Ninjin'yoeito Research Articles

Herbal medicine Ninjinyoeito inhibits RANKL-induced osteoclast differentiation and bone resorption activity by regulating NF-κB and MAPK pathway

ObjectivesOsteoporosis is a systemic bone metabolism disorder characterized by decreased bone mass and strength. Osteoclasts (OCs) are giant multinucleated cells that regulate bone homeostasis by degrading bone matrix. Excessive OC differentiation and activity can lead to serious bone metabolic disorders including osteoporosis. Current treatments, including antiresorptive drugs, exert considerable adverse effects, including jaw osteonecrosis. Herbal medicines, such as Ninjinyoeito (NYT), may also offer efficacy, but with fewer adverse effects. In this study, we investigated NYT's effects on osteoclastogenesis. MethodsTartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays were performed to examine NYT's effects on OC differentiation and function. OC-related gene expression at mRNA and protein levels was investigated to confirm NYT's inhibitory action against osteoclastogenesis. We also demonstrated involvement of signaling pathways mediated by IκBα and mitogen-activated protein kinases (MAPK) [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38] and showed nuclear translocation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and nuclear factor kappa B (NF-κB) p65 during osteoclastogenesis. ResultsTRAP staining and bone resorption assays confirmed that NYT significantly inhibited OC differentiation and function. Western blot and RT-PCR results showed that NYT ameliorated osteoclastogenesis by suppressing mRNA and protein level expression of OC-related genes. Moreover, blots and immunocytochemistry (ICC) data clarified that NYT abrogates signaling pathways mediated by IκBα and MAPK (ERK, JNK, p38), and demonstrated nuclear translocation of NFATc1 and NF-κB p65 during OC differentiation. ConclusionsThese findings suggest NYT is an alternative therapeutic candidate for treating osteoporosis.

Ninjinyoeito ameliorates anorexia and changes in peptide YY and ghrelin levels of cisplatin-treated mice

We explored the effect of Ninjinyoeito (NYT) on cisplatin-induced anorexia, which reduces cancer patient survival. Both gastrointestinal motility and plasma concentrations of gastrointestinal peptides were assessed. Nine-week-old ICR female mice received intraperitoneal cisplatin injections (10 mg/kg) and daily oral NYT doses of 300 mg/kg (NYT300) or 1000 mg/kg (NYT1000). Plasma levels of gastrointestinal peptides were measured at 3 and 6 days after cisplatin injection. Gastrointestinal motility was assessed by analyzing the concentration of phenol red marker within sections of the gastrointestinal tract. Cisplatin-injected mice showed a decrease in daily food intake, but this effect was attenuated on day 5 with NYT1000 administration. Although plasma ghrelin levels were reduced on day 3 in cisplatin-treated mice, NYT1000 administration ameliorated this decrease. However, there were no differences in ghrelin levels among all groups on day 6. Levels of peptide YY (PYY) were elevated in the plasma of cisplatin-injected mice on days 3 and 6. Administration of NYT300 and NYT1000 suppressed the increase in PYY levels on day 6 but not on day 3. Gastrointestinal motility was impaired on day 6 in cisplatin-treated mice, but NYT1000 administration attenuated this effect. Our results suggest that NYT improves cisplatin-induced anorexia by suppressing alterations in ghrelin and PYY levels and by increasing gastrointestinal motility. Therefore, NYT may be a promising candidate for alleviating cisplatin-induced anorexia.

Ninjin'yoeito Modulates Baseline and Reperfusion-Induced Changes in the Arteriole Diameter and Blood Flow in the Cerebral Cortex of Anesthetized Mice.

Intragastric administration of ninjin'yoeito (NYT), a traditional Japanese herbal medicine, reportedly prevents the decrease in baseline cerebral blood flow (CBF) in the cortex following gastric administration of water. We investigated the effect of NYT on baseline and dynamic changes in cerebral cortical arteriole diameter. Urethane-anesthetized mice were intragastrically administered 1 g/kg NYT or distilled water (DW). The artery in the left parietal cortex was imaged using two-photon microscopy. The baseline diameter of penetrating arterioles was measured before and 50-60 min after administration. Dynamic CBF and arteriole diameter changes before, during, and after transient occlusion of the left common carotid artery were measured approximately 10 min after administration. DW decreased the baseline diameter of the penetrating arterioles, whereas NYT did not. During occlusion, the increase in penetrating arteriole diameter was comparable for DW and NYT; however, during reperfusion, the return to preocclusion diameter was slower for NYT than DW. Laser-speckle contrast imaging confirmed that CBF, although comparable during occlusion, was higher during reperfusion for NYT than DW. These results suggest that NYT attenuates vasoconstriction in penetrating arterioles after intragastric administration and during cerebral reperfusion, contributing to CBF regulation.

