YOSHIKAWA INTERNATIONAL SYMPOSIUM (2ND INTL. SYMP. ON OXIDATIVE STRESS FOR SUSTAINABLE DEVELOPMENT OF HUMAN BEINGS)
Editors: F. Kongoli, H. Inufusa, C. Amatore, H. Chen, W. Huang, H. Goor.

Chronic vitamin E deficiency accelerates in vivo oxidation, which is closely related to aging. There is a significant increase in lipid hydroperoxides in the normal aged mouse brain, especially in the hippocampus compared with young controls. Accumulation of oxidative products induces risk of developing age-related disorders [1]. Normally, our bodies maintain a balance of oxidation and reduction, but the balance gradually collapses as we age. To prevent oxidation, we should always consume fresh fruits, vegetables and supplements. The supplement market is growing year by year. One antioxidant mixed supplement is Twendee X, which contains 8 substances [2]. However, the detailed beneficial effects of Twendee X have not yet been elucidated. In this study, vitamin E-deficient mice were given Twendee X and their cognitive function was measured [3]. Vitamin E-deficient mice had significantly reduced learning ability, the learning rate was comparable to normal aged mouse. Treatment with Twendee X significantly improved spatial ability, despite significantly slower swimming speeds than the untreated group. These results demonstrate that Twendee X has a potential as a powerful antioxidant supplement through protection of in vitro oxidation.

Nicotinamide adenine dinucleotide (NAD+) is involved in regulating various biological processes and decreased with aging or oxidative stress [1]. Nicotinamide mononucleotide (NMN) increases intracellular NAD+ levels and counteracts age-associated changes [2,3]. We investigated the safety and efficacy of oral NMN supplementation (250 mg/day) in older diabetic patients with impaired physical performance.
We conducted a 24-week placebo-controlled, double-blind study for male diabetic patients aged 65 years or older with reduced grip strength (less than 26 kg) or reduced walking speed (lenn than 1.0 m/sec). We studied 14 participants aged 81.1±6.4 years. NMN was tolerable without any related severe adverse events. The changes in grip strength and walking speed showed no difference between the two groups; However, there were trends toward an improved prevalence of frailty in the NMN group (p=0.066) and different changes in central retinal thickness between the two groups (P=0.051).
In conclusion, in the present study performed on older male diabetic patients with physically impaired performance, a 24-week administration of 250 mg NMN was safe. Still, it did not affect grip strength or walking speed. However, a subanalysis indicated that NMN has the potential to improve frailty status and central retinal thickness. Directions to improve frailty and retinal thickness with NMN may be a further expected issue to be investigated.

Voice and swallowing function are critical functions for human life which is supported by intriguing motion of pharynx and larynx. Swallowing function is complicated consisting of the motion of tongue, soft palate, pharyngeal muscles, laryngeal elevation, and the vocal folds.
Reactive oxygen species (ROS) affects the whole organs and their functions, which deteriorate vocal and swallowing function with age or diseases. Dysphagia causes sarcopenia, frail, and aspiration pneumonia which occasionally causes death. It is important to maintain swallowing function as well as vocal function to keep the body in healthy status.
Twendee X, the strongest anti-oxidant, can maintain the function of the pharynx and larynx by reducing ROS. Our previous data indicated that reduction of ROS leads to maintenance of the vocal folds against aging or injury. We have also confirmed that Twendee X can maintain the vocal function of professional singers.
To date, we have established a dysphagia model of guinea pig by resecting nerve branches to the thyropharyngeal muscle. This model represents motor-related dysphagia which is often observed in elderly or patients with neuromuscular diseases. In this model, the animals became unable to eat immediately after the surgery, and lost weight for about 1 week, but they recovered by compensation. When the animals were fed with Twendee x, the immediate reduction of food intake was prevented possibly because of maintenance of the muscles.
Twendee X is thought to be effective for maintenance of voice and swallowing function.

Alzheimer's disease (AD) and chronic cerebral hypoperfusion (CCH) often coexist in dementia patients in aging societies. The hallmarks of AD including amyloid-β (Aβ)/phosphorylated tau (pTau) and pathology-related events such as neural oxidative stress and neuroinflammation play critical roles in pathogenesis of AD with CCH. A large number of lessons from failures of drugs targeting a single target or pathway on this so complicated disease indicate that disease-modifying therapies targeting multiple key pathways hold potent potential in therapy of the disease. In the present study, we used a novel mouse model of AD with CCH to investigate a potential therapeutic effect of a free radical scavenger, Edaravone (EDA) on AD with CCH via examining motor and cognitive capacity, AD hallmarks, neural oxidative stress, and neuroinflammation. Compared with AD with CCH mice at 12 months of age, EDA significantly improved motor and cognitive deficits, attenuated neuronal loss, reduced Aβ/pTau accumulation, and alleviated neural oxidative stress and neuroinflammation. These findings suggest that EDA possesses clinical and pathological benefits for AD with CCH in the present mouse model and has a potential as a therapeutic agent for AD with CCH via targeting multiple key pathways of the disease pathogenesis. In a clinical setting, anti-oxidative dietary supplement TwendeeX prevented cognitive function or even improved it with MMSE (minimental score examination) in mild cognitive impairment population. These results strongly suggest a promising function of anti-oxidative approach to prevent dementia.

The decline in fertility is a major problem in many countries, especially in Japan, where the total fertility rate in 2018 is 1.42. Half of infertility couples have male causes, and the other half have female causes. Oxidative stress can be a major cause of infertility. There are many reports that the number of spermatozoa is decreased by mobile phone that emits electromagnetic waves that cause oxidative stress in the pocket near the testicle, resulting in a decrease in motility. The decline in pregnancy rates with age in women is partly due to the firmness of the egg membrane due to oxidative stress.
Administration of an antioxidant composition would increase the pregnancy rate was examined. The monitor questionnaire was conducted by EYEZ,INC., which is a third-party institution, and the permission for secondary use of the results listed on the company's website (public data) was obtained. Twendee Mtcontrol (TwM) was used as antioxidant composition, which contains L-glutamine, ascorbic acid (VC), L-cystine, coenzyme Q10, succinic acid, fumaric acid, vitamin B2, niacin, Pantothenic acid, thiamine (VB1), pyridoxamine (VB6), folic acid, biotin, vitamin B12, lactoferrin. Twenty-eight couples who had been infertile for more than 2 years and wanted to raise their children joined the monitor questionnaire. For 1 year, both couples took TwM and their pregnancy was monitored. As a result, 9 pairs became pregnant, and the rate was 32% (9/28). Pregnancy rate is about 16% for a couple of more than two years of infertility when they receive infertility treatment at hospital. TwM showed almost 3 times higher pregnancy rate than infertility treatment at hospital. The possibility of antioxidant treatment for infertility was suggested.

