionized during gastrointestinal absorption. Thus, the analysis of change in physical forms of silver nanoparticles after absorption in vitro showed that the silver nanoparticles were hardly ionised in the collected blood. On the other hand, the analysis of change in physical forms of silver nanoparticles during the absorption process in vitro showed that the silver nanoparticles were ntracellularly ionised. The findings suggested that the absorption process may contribute to the ionization of orally exposed silver nanoparticles.

30－05

Analysis of the Mechanism of Color Change in Black Tea Due to pH Variation

Yosuke Matsuo
Graduate School of Biomedical Sciences, Nagasaki University

　Black tea is a widely consumed beverage worldwide. During its production, tea catechins undergo enzymatic oxidation, generating various oxidation products, including pigments characteristic of black tea. Lemon tea, made by adding lemon to black tea, exhibits a color change from red to light orange due to pH reduction caused by citric acid in lemon. To elucidate the chemical mechanism of this color change, an analysis of key pigments was conducted.
　HPLC analysis with PDA detection of commercial black tea extracts revealed theaflavins and tricetinidin as major peaks at 490 nm, suggesting their significant contribution to black tea's color. UV/vis spectra of theaflavin and tricetinidin were measured in buffer solutions of various pH levels. Both compounds showed changes in their visible light region spectra with pH alteration. These results indicate that theaflavins and tricetinidin contribute to the color change of black tea associated with pH variation.

30－06

Development of an analytical method for determination of furan and its derivatives in processed foods using solid-phase microextraction

Tomoaki Tsutsumi
Division of Foods, National Institute of Health Sciences

　We developed an analytical method for four furans (furan, 2-methylfuran, 3-methylfuran and 2,5-dimethylfuran) in processed foods using headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME/GC-MS). Here we evaluated the analytical method for determining the concentrations of the four furans in five processed foods (infant formula, fermented soybean paste, rice cracker, coffee, soy sauce). The trueness and precision of the method were assessed by performing replicate analyses of the five processed foods spiked with the four furans at two different concentration levels. The four furans found were 76%-103% of the spiked concentrations, with intra-day precisions below 6.8% and inter-day precisions below 12%. Two quality control coffee samples were analyzed by the method, and the concentrations of the four furans were within acceptable limits. Finally, the developed method was used to determine concentrations of the four furans in the five processed foods (30 samples in total) on the Japanese market. Although chromatographic interference was observed in the vicinity of furan-d₄ in fermented soybean paste samples, the selectivity of the method was generally satisfied. Overall, these results suggested the present method is useful for analysis of the four furans in processed foods.

30－07

Development of an Efficient Enzymatic Supply Method for Imino Sugars from Mulberry Leaves as Novel Healthy Sweeteners with Anti-Hyperglycemic Effects

Soo Takasu
Gifu Pharmaceutical University

　Iminosugars, sugar analogues with anti-hyperglycemic properties, are promising novel sweeteners. Iminosugars attenuate postprandial glucose spikes and, when combined with traditional sweeteners such as sucrose, provide potential for diabetes prevention. Mulberry leaves, which are rich in iminosugars, are a primary natural source, although their content varies with species and growing conditions, requiring a stable method of supply for food applications. Previous research has shown that enzymatic hydrolysis of iminosugar glycosides increases the bioavailability and bioactivity within mulberry leaves, thereby facilitating iminosugar utilization. This study aimed to develop enzymatic treatments to increase the utility of mulberry leaves. Initially, I focused on establishing an analytical method for the quantification of iminosugars in mulberry leaves. While various iminosugars were successfully detected, further refinement is required to achieve accurate quantification. In addition, an isomer analysis method using ion mobility spectrometry was developed. Subsequently, food grade enzymes were used to hydrolyze specific glycosides in mulberry leaves and partial degradation was achieved. I am planning to evaluate the increase in iminosugar content in mulberry leaves by enzymatic degradation.

30－08

Construction of an evaluation system for the safety and efficacy of food additives using embryo-environmentally manipulated mice and non-digestible oligosaccharides

Shiori Ishiyama
Faculty of Life and Environmental Sciences, Graduate Faculty of Interdisciplinary Research, University of Yamanashi