Relationship between the severity of pre-frailty and the degree of adaptation of Ninjin'yoeito (NYT) on pre-frailty.

With the global trend towards longer life expectancies, there's an increasing emphasis on not just living longer, but also maintaining health and wellbeing into older age. This study explores the efficacy of Ninjin'yoeito (NYT) in the early stages of frailty, a critical period for preventive interventions. Taking account of the knowledge gap regarding the association between early frailty and NYT, we use data from workplace health checkups to examine the relationship between pre-frailty severity and NYT adaption. The objective of our research is to enhance the comprehension of early treatments using NYT to prevent the progression of frailty. A total of 314 employees of the Kyoto Industrial Health Association who received workplace health checkups between November 2021 and March 2023 and consented to this study were included in the analysis. Information on gender, age, body mass index (BMI), NYT-specific symptoms assessment, the Japanese version of the General Health Questionnaire-12 (GHQ-12), and the Kihon Checklist (KCL) were obtained. The correlation analysis revealed that there was a strong positive correlation between the number of applicable NYT indications and the GHQ-12 score (r = 0.5992, p < 0.0001). Similarly, a moderate positive correlation was observed between the number of applicable NYT indications and the KCL score (r = 0.5030, p < 0.0001). In the multivariate analysis, both GHQ-12 (β = 0.49, SE = 0.06, t = 7.66, 95% CI: 0.36 to 0.62, p = 0.000) and KCL (β = 0.54, SE = 0.12, t = 4.29, 95% CI: 0.29 to 0.79, p = 0.000) showed significant positive associations with the variance in the number of applicable NYT indications, indicating that higher scores on these measures were related to a greater number of indications. NYT has the potential to be utilized not only as a therapeutic intervention for frailty, but also as a preventive measure.

The protective effects of Ninjin’yoeito against liver steatosis/fibrosis in a non-alcoholic steatohepatitis model mouse

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic form of non-alcoholic fatty liver disease. Liver fibrosis leads to liver cancer and cirrhosis, and drug therapy for NASH remains lacking. Ninjin’yoeito (NYT) has shown antifibrotic effects in a model of liver fibrosis without steatosis but has not been studied for NASH. Therefore, we evaluated the efficacy of NYT in mice fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a NASH model. Compared with the normal diet group, mice fed CDAHFD showed decreased body weight and increased white adipose tissue, liver weight, and triglyceride content in the liver. Furthermore, a substantial increase in the hepatic concentration of hydroxyproline, expression of α-smooth muscle actin (α-SMA), and transforming growth factor-β was observed in CDAHFD-fed mice. Masson’s trichrome and Picro-Sirius red staining revealed a remarkable increase in collagen fiber compared with the normal diet group. Compared with mice that received CDAHFD alone, those supplemented with NYT exhibited reduced hepatic triglyceride and hydroxyproline levels and α-SMA expression. Additionally, compared with the group fed CDAHFD alone, the stained liver tissues of NYT-treated mice exhibited a reduction in Masson’s trichrome- and Picro-Sirius red-positive areas. Locomotor activity was significantly reduced in the CDAHFD-fed group compared with the normal diet group. In the NYT-treated group, the CDAHFD-induced decrease in locomotor activity was significantly suppressed. The findings indicate that NYT inhibited fatty and fibrotic changes in the livers of NASH mice and alleviated the decrease in locomotor activity. Therefore, NYT may serve as a novel therapeutic approach for NASH.Graphical abstract

Evaluating the Practicality of Causal Inference From Non-randomized Observational Data in Small-Scale Clinical Settings: A Study on the Effects of Ninjin'yoeito.