COVID-19 infection produces large amounts of Reactive Oxygen Species (ROS) in the human body, and the attack on the immune system by ROS causes a cytokine storm. Therefore, COVID19 infection generally does not cause severe disease in children with low ROS levels, but often causes severe disease and death in the elderly or those with underlying disease already ROS levels are high. The major problem with after COVID-19 pandemic is not only Long-COVID, but also the large number of patients who suffer from the tremendous side effects caused by mRNA vaccines. Pfizer and other genetic vaccines produce spike proteins and antibodies against them. Not only the spike protein itself causes ROS, but also pseudouridine and Lipid Nanoparticles (LNP) contained in the vaccine induce inflammation and increase ROS. In other words, both diseases are oxidative stress diseases. Twendee X (TwX) is a very strong antioxidant composition and reduces inflammation which L-glutamine, ascorbic acid (VC), L-cystine, coenzyme Q10, succinic acid, fumaric acid, vitamin B2, and niacin was used for patients. Patients took 3 tablets of TwX once a day for 4 weeks and answered a web-based questionnaire about their symptoms before and after taking TwX. A score of 0 on the questionnaire indicated no symptoms, while a score of 5 indicated severe symptoms. A total of 149 patients with mRNA vaccine sequelae completed the monitoring questionnaire completely. The monitored symptoms were Fatigue, Breathing difficulty, Chest pain, Smell or taste disorder, Headache, Brain fog, Joint pain, and Dizziness. All monitored symptoms showed significant post-medication score reductions and symptom improvement. Especially in patients with moderate to severe disease with a score of 3 or higher, the mean score dropped to less than half of the pre-medication level for all items. TwX were thought to be effective in relieving symptoms in these patients, many of whom suffer from mRNA vaccine sequelae worldwide.

The number of severely ill patients requiring treatment for sleep apnea syndrome (SAS) is estimated to be over 9 million in Japan and 400 million worldwide. The Space Shuttle Challenger tragedy and the Exxon Valdez maritime accident were caused by SAS, resulting in significant global economic losses. The only effective treatment currently available is CPAP, a forced-air ventilator worn during sleep, which is often discontinued by patients due to its complexity and noise.
About 70% of SAS patients have elevated mCRP and oxidative stress in their blood, indicating inflammation somewhere in the body.　Twendee X (TWX) is a very strong antioxidant composition and reduces inflammation which contains L-glutamine, ascorbic acid (VC), L-cystine, coenzyme Q10, succinic acid, fumaric acid, vitamin B2, and niacin. Clinical research of SAS using TWX for SAS was conducted at Takashima Clinic, Utsunomiya Japan. A survey of SAS patients was also conducted at Eyez Inc.The number of snoring as a major symptom of SAS was monitored. 2 weeks of treatment with TWX significantly reduced the number of snoring in 3 out of 5 patients, and 4 weeks of treatment significantly reduced the number of snoring in all 5 patients. 4 weeks of treatment with TWX showed the following effects in 30 SAS patients. TWX reduced snoring by 85%, nighttime waking phenomenon by 84%, deep sleep by 91%, daytime sleepiness by 84%, apnea by 85%, and headache upon awakening by 96%.
TWX is a supplement with no side effects and can be safely taken by children, suggesting that it may be effective for about 80% of SAS patients.

Oxidative stress is involved in over 150 different diseases, and cancer is one of the most common. High oxidative stress can damage proteins and DNA that are essential to the body, leading to the formation of cancer over time. Oxidative stress in the body is also elevated in carcinoma carriers. When the body's oxidative stress is elevated, inflammatory substances increase, and the body's cancer immunity is reduced, which is thought to promote cancer cell growth and metastasis. Since antioxidants are thought to play an effective role in breaking this vicious cycle, we used the antioxidant compound Twendee X (TwX) to verify the effect of TwX on cancer cells through in vivo and in vitro experiments.
Human colon cancer RPMI4788 cells are used as cancer cells.
In in vitro experiments, the addition of 60 μg/ml of TwX to RPMI4788 cell culture medium slowed cell proliferation from 1 hour after addition, whereas the same concentration of the antioxidant Vitamin C did not inhibit cell proliferation. The inhibition of cell proliferation by the addition of TwX was not observed in normal cells.
A mouse model of lung metastasis was created by transplanting RPMI cells (2 x 104 cells/ml) into nude mice via intravenous tail injection. After 6 weeks of treatment with TwX (40 mg/kg/day), blood samples were taken, and oxidative stress in the plasma, lung　metastasis, and NK activity was measured. The results showed that the oxidative stress level of the tumor-bearing mice was twice as high as that of normal mice, but TwX suppressed the increase from the normal level to less than half. Lung weights were also significantly lighter than controls. NK activity was significantly lower NK activity than normal mice, while the TWX group had twice higher NK activity as the carriers and 1.5 times higher than the normal mice.
These results suggest that TwX may suppress cancer metastasis by suppressing elevated oxidative stress, thereby increasing NK activity, which is cancer immunity. However, similar results may not be obtained in the case of single-component antioxidants. We believe that the results of this study will help antioxidants in cancer treatment and prevention.