　Objective: Fructooligosaccharide is currently the most widely used non-digestible oligosaccharide in Japan. However, little is known about the effects of fructooligosaccharide in animal models with organ damage, and its safety and efficacy in humans with organ damage is unknown. In this study, we investigate whether fructooligosaccharide is safe and effective (suppression of organ damage) in animal models with diabetic kidney disease as organ damage. Methods: 5-Week-old NSY mice with spontaneous type 2 diabetes mellitus were fed a high-fat, high-sucrose diet and a high-fat, high-sucrose diet with 5% fructooligosaccharide, respectively, for 23 weeks, and pathological evaluation in the kidney and intestine was conducted. Results and discussion: Feeding type 2 diabetic NSY mice the diet including fructooligosaccharide suppressed an increase in the mesangial area in the kidney, and in the number of goblet cells, which contribute to the intestinal barrier by secreting mucins, in the jejunum, ileum, and cecum. In particular, the inverse correlation between the renal mesangial area and the number of intestinal goblet cells was strongest in the ileum, in which intestinal bacteria were less than in the large intestine (cecum and colon). The expression of a dendritic cell surface protein gene (Cd11) was reduced in mesenteric adipose tissues, which includes lymph nodes, around the small intestine, by the fructooligosaccharide. Fructooligosaccharide may uppress diabetic kidney disease by increasing the barrier functions in the small intestine and repressing the activation of immune cells such as dendritic cells. Conclusion: Fructooligosaccharide could inhibit diabetic kidney disease by enhancing the barrier functions independent of intestinal bacteria.

30－09

Functional metabolites of tannins listed as existing food additives

Hideyuki Ito
Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University

　Polyphenols are found widely in nature, such as in foods and medicinal plants, and have been shown to have various biological activities including antioxidant and anti-inflammatory effects. Bioavailability of hydrolyzable tannins including ellagitannins and allotannins has been studied in recent years, and various biological activities as antiglycation and anti-aging effects of metabolites such as urolithins have also been reported. However, there are many unclear points about bioavailability such as absorption, distribution, metabolism, and excretion after hydrolyzable tannins ingestion. An appropriate intake of functional foods can be suggested to establish by understanding in vivo behavior, but there are few reports on bioavailability after ingesting polyphenol-rich sources. In this study, we investigated the in vivo behavior of hydrolyzable tannins and the related metabolites after administration of polyphenol-rich Trapa bispinosa pericarp extract (TBPE) listed as existing food additives to rats.
　The collected plasma and urine samples after oral administration of TBPE to rats were deconjugated and then extracted with ethyl acetate to prepare samples for quantitative analysis. Eighteen metabolites, including 12 urolithins and ellagitannin related metabolites, and six gallotannin related metabolites were quantified by HPLC-ESI-MS/MS. Although ellagitannin metabolites take time to be absorbed and excreted, gallotannin metabolites are shown to be rapidly absorbed and excreted in the body, indicating to clarify that each type of metabolite exhibits different in vivo behavior. The urinary excretion rates of ellagitannin and gallotannin related metabolites were calculated to be approximately 68% and 10%, respectively, suggesting that ellagitannins showed higher bioavailability than gallotannins after administration of TBPE to rats. These results provide basic data on the pharmacokinetics of hydrolyzable tannins contained in TBPE, which is expected to lead to the demonstration of the functionality of hydrolyzable tannins as well as Trapa ingredients.

30－10

Analysis of pharmacokinetics and mechanisms of action of phenethylamine, a food flavor ingredient

Yusuke Hirata
Graduate School of Pharmaceutical Sciences, Tohoku University

　Phenethylamine, a monoamine alkaloid, is an aroma component found in various foods, including cheese, wine, cabbage, processed fish products, and beer. It is also used as a food additive in products like confectionery, meat products, frozen dairy items, and soft drinks to enhance their aroma. However, its biological effects and efficacy are not well understood. This study aims to elucidate the mechanisms of biological actions of phenethylamine and its derivatives. Detailed analysis revealed that phenethylamine potently inhibits non-apoptotic regulatory cell death at low concentrations (in the nanomolar range). While phenethylamine metabolites exhibited antioxidant activity, phenethylamine itself did not. Interestingly, the compound significantly inhibited the cation influx and efflux (potassium and sodium ions) into and out of cells just before cells undergo cell death. These findings suggest that phenethylamine may exert beneficial effects, such as hepatoprotection, by suppressing regulated cell death through the inhibition of cation channels on the plasma membrane.