Objective The primary goal of this study was to demonstrate the practical application of causal inference using non-randomized observational data, adapting this approach to smaller populations, such as those in hospitals or community healthcare. This adaptation seeks a more effective and practical research method than randomized controlled trials (RCTs), with the goal of revealing novel insights unexplored by traditional research and enhancing understanding within the realm of causal inference. Methods This study evaluated the effects of Ninjin'yoeito (NYT), a traditional Japanese Kampo medicine, on Overactive Bladder Symptom Score (OABSS) and the frailty scores. Employing new statistical methods, this study sought to illustrate the efficacy of estimating causal relationships from non-randomized data in a clinical setting. The database included 985 women aged 65-90 years who visited a clinic between November 2016 and November 2022. By utilizing various statistical techniques, including regression analysis, inverse probability of treatment weighting (IPTW), instrumental variable (IV), and difference-in-differences (DiD) analysis, this study aimed to provide insights beyond traditional methods, attempting to bridge the gap between theory and practice in causal inference. Results After applying propensity score matching, the NYT treatment group (220 participants) and non-treatment group (182 participants) were each adjusted to two groups of 159 individuals. NYT significantly improved OABSSand frailty scores. IPTW analysis highlighted that on average, the NYT treatment group showed an improvement of 0.8671 points in OABSS and 0.1339 points in the frailty scores, surpassing the non-treatment group (p<0.05). IV analysis indicated that NYT treatment is predicted to increase ΔOABSS by an average of approximately 4.86 points, highlighting its significant positive impact on OABSS improvement. The DiD analysis showed that the NYT treatment group demonstrated an average improvement of 0.5457 points in OABSS, which was significantly higher than that of the control group.The adjusted R² value for the model is 0.025. Conclusion This study successfully implemented a practical application of causal inference using non-randomized observational data in a relatively small population. NYT showed a significant improvement in OABSS and vulnerability, and this result was confirmed using a new statistical method. The relatively low adjusted R² of the model suggests the existence of other unmeasured variables that influence OABSS and vulnerability improvement. In particular, the use of diverse statistical techniques, including IPTW, IV, and DiD analysis, is an important step toward revealing the effectiveness of inferring causal relationships from non-randomized data and narrowing the gap between theory and practice. This study provides a valid and practical alternative to RCTs and reveals new insights that have not been explored in traditional research.

Review of frequently used Kampo prescriptions: Part 4, Ninjin'yoeito

AbstractBackgroundThe source of ninjin'yoeito (NYT) is considered the ‘Formulary of Bureau of Taiping People's Welfare Pharmacy’, written around 1241–52 AD. NYT is used to treat patients with chronic fatigue, malaise with qi deficiency, and exhaustion of all five parenchymatous viscera with difficulty in recovering.Key FindingsNYT formula consists of 12 crude drugs. NYT extract for medical use is covered by the National Health Insurance system of Japan for convalescence after recovery from diseases, fatigue and malaise, anorexia, perspiration during sleep, cold limbs, and anemia. Studies have shown clinical effects of NYT: treatment of anemia, cancer treatment support, frailty, cognitive dysfunction, apathy, fatigue/malaise, anorexia, and lung diseases. Preclinical studies support clinical studies; activating ghrelin‐neuropeptide Y pathway‐mediated appetite‐enhancing effects, inhibiting muscle volume loss through the Akt/mTOR pathway‐mediated hyperphosphorylation of 4E‐BP1 and FoxO1‐mediated Atrogin‐1, suppressing the hypothalamic–pituitary–adrenal axis by downregulating the sympathetic‐adrenal‐medullary axis and GABA neurons for improving sociability, promoting recovery from skeletal muscle atrophy through the activation of PGC‐1α, hematopoiesis‐promoting effects, and increasing the number of immune cells in immunocompromised models. The incidence of adverse events of NYT was reported as 3.09 or 2.03%. The most significant adverse effects of NYT are gastrointestinal disorders.ConclusionRecently, NYT has been widely used to treat several symptoms and conditions under the National Health Insurance System of Japan. Clinical studies showed the efficacy of NYT to treat anemia, frailty, cognitive dysfunction, anorexia, apathy, and fatigue/malaise, as well as for long‐term cancer treatment support. The pharmacological mechanisms supporting its effects are also being reported.

Ninjin'yoeito Improves Genitourinary Symptoms in Patients With Frailty.