The placenta and umbilical cord roles during pregnancy are pivotal for fetal development and maternal health. The placenta is a unique immunological site responsible for maternal tolerance to the fetus and maternal and fetal defence against possible pathogens. Alterations on these organs can lead to dramatic outcomes, including maternal and fetal death. During pregnancy, their correct functioning also influences the newborn's development and the onset of future chronic diseases. Therefore, investigating them in different diseases can give a real picture of health at the time of birth. Among the maternal diseases, pre-eclampsia (PE) is one of the most important, and despite incomplete understanding, oxidative stress and inflammation are recognised as their biochemical basis [1]. Another important aspect related to maternal-child health during pregnancy is related to ZIKA Virus (ZIKV), which can be vertically transmitted and infects the fetus, which may develop congenital Zika syndrome, characterized by stillbirth/miscarriage, fetal growth restriction, microcephaly, ocular abnormalities, ventriculomegaly and other brain malformations [2]. As such, the aims of the present work are divided into two parts. The first one is related to the comparison of the redox imbalance and inflammation biomarkers in the placenta and umbilical cords of pregnancies with and without preeclampsia (PE), while the second one addresses the investigation of natural products, which may inhibit ZIKV infection of trophoblast cells, a good model to test ZIKV molecular interactions and potential interveners [3]. In the first part, a controlled cross-sectional study was conducted in Maceió-Alagoas-Brazil with pregnant women diagnosed with PE and healthy pregnant women (control group). After delivery, placenta and umbilical cords’ samples were collected to measure redox imbalance (antioxidant defence and oxidative damage) and inflammation biomarkers: myeloperoxidase (MPO), interleukin (IL) -6, IL-8, IL-10 and tumour necrosis factor-alpha (TNF-α). A total of 100 pregnant women with PE and 50 healthy pregnant women were studied. Higher placental levels of catalase (p = 0.018), SOD (p = 0.031), the GSH/GSSG ratio (p = 0.019) and IL-6 (p = 0.010) and lower GSSG (p = 0.001) were observed in pregnant women with PE than in the control group. As results, PE-derived placentas had high concentrations of some antioxidants (enzymes and thiols), which might be a compensation mechanism against oxidative stress. In the second topic, the potential of some natural extracts against ZIKV infection was evaluated using an in vitro method. Ethanolic extracts obtained from peels and complete fruits of pink pepper (Schinus terebinthifolius) could reduce ZIKV infection and modulate placental response to protect placentas from potential harms provoked by ZIKV infection. In conclusion, both studies unveil the role of specific antioxidants in protecting maternal-child health and provide extra value to products from family farming.

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, senescent decline and ageing is broadly influenced by genetic and extrinsic factors. Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Key determinants of this capacity are the molecular mechanisms that link ageing to main stress response pathways, and mediate age-related changes in the effectiveness of the response to stress. How each pathway contributes to modulate the ageing process is not fully elucidated. A better understanding of the dynamics and reciprocal interplay between stress responses and ageing is critical for the development of novel therapeutic strategies that exploit endogenous stress combat pathways against age-associated pathologies. Mitochondria, the indispensable and highly dynamic, energy-generating organelles in all eukaryotic cells, play essential roles in fundamental cellular processes. Neuronal cells depend, perhaps more than any other cell type, on proper mitochondrial function. Mitochondrial impairment is a major hallmark of several age-related neurodegenerative pathologies, including Alzheimer’s disease. Interestingly, accumulation of damaged mitochondria has been observed in post-mortem brain of Alzheimer’s disease patients. Mitophagy is a selective type of autophagy mediating elimination of damaged mitochondria, and the major degradation pathway, by which cells regulate mitochondrial number in response to their metabolic state. However, little is known about the role of mitophagy in the pathogenesis of Alzheimer’s disease. Although disease-associated tau and amyloid β are known to deregulate mitochondrial function, it remains elusive whether they also directly influence the efficiency of mitophagy. To address this question, we developed an in vivo imaging system to monitor mitophagy in neurons. We demonstrated that neuronal mitophagy is impaired in C. elegans models of Alzheimer’s disease. Urolithin A- and nicotinamide mononucleotide-induced mitophagy ameliorates several pathological features of Alzheimer’s disease, including cognitive defects. Mitophagy stimulation restores memory impairment. Age-dependent decline of mitophagy both inhibits removal of dysfunctional or superfluous mitochondria and impairs mitochondrial biogenesis resulting in progressive mitochondrial accretion and consequently, deterioration of cell function. Our findings suggest that impaired removal of damaged mitochondria is a pivotal event in Alzheimer’s disease pathogenesis highlighting mitophagy as a potential therapeutic intervention.

Our group focuses on developing new devices and analytical methods for tracking of chemical and electrical signals simultaneously in the brain of free-moving animals. Systematic and in-depth research has been carried out on breaking the key bottleneck of real-time monitoring and quantification of from reactive oxygen species and oxidative stress related species in physiological and pathological processes with high selectivity, high accuracy, long-term stability, and designing new devices for brain imaging with high temporal resolution.[1-2] Firstly, the synergy strategy of molecular recognition and electrochemical recognition was proposed for achieving highly selective determination, and new approaches based on rational design of specific molecules with built-in calibration were established for accurate quantification in living brains.[3] Secondly, to challenge the real-time determination in the complicated brain, Au-S, Au-Se and Au-C≡C bonds were systematically investigated. The Au-C≡C bond showed the highest stability under thiol-rich biological conditions and the best electrochemical performance compared to the others. Furthermore, a more reliable sensing platform with long-term stability and anti-biofouling was constructed through rational integrating highly stable graphene-layered molecular interface, remarkably elongating the time dimension for real-time tracking of the dynamic changes in free-moving animals up from several hours to a record-long 60 days.[4] Thirdly, a SERS optophysiological probe was created for real-time mapping and recording of chemical and electrical signals without cross-talk in the live brain. Using this powerful tool, three new routes that causes Cu+ and Cu2+ change were discovered during ischemia: export from neurons; release from digested copper-containing proteins; conversion from Cu+ to Cu2+. Moreover, it was the first time that a Raman fibre photometry was built up for real-time tracking and simultaneous quantitation of multiple molecules in mitochondrial across the brain of free-moving animals. Meanwhile, a highly selective non-metallic Raman probe was created through triple-recognition strategies of chemical reaction, charge transfer, and characteristic fingerprint peaks, for monitoring and quantifying of local mitochondrial O2•-, Ca2+ and pH in six brain regions upon hypoxia. It was discovered that hypoxia-induced mitochondrial O2•- burst was regulated by ASIC1a, leading to mitochondrial Ca2+ overload and acidification.[5-7]

Cancer is a conundrum in modern medicine and has become a serious threat to human health and life [1], thus the the early diagnosis and precise therapy are crucial to improve the cancer cure rates [2]. Taking advantage of the DNA nanotechnology, an RNA aptamer-based near-infrared gated nanodevice for low-background imaging of mRNA in living cells have been first developed based on the fluorescence enhancement property of RNA aptamer and the catalytic hairpin assembly reaction-mediated signal amplification strategy. Subsequently, DNA nanocarrier with excellent biological stability, strong enzymatic resistance and collaborative drugs delivery is constructed by functionalized circular DNA, which can effectively combat the multidrug-resistant human leukemia. Moreover, a multifunctional DNA-Au nanomachine integrating both diagnostic and therapeutic agents is proposed, which can be triggered by the endogenous mRNA, leading to the activation of multi-mode synergistic therapy and in situ imaging. These researches conducive to the accurate diagnosis and image-guided precise treatment of cancers, which may provide a new route for the construction of intelligent responsive theranostic nanoplatform.