30－11

Development of a method of liquid chromatography coupled with tandem mass spectrometry for simultaneous determination of hyaluronan oligosaccharide

Yuki Sato
Faculty of Pharmaceutical Sciences, Hokkaido University

　Hyaluronan (HA) is a repeating disaccharide of glucuronic acid and N-acetylglucosamine and has recently been used in foods and food additives, as well as in medicines and cosmetics. In this study, we focused on low-molecular-weight HA oligosaccharides and aimed to establish a method for their simultaneous determination. First, carboxy groups of HA oligosaccharides were derivatized with a condensing reagent (DMTMM) and Girard's reagent P. After pretreatment by solid-phase extraction using hydrophilic interaction, peaks of HA 2, 4, 6 and 8-mer were detected by LC/MS/MS. Validation studies were conducted for this method. Additionally, plasma concentrations of HA were determined after oral administration of the HA formulation to rats. We confirmed by MS scan that the derivatized HA oligosaccharides could be analyzed by the developed quantitative method and identified the peak of derivatized HA oligosaccharides. The MS/MS conditions were optimized, and a calibration curve was generated, which showed good linearity for HA 4, 6 and 8-mer. This method enabled measurement of plasma concentrations of HA 4, 6 and 8-mer after oral administration of HA formulations. All points in the validation study were within the specified range, indicating that this method is valid for simultaneous determination of HA 4, 6 and 8-mer.

30－12

Evaluation of the effect of food additives on the differentiation and fusion of trophoblast cells essential for placentation using a dual-split biosensor

Mikihiro Yoshie
Department of Endocrine Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences

　The placenta, which acts as an interface between fetal and maternal circulations, is an indispensable organ for fetal growth in mammalian pregnancy. It mediates the transportation of nutrient, the exchange of gases such as oxygen and carbon dioxide, and the excretion of waste products between the fetus and mother. The surface of placental villi is covered by two layers of mononuclear undifferentiated cytotrophoblasts and their fused multinucleated syncytiotrophoblasts. Syncytiotrophoblast function not only as the placental barrier to separate maternal blood from fetal tissue but also as the main source of human chorionic gonadotropin (hCG) and progesterone (P4) during pregnancy. The safety of food additives on pregnancy has been ensured by teratogenicity evaluations in experimental animals, but there are few reports on the effects of food additives on placentation. In this study, we screened and evaluated the effects of food additives by creating a novel biosensor assay system that can quantitatively assess the fusion of trophoblast cells. We found that several food additives affect trophoblast cell fusion and that butyric acid, a short-chain fatty acid, promotes trophoblast cell differentiation and fusion and contributes to the formation and maintenance of placental villi.

30－13

Bioregulatory Function of An Inclusion Complex of Myricitrin-rich Red Bayberry (Myrica rubra) and γ-Cyclodextrin Considering Pharmacokinetics

Hitoshi Ashida
Faculty of Food Science and Nutrition, Mukogawa Women's University

　Myricitrin (Myr) is a flavonol glycoside which prevents hyperglycemia. However, its functionality is low due to its low water solubility and poor stability. In this study, water-soluble myricitrin (W-Myr), which is an inclusion complex of Myr and γ-cyclodextrin, was used to enhance stability and water solubility. We investigated that comparison of pharmacokinetics between Myr and W-Myr in mice, then the anti-hyperglycemic effect of W-Myr. The amounts of Myr and myricetin (Mce), an aglycone of Myr, were quantified by HPLC in serum and issues of mice. Our results demonstrated that the amounts of Myr and Mce in the ileal and cecal epithelium of W-Myr-dosed mice were higher than those in Myr-given ones. Oral administration of these compounds suppressed postprandial hyperglycemia, and W-Myr showed higher effect than Myr. The suppression of postprandial hyperglycemia was canceled by pre-administration of a GLP-1 receptor antagonist, and W-Myr increased blood GLP-1 and insulin levels, indicating that W-Myr has effect on hyperglycemia by promoting secretion of GLP-1. In cultured cell studies, Myr promoted the phosphorylation of cAMP response element-binding protein. But Myr didn't change intracellular Ca²⁺ response and cAMP levels.

30－14

Ascorbic acid protects glycocalyx to regulate heparanase 1 in brain vascular endothelial cells

Masahiro Komeno
Tokyo University of Science

　We investigated the function of ascorbic acid (AsA) and its derivatives to inhibit the degradation of glycocalyx on the surface of vascular endothelial cells. First, we investigated the inhibitory effect on the expression of heparanase 1 (HPSE1), which is responsible for the degradation of the vascular endothelial glycocalyx. The induced HPSE1 gene expression level was measured in a reporter assay to use a plasmid composed of the human HPSE1 gene promoter region with luciferase gene transfected into EA.hy926 cells;a human vascular endothelial cell line. AsA significantly suppressed the promoter activity of the HPSE1 gene, however AsA-glucoside and -palmitate did not alter the promoter activity of the HPSE1 gene. Next, we examined the effect of AsA on cerebral infarction. We administered AsA to mice after inducing cerebral infarction. Then, cerebral infarct area was decreased in AsA treated group, and decrease TNF-α production in LPS stimulated macrophage cell line. Therefore, it was suggested that AsA may improve the prognosis of cerebral infarction by suppressing the inflammatory response of macrophages.