Introduction In geriatric medicine, there is currently significant attention on frailty, a condition commonly associated with aging and characterized by muscle weakness and other age-related changes. Within the fields of urology and gynecology, conditions such as overactive bladder (OAB) and genitourinary syndrome of menopause (GSM) have been identified as crucial concerns due to their negative impact on the quality of life of elderly individuals. In this study, we investigated the potential of Ninjin'yoeito (NYT), a traditional Chinese herbal medicine, as a viable treatment option for frailty. Additionally, we hypothesized that NYT may also contribute to the improvement of symptoms associated with OAB and GSM, and potentially help in reducing the dosage of OAB medications. Methods In this retrospective cohort study conducted from November 2016 to November 2022, we created a website describing the relationship between frailty and genitourinary symptoms in frail patients aged ≥ 65 years with GSM who underwent pelvic floor muscle training. The patients were divided into two propensity score-matched groups: NYT group (received NYT for one year) and no-NYT group (did not receive NYT), based on their wishes. The fatigue, resistance, ambulation, illnesses, and loss of weight (FRAIL) scale was used to assess frailty status. Urinary symptoms were evaluated using the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) and Overactive Bladder Symptom Score (OABSS). Genital symptoms were investigated using the vaginal health index score and vulvodynia swab test. The value of each score was obtained before (T0) and 12 months after (T12) treatment, and the difference (ΔT0/T12) was calculated. Results During the study period, 985 outpatients visited our clinic, of whom 725 were considered frail/pre-frail; 402 women with frailty/pre-frailty (mean age 77.5 ± 6.49 years) were included, with a median follow-up of 14.5 months. The NYT and no-NYT groups had 220 and 182 patients, respectively. After propensity score matching, each group had 159 patients. ΔT0/T12FRAIL scale score was significantly higher in the NYT group (0.13 ± 0.37) than in the no-NYT (0.01 ± 0.10) group (p=0.001.) However, urinary symptoms improved in the NYT group more than in the no-NYT in terms of the following parameters: ΔT0/T12OABSS (NYT: 0.89 ± 1.65; no-NYTl: 0.36 ± 1.14, p=0.001) and ΔT0/T12ICIQ-SF score (NYT: 1.51 ± 1.75; no-NYT: 0.42 ± 1.18, p<0.001). Genital symptoms were better in the NYT group in terms of ΔT0/T12VHIS (NYT: 0.58 ±1.08; no-NYT: 0.21 ±0.65, p<0.001). The vulvodynia swab test showed improvements in left para-hymen evoked pain in both groups. In the NYT group, 5% of the patients underwent antimuscarinic drug dose reduction for overactive bladder treatment. NYT use was not associated with significant side effects, and only 0.6% of patients reported drug allergies. Conclusion NYT improved activity levels in frail/pre-frail patients. Moreover, NYT use improved various genitourinary symptoms experienced by patients with frailty/pre-frailty. Treatment with NYT may reduce the dose of overactive bladder medications. The anticholinergic load-reducing effect of NYT may help solve the problem of polypharmacy.

Frailty Improvement by Multicomponent Drug, Ninjin'Yoeito, in Mild Cognitive Impairment and Mild Alzheimer's Disease Patients: An Open-Label Exploratory Study (FRAMINGO).

Alzheimer's disease (AD) and dementia have increasingly been conceived of as "complex diseases of aging", determined by multiple, simultaneous, interacting pathophysiological processes. The condition known as frailty is a phenotype of aging and its comprehensive pathophysiology is thought to be closely related to the incidence of mild cognitive impairment (MCI) and the exacerbation of dementia. This study aimed to investigate the effect of the multicomponent drug, ninjin'yoeito (NYT), on frailty in MCI and mild AD patients. This study was an open-label trial. A total of 14 patients, including 9 with MCI and 5 with mild AD, were enrolled. Among them, 11 were frail while 3 were prefrail. NYT (6-9 g/day) was administered orally for 24 weeks, and assessments were carried out at baseline (week 0), and at 4, 8, 16, and 24 weeks. In the primary endpoint, significant early improvements were observed in the anorexia scores according to the Neuropsychiatric Inventory after four weeks of treatment with NYT. The Cardiovascular Health Study score was significantly improved, and no frailty was observed after 24 weeks. The fatigue visual analog scale scores also significantly improved. The Clinical Dementia Rating and the Montreal Cognitive Assessment scores remained at baseline levels during the NYT treatment period. The results suggest that NYT may be effective in the treatment of frailty, especially for anorexia and fatigue, in both MCI and mild AD patients, which would be beneficial for the prognosis of dementia.