It is known that when a living organism is exposed to radiation, water molecules in the body ionize, producing reactive oxygen species, which in turn cause inflammation.
Twendee X (TwX) is an antioxidant supplement consisting of eight ingredients (coenzyme Q10, vitamin C, vitamin B2, niacin, L-cystine, succinic acid, fumaric acid, and L-glutamine). We investigated whether TwX and other substances can inhibit oxidative stress generated by radiation.
C57BL/6J mice were orally administered the test substance TwX (15 mg/kg) for 3 days at 24-hour intervals, irradiated with 6 Gy of radiation on the fourth day, and blood samples were taken 72 hours later, and survival was observed as is. Vitamin C (15 and 150 mg/kg), Resveratrol, Astaxanthin, Protandim, and Pycnogenol were used as antioxidants for comparison to TwX. Plasma hydroperoxide level was measured as blood oxidative stress (d-ROMs test).
Increased d-ROMs test value by radiation was reduced by one third in the group treated with TwX (15 mg/kg) before irradiation for 3 days (t-test, p<0.05), and the oxidative stress level was lower in the group treated for 7 days than in normal mice. In addition, the administration of TWX suppressed radiation-induced deaths.
Vitamin C (150 mg/kg) and Astaxanthin treatment groups reduced d-ROMs test values, but the difference was not statistically significant. The low concentrations of vitamin C (15 mg/kg), Pycnogenol, and Protandim did not change the d-ROMs test values compared to the control group, and for the Resveratrol group, the d-ROMs test values increased compared to the control group. Furthermore, Twendee Mtcontrol (TwM), which added seven active ingredients to TwX, reduced radiation-related deaths with fewer doses than TwX.
TwX significantly reduced radiation-induced oxidative stress at a small dose of 15 mg/kg, while other single antioxidants with normal amount have no significant improvement in animal radiation model. High dose antioxidants may cause side effects. TwX, TwM are composed only of substances that exist in the human body, and a small dose of them showed high antioxidant activity. This may have reduced inflammation caused by oxidative stress, ultimately leading to an increase in survival rate. The antioxidant potential of Twendee series in this experiment suggests the possibility of future applications for various oxidative stress-related diseases.

[Purpose]
Many studies have shown the importance of keeping oral health to prevent various diseases. Neutrophils (PMNs) play an important role to keep oral health by its bactericidal activity such as reactive oxygen species (ROS) production and phagocytosis. We investigated the effects of lysed and heat-treated Enterococcus faecalis FK-23 (LFK) on ROS production and phagocytosis of human PMNs and its mechanism.
[Method]
I. PMNs (1x10⁶ cells/ml) isolated from peripheral blood were stimulated with water-soluble LFK (5 mg/ml) and/or phorbol 12-myristate 13-acetate (PMA). Furthermore, the mechanism of ROS production was examined using each inhibitor. Then, ROS production was measured by MCLA-dependent chemiluminescence for a total 300s time. II. PMNs (1x10⁶ cells/ml) were incubated for 1h at 37℃ with/without LFK. Then, carboxylate-modified fluorescent beads were added to PMNs suspension and incubated for 30 min at 37℃. After that, phagocytosis was measured by flow cytometry.
[Results and Conclusion]
LFK transiently increased ROS production from PMNs without change in total amounts. This effect was decreased by some inhibitors of mitochondrial complex and protein kinase C (PKC) inhibitor. Thus, LFK might increase the reactivity of PMNs via PKC by enhancing mitochondrial functional ability or phagocytosis ability. These results suggest that LFK may increase bactericidal activity of PMNs to improve oral health.

Free radical reactions play an important role in the biological system. In particular, oxygen-derived free radicals are sometimes used as an energy source for organisms that use oxygen to live, and also as a weapon to attack foreign objects at one stage.
This reaction that works in the body is called ‘oxygen stress’, and I have devoted my life to this research. The contents of the research can be broadly divided into two. One of them is pathophysiological research as a cause of disease, and I have been researching disease prevention with antioxidants. Contrarily, the other research is to use this powerful oxidation reaction to defeat external enemies such as cancer, viruses, and bacteria.
We still cannot prevent many diseases and cure them even with these series of studies. I shall continue my research toward the solution of this proposition.

Many diseases and physical disorders are caused by both external and internal factors, but the common denominator is inflammation and oxidative stress. Suppressing oxidative stress on a daily basis and maintaining a high level of self-immunity can help prevent and treat disease and maintain good health.
Intestines are involved in about 60% of our immune system. There are over 100 species of, or 100 trillions of bacteria in our intestines, and their ideal balance is 20% of good, 10% of bad, and 70% of opportunistic bacteria. This balance is lost by disturbed life rhythm and various diseases, in other words “oxidative stress”, being said to decrease bacterial diversity. Reducing oxidative stress and increasing diversity of intestinal bacteria is important for maintaining healthy life. Composed of vitamins, CoQ10, and amino acids, Twendee X (TwX) is an antioxidant which is confirmed to have a dementia-prevention effect. Effects of oxidative stress reduction by taking antioxidants on the bacterial diversity of the intestinal microflora and the balance of the intestinal microflora are examined.
Rats were given an antifungal agent OPP for 2 weeks, then blood and feces samples were collected to analyze blood oxidative stress and intestinal bacterial diversity. TwX was administrated from 1 week before OPP initiation. Although OPP rat group increased blood oxidative stress by 5%, no significant change was observed in microbial flora diversity. A rat group given TwX since 1 week before decreased oxidative stress by 13.3% and increased bacterial diversity 1.5 times higher, compared to rats which wasn’t given, and the number of Akkermansia, anti-inflammatory bacteria, was increased 3.5 times more. In the questionnaire survey for healthy participants who were given TwX for 1 month, the number of good bacteria and intestinal bacterial diversity were increased. Their defecation status and QOL were improved with that.
TwX tends to reduce oxidative stress, to increase the number of bacteria and diversity in intestinal microflora. It was suggested that this enables to protect human body from harmful substances invaded inside, improve immune system, and improve daily defecation status and QOL.

