Ayaka Nakashima

Background: Dietary approaches to support immune function have emphasized the importance of dietary fiber, including non-digestible polysaccharides, although intake remains below recommended levels in many populations. In this context, microalgae have attracted attention as sustainable sources of these components. Euglena gracilis , a microalga used in foods, contains paramylon, a β-1,3-glucan with reported immunomodulatory properties; however, its effects on plasmacytoid dendritic cell (pDC) activation in humans within a dietary context remain incompletely understood. Methods: This randomized, double-blind, placebo-controlled trial investigated the effects of 8-week E. gracilis intake (1,000 mg/day) on immune function and common cold symptoms in healthy Japanese adults. A total of 200 participants were randomized, of whom 181 (91 in the Euglena group and 90 in the placebo group) were included in the per-protocol analysis. The primary endpoint was the change in the log-transformed median fluorescence intensity (MFI) of CD86 on pDCs at Week 8. Results: Analysis using a linear mixed model revealed that the mean ± standard deviation of the log-transformed CD86 MFI at Week 8 was higher in the Euglena group than in the placebo group (placebo: 7.0 ± 2.1; Euglena : 7.7 ± 0.4; group difference 0.7; 95% confidence interval 0.2–1.1; P = 0.004). As exploratory secondary outcomes, the circulating monocyte percentage was higher and the incidence of common cold symptoms—including chills, fatigue, sneezing, rhinorrhea, nasal congestion, sore throat, cough, and headache—was lower in the Euglena group than in the placebo group. Conclusions: These findings suggest that daily consumption of E. gracilis may support immune function and be associated with a reduced incidence of common cold-like symptoms. The intervention was well tolerated, with no adverse events reported and no clinically meaningful changes in hematological or urinalysis parameters. These findings support the potential of E. gracilis consumption as a viable dietary strategy. Trial registration: UMIN Clinical Trials Registry, UMIN000056125.

The immune system functions to eliminate pathogens, such as viruses, and regulate physiological responses and pathways. General immunity is affected by various factors, including fatigue, stress, and aging. A weakened immune system reduces the ability of the body to eliminate pathogens, thereby increasing the risk of infections, including the common cold. Euglena gracilis is a microalga rich in various nutrients, including β‐1,3‐glucan paramylon, which aids in maintaining and regulating immune function. In the present study, we aimed to investigate the effects of E. gracilis administration on cold symptoms in healthy individuals. Euglena gracilis was administered orally in capsule form (once daily, in the morning) continuously for 8 weeks to healthy adult Japanese men and women. Evaluation of the effects of E. gracilis administration on alleviating cold symptoms revealed significantly fewer cumulative days with cold‐like symptoms over 8 weeks in the E. gracilis group than those in the placebo group. Participants in the E. gracilis group also demonstrated significantly fewer severe cold‐like symptoms, such as nasal congestion, sore throat, fatigue, and myalgia, than the placebo group in the second half of the trial (weeks 5–8). In conclusion, this study demonstrated that the continuous intake of E. gracilis could suppress cold‐like symptoms. Moreover, the consumption of the test food under the conditions used in this study was deemed safe.

Mycotoxins are synthesized by various fungal species and are known to exert toxic effects on vertebrates and other animals, even at low concentrations. However, the current countermeasure for mycotoxin contamination is random inspection of samples prior to shipment. In this study, we focused on Chlorella sorokiniana (CS) from Ishigaki Island, Japan, and examined its ability to detoxify deoxynivalenol (DON), ochratoxin A (OTA), and fumonisin B1 (FB1) in vitro and in vivo. The binding of CS to DON, OTA, and FB1 was evaluated in vitro. The detoxification of CS was demonstrated by monitoring its concentrations in the plasma and urine samples of male ICR mice. Plasma and urine samples were collected 30 min, 2 h, and 24 h after an oral administration of 5 mg/kg mycotoxins and/or 500 mg/kg CS. CS bound to more than 80% and 40% of DON and OTA, respectively, whereas the binding of CS to FB1 was less than 10%. The concentrations of DON and OTA in plasma and urine samples were substantially reduced by CS co-administration, whereas CS did not affect FB1 absorption. The co-administration of CS substantially inhibited the systemic absorption of DON and OTA.