The Effects of Ninjinyoeito on Impaired Spatial Memory and Prefrontal Cortical Synaptic Plasticity through α-Amino-3-hydroxy-5-4-isoxazole Propionic Acid Receptor Subunit in a Rat Model with Cerebral Ischemia and β-Amyloid Injection.

Ninjinyoeito (NYT), a traditional Japanese medicine, is effective for improving physical strength and treating fatigue and anorexia. Recently, a clinical report revealed that NYT ameliorates cognitive dysfunction in Alzheimer's disease (AD) patients, although the mechanisms remain unclear. AD is a neurodegenerative disorder accompanied by a progressive deficit in memory. Current therapeutic agents are largely ineffective in treating cognitive dysfunction in AD patients. In this study, we investigated the effects of NYT on spatial memory impairment in a rat model of dementia. Rats were prepared with transient cerebral ischemia and intraventricular injection of β-amyloid1-42 for 7 days (CI + Aβ). NYT was orally administered for 7 days after cerebral ischemia. We evaluated spatial memory using the Morris water maze and investigated the expression of α-amino-3-hydroxy-5-4-isoxazole propionic acid receptor subunits, the phosphorylation level of glutamate receptor A (GluA)1 at serine sites S831 and S845, and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the hippocampus and prefrontal cortex of CI + Aβ rats. In the CI + Aβ rats, NYT treatment shortened the extended time to reach the platform. However, NYT did not restore the decrease in the hippocampal GluA1, GluA2, or CaMKII expression but increased prefrontal cortical phosphorylation levels of S845-GluA1 and CaMKII. Therefore, NYT may alleviate spatial memory impairment by promoting glutamatergic transmission involved in the phosphorylation of S845-GluA1 and CaMKII in the prefrontal cortex of CI + Aβ rats. Our results suggest that NYT is a valuable treatment for AD patients.

Effects of Ninjin'yoeito on Human CYP3A and Mouse CYP3A Activity.

Ninjin'yoeito (NYT) is widely used clinically for the management of patients with frailty and other multiple symptoms. NYT is often administered with other drugs; however, little information is available on its drug interactions. Previous studies using human liver microsomes have reported that constituents of NYT either inhibit (schisandra fruit, cinnamon bark, glycyrrhiza, and poria sclerotium) or induce (schisandra fruit and glycyrrhiza) CYP3A4 expression. Herein, we conducted in vitro and in vivo studies targeting human CYP3A and mouse CYP3A to elucidate the effects of NYT coadministration with other drugs on hepatic drug metabolism. In an inhibition study using human liver microsomes, NYT showed concentration-dependent reversible inhibition and time-dependent inhibition. Furthermore, in an induction study using frozen human hepatocytes, the addition of 0.01-0.1 mg/mL NYT resulted in a concentration-dependent increase in CYP3A gene expression. Contrarily, no significant changes in CYP3A substrate blood concentrations were observed between untreated mice and mice that received either a single dose of NYT or repeated doses for 15 days. These results demonstrate that NYT has inhibitory and inductive effects on hepatic CYP3A in vitro, but orally administered NYT does not affect drug metabolism mediated by hepatic CYP3A in vivo in the mouse model. Although there is a little information about drug interactions of NYT, this study provides new evidence for that.

Ninjinyoeito Prevents Onset of Depression-Like Behavior and Reduces Hippocampal iNOS Expression in Senescence-Accelerated Mouse Prone 8 Mice.