Glutamate (Glu) is a crucial fundamental excitatory neurotransmitter released through vesicular exocytosis in the central nervous system. Dysregulation of the glutamate uptake by neurons and glial cells result in increase of the glutamate extracellular concentration leading eventually to excitotoxicity associated with increased oxidative stress and neurodegeneration [1]. Hence, quantitative measurements and interpretation of intravesicular Glu and of transient exocytotic release contents directly from individual living neurons are highly desired for understanding the mechanisms (full or sub-quantal release?) of synaptic transmission and plasticity. However, this could not be achieved so far due to the lack of adequate experimental strategies relying on selective and sensitive Glu nanosensors.
We will show that a novel electrochemical Glu nanobiosensor based on a single SiC nanowire [2] is prone to selectively measure in real-time Glu fluxes released via exocytosis by large Glu vesicles (ca. 125 nm diameter) present in single hippocampal axonal varicosities as well as their intravesicular content before exocytosis by IVIEC.
Combination of these two series of measurements revealed a sub-quantal release mode in living hippocampal neurons, viz., only ca. one third to one half of intravesicular Glu molecules are released by individual vesicles during exocytotic events.
Importantly, this fraction remained practically the same when hippocampal neurons were pretreated with L-Glu-precursor L-glutamine, while it significantly increased after zinc treatment, although in both cases the intravesicular contents before release were drastically affected.
Finally, the simulations of the electrochemical monitoring of the glutamate release events will be presented. The obtained theoretical results support the quantitative measurements with the enzymatic electrode. In addition, simulation results will also serve to discuss the meaning and adequacy of pre-calibrations performed in bulk solutions [3] to assess the analytical properties of enzyme-based electrochemical nanosensors aimed to detect fast transient release events.

Monitoring of key biomolecules and/or oxidative stress at cellular or sub-cellular levels by means of electrochemistry requires electrodes with good selectivity and sensitivity. These both characteristics often achieved by employing enzymatic electrodes. At these electrodes the enzymes are generally dispersed within a polymer layer covering electrode surface, where product(s) of the enzymatic conversion are detected. Rationalization of the experimental data imply understanding mass transport towards an enzymatic electrode which is a complicated process due to random distribution of the enzymes along the electrode surface. This process can be considered through the framework of random arrays, that is a set of active sites distributed randomly, which is also useful for description of many practical micro- and nanoscale systems [1]. As shown previously these systems can be efficiently addressed theoretically by using Voronoi diagrams [1, 2] which allows facile tessellation of the system into the unit cells around each active sites. The overall current flowing in the system can then be evaluated by modelling diffusion-reaction processes inside every unit cell and summing the contributions from individual active sites. Although this approach is tempting by its simplicity and efficiency [1] one should bear in mind that Voronoi diagram representing the unit cells by polygonal prisms remains approximation and as each approximation remains valid only under certain conditions. We have shown [3] that even for the case of diffusion limited electron transfer (ET) the actual shapes of the unit cells are more complicated and depend on the local configuration of the neighbouring active sites. This was exemplified on the small patches of the random arrays with band-like and disk-like active sites via simulations and analytical derivations.
Importantly, by comparing the total and individual electrode currents obtained by employing Voronoi tessellation and simulation of the system without any approximations we found that the former are reproduced with a good accuracy while the latter are evaluated with a much larger relative error [3], thus demonstrating the limits of Voronoi tessellation for representation of such systems.
Moreover, diffusion interaction between the neighbouring sites compensate the differences in unit cell sizes leading to a more uniform unit cell sizes then predicted by Voronoi tessellation [4]. This, in particular explains why the early theory of random arrays using uniform representation of the system were quantitatively successful [5].

Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g., phagocytosis) and pathological conditions (e.g., cancer) during episodes involving Oxidative Stress conditions. The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques. Nanometer-sized electrochemical probes are especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this paper, we will survey recent advances in localized measurements of ROS/RNS inside single cells and discuss several methodological issues, including optimization of nanoelectrode geometry, precise positioning of an electrochemical probe inside a cell, and interpretation of electroanalytical data.
Application of this method will be presented for detection of ROS/RNS in human cancer cells and for demonstrating the existence of homeostatic control in phagolysosomes during phagocytosis by macrophages.
We will also show using these methods that remediation of Oxidative Stress in neurons artificially placed under Parkinson Disease conditions avoids the impeachment of synaptic communication when the neurons are pre-treated with Harpagide, a natural sugar derivative which alleviate the oxide stress borne by mitochondrions.

Oxidative stress conditions are encountered by all aerobic organisms during their whole life. Indeed, aerobic cells mostly derive their energy from the intracellular enzyme-catalyzed oxidation of fat and sugars to CO2. Also, metalloenzymes which are central actors of the respiratory chain in mitochondria are generally good reducing agents, prone to open side routes leading to O2 reduction to superoxide ion (O2•-) that is the precursor of a series of hazardous species collectively named as “reactive oxygen species (ROS)” and “reactive nitrogen species (RNS)” [1,2]. ROS and RNS may induce molecular damages to almost all organic compounds performing biological functions (nucleic acids, proteins, cells carbohydrates and lipids, etc.) – a situation termed “oxidative stress” when it runs out of control. Even without exposure to radiation or other photo-biological effects, oxidative stress can bring about such pathological conditions as inflammation, carcinogenesis, Parkinson and Alzheimer diseases, and various autoimmune illnesses, as well as accelerated ageing.
The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques [3].
Nanometer-sized electrochemical probes with cylindrical shapes do not experience this problem since they can penetrate across the cell membranes that reseal around their shaft (7). They are then especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this paper, we will survey recent advances in localized measurements of ROS/RNS inside single cells.
Application of this method will be presented for detection of ROS/RNS in phagolysosomes during phagocytosis by macrophages (4,5). We will also evidence using these methods that remediation of Oxidative Stress in neurons artificially placed under Parkinson Disease conditions avoids the impeachment of synaptic communication when the neurons are pre-treated with Harpagide, a natural sugar derivative which alleviate the oxide stress borne by mitochondria (9).