Neuronal cell death is a key mechanism involved in the development and exacerbation of Parkinson’s disease (PD). The excessive production of reactive oxygen species (ROS) is a major cause leading to neuronal death; therefore, compounds that prevent oxidative stress-dependent neuronal death may be promising as a preventive method for PD. Ergothioneine is a natural amino acid with antioxidant properties, and its protective functions in the body are attracting attention. However, there has been no investigation into the protective functions of ergothioneine using in vivo and in vitro PD models. Thus, in this study, we analyzed the efficacy of ergothioneine against 6-hydroxydopamine (6-OHDA)-dependent neuronal cell death using immortalized hypothalamic neurons (GT1-7 cells). First, we found that ergothioneine prevents 6-OHDA-dependent neuronal cell death by suppressing ROS overproduction in GT1-7 cells. The cytoprotective effect of ergothioneine was partially abolished by verapamil, an inhibitor of OCTN1, which is involved in ergothioneine uptake. Furthermore, ergothioneine-rich Rice-koji (Ergo-koji) showed cytoprotective and antioxidant effects similar to those of ergothioneine. Taken together, these results suggest that ergothioneine or foods containing ergothioneine may be an effective method for preventing the development and progression of PD.

Wild watermelon contains various nutrients, but the effect of its acute ingestion on arterial stiffness is unclear. This study aimed to investigate whether a single bout of acute ingestion of wild watermelon-extracted juice decreased arterial stiffness concomitant with an increase in nitric oxide (NO) production. Twelve healthy young female participants were tested under two conditions in a randomized, double-blind crossover study: (1) a beverage containing 90 g of wild watermelon extract and (2) a control beverage: a placebo. Pulse wave velocity (PWV), an index of arterial stiffness, blood flow, and plasma nitrate/nitrite (NOx) levels were measured in the supine position at 30, 60, and 90 min after the intake of each beverage. The changes in femoral-ankle PWV were significantly reduced after wild watermelon-extracted juice intake compared to those in the placebo group. Additionally, the changes in blood flow in the posterior tibial artery and plasma NOx levels after intake of wild watermelon-extracted juice were significantly increased compared to those in the placebo group. These data show that acute ingestion of wild watermelon-extracted juice reduces peripheral (lower limb) arterial stiffness and increases NO bioavailability. To confirm these associations, more detailed investigations of the nutrients that influence these effects should be conducted.

A water extract derived from the isolated cell walls of Chlorella sorokiniana (C. sorokiniana, Chlorella water extract, CWE) was analyzed for the presence of lipopolysaccharide (LPS)-related material via the Limulus amebocyte lysate (LAL) assay and evaluated for its growth stimulation effect on the bone marrow cells and splenocytes in vitro cell cultures. The extract contained low levels of LPS-related material, and a mass spectrum suggested that the extract contained many components, including a low level of a lipid A precursor, a compound known as lipid X, which is known to elicit a positive response in the LAL assay. Treatment with the CWE dose- and time-dependently stimulated the growth of mouse bone marrow cells (BMCs) and splenocytes (SPLs). Treatment with the CWE also increased specific BMC subpopulations, including antigen-presenting cells (CD19⁺ B cells, 33D1⁺ dendritic cells and CD68⁺ macrophages), and CD4⁺ and CD8⁺ T cells, but decreased the number of LY6G⁺ granulocytes. Treatment with the CWE also increased cytokine mRNA associated with T cell activation, including TNFα, IFNγ, and granzyme B in human lymphoblasts. The present study indicates that the cell wall fraction of C.sorokiniana contains an LPS-like material and suggests a candidate source for the bioactivity that stimulates growth of both innate and adaptive immune cells.

The antitumor effects of a partially purified water extract from Euglena gracilis (EWE) and EWE treated by boiling (bEWE) were evaluated using orthotopic lung cancer syngeneic mouse models with Lewis lung carcinoma (LLC) cells. Daily oral administration of either EWE or bEWE started three weeks prior to the inoculation of LLC cells significantly attenuated tumor growth as compared to the phosphate buffered saline (PBS) control, and the attenuation was further enhanced by bEWE. The intestinal microbiota compositions in both extract-treated groups were more diverse than that in the PBS group. Particularly, a decrease in the ratio of Firmicutes to Bacteroidetes and significant increases in Akkermansia and Muribaculum were observed in two types of EWE-treated groups. Fecal microbiota transplantation (FMT) using bEWE-treated mouse feces attenuated tumor growth to an extent equivalent to bEWE treatment, while tumor growth attenuation by bEWE was abolished by treatment with an antibiotic cocktail. These studies strongly suggest that daily oral administration of partially purified water extracts from Euglena gracilis attenuates lung carcinoma growth via the alteration of the intestinal microbiota.