Late-life depression is a globally prevalent disorder. Ninjinyoeito (NYT), a traditional Japanese herbal medicine, attenuates depressive symptoms in older patients. However, the mechanisms underlying the antidepressive effect of NYT are unknown. In this study, we investigated the mechanism of the action of NYT using senescence-accelerated mouse prone 8 (SAMP8) mice, which exhibit accelerated aging. SAMP8 mice were treated with NYT starting at 12 weeks of age. Twelve-week-old SAMP8 mice did not show prolonged immobility time in the tail suspension test compared with age-matched SAMR1 mice (normal aging control). At 34 weeks of age, vehicle-treated SAMP8 mice displayed prolonged immobility time compared with SAMR1 mice. NYT-treated SAMP8 mice showed a shorter immobility time than that of vehicle-treated SAMP8 mice. Notably, NYT decreased hippocampal inducible nitric oxide synthase (iNOS) expression in SAMP8 mice. There was no difference in iNOS expression between SAMR1 and vehicle-treated SAMP8 mice. Subchronic (5 days) administration of an iNOS inhibitor, 1400 W, shortened the immobility time in SAMP8 mice. These results suggest that NYT prevents an increase in immobility time of SAMP8 mice by decreasing iNOS levels in the hippocampus. Therefore, the antidepressive effect of NYT in older patients might be mediated, at least in part, by the downregulation of iNOS in the brain. Our data suggest that NYT is useful to prevent the onset of depression with aging.

Ninjin'yoeito suppressed the onset of arthritis, pain, and muscle atrophy in rheumatoid arthritis model mice.

Rheumatoid arthritis is one of the most common diseases in orthopedic surgery. The main symptoms are joint pain and systemic symptoms. In recent years, rheumatoid arthritis is known to cause sarcopenia. Ninjin'yoeito (NYT), a traditional Japanese medicine, has been prescribed for patients with post-illness or post-operative weakness, fatigue, loss of appetite, rash, cold limbs, and anemia. In addition to its traditional use, NYT has been prescribed for treating frailty in gastrointestinal, respiratory, and urinary functions. Further, NYT is known to be effective in suppressing muscle atrophy in the prior literature. The present study aimed to investigate whether NYT suppresses various symptoms of the Collagen-induced arthritis (CIA) model. Long-term administration of NYT inhibited the increases in arthritis scores, decreases pain threshold, and muscle atrophy in the CIA model. In addition, NYT inhibited the elevation of the plasma IL-6 level. These results suggest that NYT may have therapeutic effects on symptoms, muscle atrophy and increase in plasma IL-6 level caused by rheumatoid arthritis.

Metallothionein synthesis increased by Ninjin-yoei-to, a Kampo medicine protects neuronal death and memory loss after exposure to amyloid β1-42

BackgroundIt is possible that increased synthesis of metallothioneins (MTs), Zn2+-binding proteins is linked with the protective effect of Ninjin-yoei-to (NYT) on Zn2+ toxicity ferried by amyloid β1-42 (Aβ1-42).MethodsJudging from the biological half-life (18-20 h) of MTs,the effective period of newly synthesized MT on capturing Zn2+ isestimated to be approximately 2 days. In the present paper, a diet containing3% NYT was administered to mice for 2 days and then Aβ1-42 was injectedinto the lateral ventricle of mice.ResultsMT level in the dentate granule cell layer was elevated 2 days after administration of NYT diet, while the administration reduced intracellular Zn2+ level increased 1 h after Aβ1-42 injection, resulting in rescuing neuronal death in the dentate granule cell layer, which was observed 14 days after Aβ1-42 injection. Furthermore, Pre-administration of NYT diet rescued object recognition memory loss via affected perforant pathway long-term potentiation after local injection of Aβ1-42 into the dentate granule cell layer of rats.ConclusionThe present study indicates that pre-administration of NYT diet for 2 days increases synthesis of MTs, which reduces intracellular Zn2+ toxicity ferried by extracellular Aβ1-42, resulting in protecting neuronal death in the dentate gyrus and memory loss after exposure to Aβ1-42.

Impact of Ninjin'yoeito on frailty and short life in klotho-hypomorphic (kl/kl) mice.