Nanopore electrochemistry refers to the promising measurement science based on elaborate pore structures, which offers a well-defined geometric confined space to adopt and characterize single entities including single cells, single particles, and even single molecules by electrochemical technology.1-3 The electrochemical confined effect within the nanopore displays the incredible ability to achieve single entity discrimination by focusing energy (e.g. electrochemical, light energies and et al.) into small areas, converting the intrinsic properties of single entities into visible electrochemical read-outs with ultra-high temporal-spatial resolution. Furthermore, the excellent resolution of confined nanopore technology also permits the possibility to resolve the transient signals for further revealing the information of single biomolecules dynamics. The chemical controlled confinement inside nanopore provides the advanced electrochemically confined effects to convert the transient single molecule difference into the enhancing signal with high temporal-spatial resolution. In our group, the nanopore electrochemistry has been further applied into disease diagnostics by identifying rare sub-populations, DNA/protein sensing by reading the sequential differences and uncovering the fundamental chemical reactions pathways by revealing the hidden intermediates. With the advents of advanced measurement mechanism, instrumentation and data algorithm, electrochemically confined nanopore is certainly an exciting and promising field. We expect the next avenue for the wide applications of nanopore electrochemistry in a variety of disciplines, leading us explore the new chemistry at a much smaller scale.

Hearing loss can occur by aging, ototoxicity of drugs, noise exposure, etc. It deteriorates the quality of life. Severe hearing loss causes isolation in human life. Tinnitus is an accompanying symptom with hearing loss, and is kind of torture for some people. Prevention of hearing loss is essential to keep the comfortable human life. Oxidative stress has been revealed to cause hearing loss caused by aging, noise exposure, and ototoxic drugs such as aminoglycoside and cisplatin (CDDP). We have revealed that reactive oxygen species (ROS) is produced in the vessels and hair cells of the inner ear. The hair cells are critical to maintain the hearing ability, and ROS causes severe and refractory damage to the hair cells. We have also revealed that Nox-3 derived ROS in the cochlea induces sensorineural hearing loss. Nox-3 is the key molecule for production of ROS. Nox knock-out mice showed maintenance of hearing ability under noise exposure or ototoxic agency.
Anti-oxidative supplement should be useful for maintaining hearing.

Oxidative stress and immune activity are closely related with disease or symptoms. Allergic reaction depending on imbalance of immune system which is easy to be damaged by oxidative stress. COVID-19 pneumonia starts at the lower and back side of lung, and CT scan shows typical interstitial pneumonia. Interstitial pneumonia is kind of allergic reaction, thus pneumonia start high blood flow area of lung and very rapidly spread to the whole lung. It is well known that interstitial pneumonia shows extremely high oxidative stress, damages lung tissue, and some patients show irreversible damage of the lung. High risks of COVID-19 infection are high age with underlying disease, especially diabetes mellitus, obesity, heart disease, hypertension, after stroke, cancer, neurogenic disorder, dementia and so on. All these risk factors show extremely high oxidative stress. Oxidative stress damages vascular endothelium and inflammation of vascular wall produces thrombosis, which are reasons of Kawasaki disease like vasculitis and cerebral infraction. Oxidative stress plays a major role in these symptoms.

Diabetes is known to be a disease caused by oxidative stress, and also known to generates its own oxidative stress, resulting in numerous complications. Twendee X (TwX) is an antioxidant combination supplement with international and Japanese patents, and has been certified as effective in preventing dementia from a randomized, double-blind clinical trial led by the Japan Society for Dementia Prevention in 2019. In recent years, we have also successfully developed Twendee Mtcontrol (TwM), an antioxidant with seven more ingredients added to TwX to increase its antioxidant effect. The antioxidants TwX and TwM were tested in animal models to see if they can treat diabetes and diabetic complications.
To examine the effect of antioxidants on diabetes, a diabetic disease model was developed. TwX or TwM was orally administered daily for 2 months. Afterwards, a glucose tolerance test was performed, and blood glucose levels were measured over time. In addition, insulin levels were measured under the same conditions. The results showed that the peak blood glucose level was significantly improved in the TwX group, and the peak level was also improved in the TwM group compared to the control group. Insulin levels also tended to be lower than controls in both groups.
In addition, to verify the effectiveness of the supplements in preventing actual diabetic complications, we tested it on SDT fatty rats, which develop diabetic complications early in the course of the disease. These rats develop elevated blood pressure and cataracts at a much earlier stage due to the progression of diabetes mellitus. SDT fatty rats (5-week-old males) were divided into three groups (n=8): control (non-treated), TwX 40 mg/kg/Day, and TwM 40 mg/kg/Day. At 17 weeks of age, most rats in the non-treated group showed severe cataracts, whereas those in the TwM-treated group had relatively mild cataracts. Systolic blood pressure also tended to increase with age in the non-treated group, but the TwX and TwM groups significantly suppressed the age-related increase in blood pressure.
These results suggest that TwX and TwM reduced blood glucose levels in an insulin-independent manner, thereby reducing diabetic complications such as hypertension and cataracts. TwX reduced blood glucose levels more, and TwM had a pronounced cataract-preventive effect. TwM may have a broader range of disease-preventive effects. however, further studies are required for the detailed mechanism of action. These results suggest the possibility of antioxidant therapy for diabetes mellitus and its complications.

COVID-19 is the shocking viral pandemics of this year which affected the health, economy, communications, and all aspects of social activities all over the world. Early diagnosis of this viral disease is very important since it can prevent lots of mortalities and care consumption.
The functional similarities between COVID-19 and COVID-2 in inducing acute respiratory syndrome lightened our mind to find a diagnostic mechanism based on early traces of mitochondrial reactive oxygen species (ROS) overproduction as lung cells’ dysfunctions induced by the virus. We designed a simple electrochemical sensor to selectively detect the intensity of ROS in the sputum sample (with a volume of less than 500µl). Comparing the results of the sensor with clinical diagnostics of more than 140 normal and involved cases resulted in a response calibration with accuracy and sensitivity both 97%. Testing the sensor in more than 4 hospitals shed promising lights in ROS based real-time tracing of COVID-19 from the sputum sample.