We previously demonstrated the anti‐influenza activity of Citrullus lanatus var. citroides (wild watermelon, WWM); however, the active ingredient was unknown. Here, we performed metabolomic analysis to evaluate the ingredients of WWM associated with antiviral activity. Many low‐molecular weight compounds were identified, with flavonoids accounting for 35% of all the compounds in WWM juice. Prenylated flavonoids accounted for 30% of the flavonoids. Among the measurable components of phytoestrogens in WWM juice, 8‐prenylnaringenin showed the highest antiviral activity. We synthesized 8‐prenylnaringenin and used liquid chromatography–mass spectrometry to quantitate the active ingredient in WWM. The antiviral activities of 8‐prenylnaringenin were observed against H1N1 and H3N2 influenza A subtypes and influenza B viruses. Moreover, 8‐prenylnaringenin was found to inhibit virus adsorption and late‐stage virus replication, suggesting that the mechanisms of action of 8‐prenylnaringenin may differ from those of amantadine and oseltamivir. We confirmed that 8‐prenylnaringenin strongly inhibited the viral entry of all the influenza virus strains that were examined, including those resistant to the anti‐influenza drugs oseltamivir and amantadine. This result indicates that 8‐prenylnaringenin may activate the host cell's defense mechanisms, rather than directly acting on the influenza virus. Since 8‐prenylnaringenin did not inhibit late‐stage virus replication of oseltamivir‐resistant strains, 8‐prenylnaringenin may interact directly with viral neuraminidase. These results are the first report on the anti‐influenza virus activity of 8‐prenylnaringenin. Our results highlight the potential of WWM and phytoestrogens to develop effective prophylactic and therapeutic approaches to the influenza virus.

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin–Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.

Euglena gracilis, a single-celled alga, is rich in nutrients and thus, used as a nutritional dietary supplement. This alga can be found in both fresh and saltwater and possesses characteristics of both plants and animals. Euglena gracilis extracts have a wide range of medicinal properties including stimulation of anticancer immunity against multiple types of cancers; however, the anticancer mechanism has not yet been fully elucidated. Therefore, a water extract from Euglena gracilis devoid of water-insoluble mature paramylons was evaluated as an anticancer agent against lung carcinoma. Two different extracts were prepared using dried powder from whole Euglena gracilis. First, partially purified water extract (EWE) was prepared by suspending dry powder in PBS and centrifugation at 10,000g for 20 min followed by a filtration using a 0.22µm pore size membrane. Second, boiled EWE (bEWE) was prepared by immersing unfiltered EWE in boiling water for 12 min followed by centrifugation at 10,000g for 10 min and filtration using 0.22µm pore size membrane. Both EWE and bEWE treatments inhibited the growth of lung carcinoma cells in vitro in a dose- and time-dependent manner. Furthermore, both extracts significantly inhibited the three-dimensional growth of Lewis Lung Carcinoma (LLC) cells. Flow cytometry analysis showed that the EWE treatment attenuates granulocytic myeloid-derived suppressor cells (MDSCs) in bone marrow cell cultures. The in vivo study was conducted using a mouse LLC orthotopic allograft model. Oral administration of EWE and bEWE (100-200 mg/kg/day) three weeks prior to LLC cell inoculation attenuated the tumor growth in the lungs of immunocompetent mice while decreasing the peripheral granulocytes. However, this attenuation was not seen for the extract treatment initiated after LLC cell inoculation. The tumor growth attenuation was more efficient with the bEWE treatment than with EWE treatment. The fecal microbiomes of the mice were analyzed by 16s rRNA gene amplicon sequencing which revealed that alpha diversity in three groups (EWE, bEWE, and PBS control) was similar, however, the microbial compositions of the EWE- and bEWE-treated mouse groups were more diversified than in the PBS group. Specifically, an increase in the ratio of Bacteroidetes to Firmicutes was observed, and a significant increase in Akkermansia and Muribaculum was detected in EWE- and bEWE- treated mice compared to PBS treated mice. These studies suggest that oral administration of partially purified water extracts from Euglena gracilis altered the intestinal microbiome and the alteration may attenuate host MDSCs, thereby preventing lung carcinoma growth. This study was supported by 2016EUGLENA-RC1 (MT), 2017EUGLENA-RC2 (MT and JC), Kansas State University College of Veterinary Medicine SMILE award (MT and JC), and NIH grant P20 RR017686 (MT). Citation Format: Deepa Upreti, Susumu Ishiguro, Mayme Loyd, Nicole Robben, Paige Cote, Morgan Phillips, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Oral administration of water extract from Euglena gracilis prevents lung carcinoma growth in mice by alteration of intestinal microbiota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2588.