With the recent aging of society, the prevention of frailty has become an important issue because people desire both a long and healthy lifespan. Klotho-hypomorphic (kl/kl) mice are known to show phenotypes of premature aging. Ninjin'yoeito (NYT) is a traditional Japanese Kampo medicine used to treat patients with vulnerable constitution, fatigue or physical exhaustion caused by aging and illness. Recent studies have reported the potential efficacy of NYT against frailty. We therefore evaluated the effect of NYT on the gait function, activity, the histopathological status of organs and survival using kl/kl mice as a model of aging-related frailty. Two sets of 28-day-old male kl/kl mice were assigned to the vehicle (non-treated; NT), 3% or 5% NYT dietary groups. One set of groups (NT, n = 18; 3% NYT, n = 11; 5% NYT, n = 11) was subjected to the analysis of free walking, rotarod, and spontaneous activity tests at approximately 58days old. Thereafter, we measured triceps surae muscles weight and myofiber cross-sectional area (CSA), and quantified its telomere content. In addition, we evaluated bone strength and performed histopathological examinations of organs. Survival was measured in the second set of groups (NT, 3% NYT and 5% NYT group, n = 8 each). In the walking test, several indicators such as gait velocity were improved in the NYT 3% group. Similar results were obtained for the latency to fall in the rotarod test and spontaneous motor activity. Triceps muscle mass, CSA and its telomere content were significantly improved in the NYT 3% group. Bone density, pulmonary alveolus destruction and testicular atrophy were also significantly improved in the NYT 3% group. Survival rate and body weight were both significantly improved in the NYT3% group compared with those in the NT group. Continuous administration of NYT from the early stage of aging improved not only gait performance, but also the survival in the aging-related frailty model. This effect may be associated with the improvements in aging-related organ changes such as muscle atrophy. Intervention with NYT against the progression of frailty may contribute to a longer, healthier life span among the elderly individuals.

Ninjin'yoeito ameliorated PPE-induced pulmonary emphysema and anxiety/depressive-like behavior in aged C57BL/6J mice.

The prevalence of chronic obstructive pulmonary disease (COPD) is increasing in the elderly. COPD is a chronic respiratory disease characterized by airway remodeling and alveolar emphysema. COPD patients are also at high risk for mental illnesses such as depression and anxiety. Ninjin’yoeito (NYT) is prescribed to patients with conditions such as post-illness and postoperative weakness, fatigue, poor appetite, skin rash, cold hands and feet, and anemia. In addition to traditional uses, NYT is also prescribed as a therapeutic drug for poor functioning of the digestive organs, respiratory organs, and urinary organs. NYT is also known to have an antioxidant effect. The objective of this study was to investigate whether NYT could ameliorate COPD-induced lung injury and anxiety/depression in aged C57BL/6J mice exposed to porcine pancreatic elastase (PPE). While intratracheal administration of PPE induced emphysema in elderly mice, long-term administration of NYT suppressed the pathology. NYT was also found to suppress the apoptosis and oxidative stress caused by PPE. In addition, long-term administration of NYT was found to ameliorate PPE-induced depressive-like behavior in three different behavioral studies. These results suggest that NYT has a therapeutic effect on emphysema and the behavioral abnormalities caused by PPE.

Ninjinyoeito improves social behavior disorder in neuropeptide Y deficient zebrafish.

Sociability is an essential component of the linkage structure in human and other vertebrate communication. Low sociability is defined as a poor social approach, including social withdrawal and apathy, and is implicated in a variety of psychiatric disorders. Ninjinyoeito (NYT), a traditional Japanese herbal medicine, has been used in the medical field. This study aimed to determine the effect of NYT on low sociality in NPY-KO zebrafish. NPY-KO zebrafish were fed a 3% NYT-supplemented diet for 4 days and subjected to behavioral tests. In the mirror test, NPY-KO zebrafish fed a control diet showed avoidance behavior toward their mirror counterparts. In contrast, the treatment of NPY-KO zebrafish with NYT significantly increased their interaction with their counterparts in the mirror. In addition, a 3-chambers test was conducted to confirm the effect of NYT on the low sociality of NPY-KO zebrafish. NPY-KO zebrafish fed the control diet showed less interaction with fish chambers, while NYT treatment increased the interaction. Phosphorylation of ERK, a marker of neuronal activity, was significantly reduced in the whole brain of NYT-fed NPY-KO zebrafish, compared to the control diet. NYT treatment significantly suppressed hypothalamic-pituitary-adrenal-related genes (gr, pomc, and crh) and sympathetic-adrenal-medullary-related genes (th1, th2, and cck) in NPY-KO zebrafish. NYT administration significantly reduced mRNA levels of gad1b compared to the control diet, suggesting the involvement of GABAergic neurons in NYT-induced improvement of low sociability. Furthermore, the expression of CREB was suppressed when NPY-KO zebrafish were fed NYT. Next, we attempted to identify the effective herb responsible for the NYT-induced improvement of low sociability. NPY-KO zebrafish were fed an experimental diet containing the target herb for 4 days, and its effect on sociability was evaluated using the 3-chambers test. Results showed that Cinnamon Bark and Polygala Root treatments significantly increased time spent in the fish tank area compared to the control diet, while the other 10 herbs did not. We confirmed that these two herbs suppressed the activity of HPA-, SAM-, and GABAergic neurons, as well as NYT-treated zebrafish, accompanied by downregulation of CREB signaling. This study suggests the potential use of NYT as a drug for sociability disorders.