Although there are genetic differences in the metabolism of alcohol, large amounts of alcohol intake can cause hangovers and, in severe cases, death due to acute alcohol poisoning. Once alcohol enters the body, it is metabolized mainly in the liver, where it is broken down into the highly hepatotoxic acetaldehyde. It is thought that excessive alcohol intake causes insufficient metabolism in the body, and the acetaldehyde induces oxidative stress, resulting in hangovers and organ damage. Therefore, we examined the effects of large amounts of alcohol on the body using Twendee S (TwS), an antioxidant formula consisting of coenzyme Q10, vitamin C, vitamin B2, niacin, L-cystine, succinic acid, fumaric acid, and L-glutamine.
Mice were intraperitoneally administered ethanol, and blood acetaldehyde and oxidative stress were measured. The results showed that acetaldehyde in the blood increased at 2 hours after ethanol administration and was hardly detectable at 24 hours. However, oxidative stress remained high even after 72 hours of ethanol administration, and antioxidant capacity was low. In contrast, TwS-treated group suppressed blood acetaldehyde and the rising oxidative stress without reducing antioxidant capacity.
In the mouse acute alcohol intoxication experiment, all mice in the Control group, to which nothing was administered, died after 12 hours. However, in the group that received 4 doses of TwS at 12-hour intervals the survival rate was 100%.
These results suggest that heavy alcohol intake increases oxidative stress in the body, and that this oxidative stress may be a factor in acute poisoning deaths. In contrast, the antioxidant compound TwS suppressed acute poisoning deaths by speeding up the metabolism of alcohol and suppressing elevated oxidative stress.
In conclusion, heavy and prolonged alcohol intake causes significant oxidative stress in the body, which may lead to aging and disease. Therefore, countermeasures against oxidative stress caused by alcohol intake are important, and TwS was shown to have the potential to reduce this risk.

Sigma S (s^s) controls the synthesis of resistance proteins in stationary pathogenic bacteria [(e.g., Escherichia coli (UPEC)]. Deletion of the rpoS gene rendered E. coli more sensitive to bactericidal antibiotics (BAs): gentamicin, nnorfloxacin and ampicillin. Proteomic analysis implicated a weakened antioxidant defense (AD). Use of the psfiA genetic reporter, 3-(p-hydroxyphenyl) fluorescein (HPF) dye, and Amplex Red showed that BAs generated more oxidative stress (OS) in the mutant. Co-administration of the antioxidant N-acetyl cysteine (NAC) and treatment under anaerobic conditions decreased drug lethality of the mutant, further indicating AD involvement. The greater OS in this strain results from impaired capacity to quench endogenous ROS, e.g, respiration- linked electron leakage. Infection by UPEC in mice showed that AD was important for UPEC antibiotic reistance also in vivo. Disruption of AD by eliminating quencher proteins, or those of pentose phosphate pathway (which provides NADPH for quenching oxygen radicals) also generated greater OS and killing by BAs. Thus, BAs kill stationary-phase bacteria also by generating OS, and targeting AD can therefore enhance their efficacy. Using bioinformatics, small molecule compounds were identified towards this end, and initial results have given promising results. In space flights, astronauts often suffer from UPEC infection. The EcAMSat mission, using a highly sophisticated microfluidic system showed that UPEC missing s^s had increased sensitivity to gentamicin also in space. We have also developed method for determining resistance at single cell level. Together, these results promise to provide powerfull means to combat bacterial antibiotic resistance.

Cells in the body reside in dynamic mechanical milieus and can sense mechanical forces and then translate them into biochemical signals via mechanotransduction. Reactive oxygen/nitrogen species (ROS/RNS) are closely involved in cellular oxidative stress and physiopathological states, and real-time acquiring the information of ROS and RNS signaling during cell mechanotransduction is vitally important to reveal their complicated roles. Owing to rapid response and excellent sensitivity, electrochemical sensing has been extensively used for tracking ROS/RNS signaling in living cells. However, conventional electrochemical sensors are rigid and fail to comply with the shape changes of soft cells, and this greatly limits the accurate measurement of ROS/RNS signaling during cell mechanotransduction.
To this end, we have designed a series of stretchable electrodes based on gold nanotubes, carbon nanotubes and conductive polymer nanofibers [1,2]. These sensors possess good electrochemical stability against mechanical deformations and can be easily deformed to achieve the dynamic stretching of cells. Further, we have also introduced high-performance catalysts (e.g, platinum nanoparticles and biomimetic catalysts) to confer the stretchable sensing interfaces with prominent electrocatalytic property toward ROS and RNS [3].
Based on these stretchable electrochemical sensors, we have successively achieved the real-time monitoring of stretch-induced NO and H2O2 molecules [3,4], and the simultaneous monitoring of them by a single device during endothelial mechanotransduction [5]. Besides, the developed stretchable electrochemical sensors have been also employed to explore the ROS and RNS signaling during mechanotransduction of chondrocytes and lung cells, which demonstrate excessive or acute mechanical loading can evoke severe oxidative stress [2]. These researches present efficient platforms to evaluate the oxidative stress level in dynamic mechanotransduction, which benefit to the understanding of the role of mechanical cues in cell biology.

As examples of cancer treatment, immunotherapy is now widely recognized as a powerful cancer therapy in addition to conventional therapies such as operation, chemotherapy and radiotherapy. Although they are all effective treatments, chemotherapy and radiotherapy, which take advantage of small differences between cancer cell (tumor cell) and normal cell, damage normal cells as well as cancer cells. As a result, adverse events are almost inevitable and are often difficult to continue treatment depending on the degree. 　As these treatments are administered over months or years, physical and mental damage accumulate, leading to a decline in physical strength and immunity.
These adverse events are largely due to oxidative stress. That is, the tumor itself, and reactive oxygen species (ROS) generated by treatment for it, can cause damage to various cells due to oxidative stress. And oxidative stress can cause a decline in physical strength and immunity, which leads to tumor growth and a negative spiral. Thus, suppression of oxidative stress in tumor and cancer treatment may be very effective in not only alleviating symptoms but also prolonging prognosis. Since oxidative stress-mediated mechanisms of carcinogenesis also exist, suppression of oxidative stress may help to prevent cancer.
An antioxidant compound Twendee X (TWX) which composed of vitamins, amino acids, and CoQ 10 is rated as the strongest and safe antioxidant in the world by ICDD (France). Cytoprotective effects of TWX against radiation exposure were examined using murine alveolar epithelial cells (LA-4). After allowing LA-4 cells to adhere, 25ug/ml of TWX was added in the medium. The cells were irradiated with 30Gy of radiation 2 hours after the addition, and after 72 hours, the effect on cell proliferative capacity was examined. The result showed that TWX significantly inhibited the radiation-induced decrease in cell proliferative capacity.
In addition, TWX can be used in single and continuous doses in both healthy and pregnant mice and also increased NK activity, which is tumor immunity. In summary, TWX not only reduced adverse events during cancer treatment but also increased tumor immunity. We believe TWX can be an aid in the treatment and prevention of cancer.

Dysphagia is one of the significant concerns in the aging society. In particular, the elderly with swallowing impairment caused by either central or peripheral nervous systems has difficulty in oral intake, possibly resulting in fatal pneumonia. As such, the development of a therapeutic approach that decreases in the risk of aspiration in dysphagic patients remains a pressing issue for medical doctors. The purpose of our study was to establish the animal model of dysphagia and test the hypothesis that decreased pharyngeal constriction caused by the denervation of the vagal efferent nerves can be improved by the application of the anti-oxidant agent. Thus, we developed the animal model in guinea pigs in which the pharyngeal branch of the vagus nerve was cut and initially evaluated swallowing function using videofluoroscopy. We then tested the effect of oral application of the anti-oxidant agent Twendee X on swallowing function in the dysphagia model animals. Insufficient pharyngeal constriction and pharyngeal residue were found in the denervated animals. The swallowing dysfunction was less pronounced in dysphagic animals with the administration of Twendee X. In conclusion, Twendee X could have a possible role in the improvement of swallowing function in dysphagic patients with decreased swallowing muscle strength caused by neuromuscular atrophy.

Reactive oxygen species may attack several living organs and gradually accumulate oxidative products such as lipid hydroperoxides. Recently, several kinds of evidence have been demonstrated that accumulation of oxidative products induces onset and progression of several severe diseases such as inflammations, cardiovascular events and more [1]. One severe disease is dementia, and it also deeply relates to senescence. To prevent age-related neurodegenerative disorders, treatment with antioxidant is effective. Twendee X, a mixed antioxidant supplement contains 8 substances [2] and anyone can buy it. However, the detailed scientific evidence is not enough. In this study, we fed Twendee X to vitamin E-deficient mice and check cognitive function.
We created vitamin E-deficient mice and Twendee X treated for one month. Cognitive function was measured by the Morris Water maze task [3].
Vitamin E deficient mice were significantly decrease learning ability. Treatment with Twendee X was significantly improved it. Brain neurotrophic factors in Twendee X-treated mouse were significantly increase compared to the untreated groups.
Twendee X has a potential as a powerful antioxidant supplement through protection against neuronal dysfunction.

Oxidative stress is an essential metabolic outcome in aerobic organisms due to the activity of mitochondria in providing the basic energy of cells or during the operation of several enzymatic pools. It also serves to regulate the size and shape of organs or restructure them during foetal development by apoptosis. Oxidative stress is also indispensable to the immune system by allowing macrophages to eliminate virus, bacteria and impaired or dead cells through phagocytosis [1]. In fact, no aerobic organism could live without oxidative stress, a fact that explains why evolution maintained such unsafe mechanisms in aerobic organisms. Though, they are associated to highly negative issues.
Indeed, oxidative stress mechanisms provide a variety of life-harmful radicals and species called generically Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) whose fluxes need to be finely controlled to avoid the destruction of most organic molecules (e.g., lipids in cell membranes, enzymes, etc.) and biological ones (DNA, proteins, etc.) in cells. Thus, under normal conditions, a panoply of antioxidants and enzymatic systems ensures a fine homeostatic balance. However, rupture of this delicate balance is frequent and may provoke severe damages leading to human pathologies (aging, cancers, AIDS, hearth and brain strokes, Parkinson and Alzheimer’ diseases, etc.).
Using platinized carbon fiber ultramicroelectrodes we could establish that the composition of the primary oxidative stress in macrophages [1,2] and characterize the nature of functional hyperemia in the brain.3 This led us to formulate an alternative hypothesis about the onset of Alzheimer disease when Amyloid-β and ascorbate molecules are present [4,5].

Stress granules (SGs) are membrane-less organelles that assemble in the cytoplasm under unfavorable conditions, which is closed related with cancers and neurodegenerative diseases etc. To understand how SGs contribute to physiological functions, we used electrochemical measurements to detect oxidative stress-induced granules and investigate the effects of them in neurotransmission. We found that reactive oxygen species (ROS) are encapsulated inside arsenite-induced SGs, and H2O2 is the main species,which implicates SGs as communicators of the cellular stresses rather than a simple endpoint. In addition, the number of transmitter molecules released from single vesicles of Chromaffin cells decrease significantly while the production doesn’t show obvious alteration. This may explain how SGs regulate cellular metabolism and stress responses,which helps better understanding their cytoprotective functions in pathological conditions associated with SGs such as neurodegenerative diseases (NDs), cancers and viral infections.

In Chronic Fatigue syndrome (CFS), a complex combination of factors such as infections and excessive stress in daily life causes abnormalities in the immune system, endocrine system, brain, and neurological functions. The pathogenesis of the syndrome is not yet clear, and no treatment has been established. Recently, it has been reported that CFS-like symptoms are also observed in COVID-19 sequelae and SARS-CoV-2 vaccine sequelae, and there is an urgent need to clarify the pathogenesis mechanism and establish prevention and treatment methods. Abnormal energy metabolism due to mitochondrial dysfunction and inflammation in the brain have been reported as one of the causes of CFS. Since oxidative stress is highly related to both of these factors, it was thought that suppressing oxidative stress may lead to improvement of various symptoms of CFS. Therefore, we analyzed the symptom changes before and after the use of an antioxidant combination supplement, Twendee Mtcontrol (TwM), taken by people who had been diagnosed with chronic fatigue syndrome. 2 months of TwM intake resulted not only in an anti-fatigue effect, but also in improvement of pains and QOL, compared to before intake.
Although the effects of various antioxidants have been reported for CFS, most of them are mainly for fatigue, and there are no reports showing significant effects on various symptoms. This may be due to differences in the combination of antioxidants and their antioxidant capacity. The results of this study may be useful for future treatment and prevention of CFS.

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