Unkeito Suppresses RANKL-Mediated Osteoclastogenesis via the Blimp1–Bcl6 and NF-κB Signaling Pathways and Enhancing Osteoclast Apoptosis

Osteoporosis is a common bone disease, particularly in menopausal women. Herein, we screened four Kampo medicines (Unkeito (UKT), Kamishoyosan (KSS), Kamikihito (KKT), and Ninjinyoeito (NYT)), frequently used to treat menopausal syndromes, for their effects on receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast differentiation in RAW 264 cells. Considering that UKT exhibited the most potent effect, we examined its effect on RANKL-induced osteoclastogenesis, the induction of osteoclast apoptosis, and the mechanisms underlying its effects. UKT inhibits RANKL-induced osteoclast differentiation in the early stage and decreases osteoclast-related genes, including tartrate-resistant acid phosphatase (Trap), dendritic cell-specific transmembrane protein (Dcstamp), matrix metalloproteinase-9 (Mmp9), and cathepsin K (Ctsk). Specifically, UKT inhibits the nuclear factor of activated T cells 1 (NFATc1), which is essential for osteoclastogenesis. UKT increases Bcl6, which antagonizes NFATc1 and Dc-stamp, thereby blocking the progression of osteoclasts to maturation. UKT also decreased nuclear translocation by downregulating the activity of p65/NF-κB. In addition, UKT enhances mononuclear osteoclast apoptosis via activation of caspase-3. Herein, we demonstrate that UKT suppresses RANKL-mediated osteoclastogenesis via the Blimp1–Bcl6 and NF-κB signaling pathways and enhances mononuclear osteoclast apoptosis. Furthermore, UKT prevents bone loss in OVX mice. Thus, UKT might be a potential therapeutic agent for postmenopausal osteoporosis.

Safety and efficacy of Ninjin’yoeito along with iron supplementation therapy for preoperative anemia, fatigue, and anxiety in patients with gynecological disease: an open-label, single-center, randomized phase-II trial

BackgroundPreoperative anemia affects perioperative outcomes and often causes fatigue and psychological disorders. Therefore, anemia should be treated before a patient undergoes surgery. Ninjin’yoeito (NYT), a Japanese Kampo medicine composed of ginseng and Japanese angelica root with the other 10 herbs, is administered for anemia, fatigue and anxiety; however, there are a few reports that have prospectively examined the effects of NYT before surgery for gynecological diseases. Hence, we tended to investigate its efficacy and safety.MethodsIn this open-label randomized trial, women with gynecological diseases accompanied by preoperative anemia (defined as < 11.0 g/dL Hemoglobin [Hb]) were randomly assigned (1:1) into the iron supplementation and NYT groups. Patients of the iron supplementation group and the NYT group received 100 mg/day iron supplementation with and without NYT (7.5 g/day) for at least 10 days before surgery. The primary endpoint was improvement in Hb levels before and after treatment, and Cancer Fatigue Scale (CFS) and Visual Analogue Scale for Anxiety (VAS-A) scores between groups. Statistical analyses were performed with Wilcoxon signed rank test, Wilcoxon rank sum test, and Fisher’s exact test as appropriate.ResultsForty patients were enrolled of whom 30 patients were finally analyzed after allocating 15 to each group. There was no difference in the characteristics between both groups. Hb significantly increased in both groups (iron supplementation group, 9.9 ± 0.8 g/dL vs. 11.9 ± 1.6 g/dL; NYT group, 9.8 ± 1.0 g/dL vs. 12.0 ± 1.0 g/dL); the difference in the elevations in Hb between both groups was statistically insignificant (P = 0.72). Contrarily, CFS (17.9 ± 10.2 vs. 8.1 ± 5.2) and VAS-A (56 mm (50–70) vs. 23 mm (6–48)) scores were significantly decreased only in the NYT group and these changes were greater in the NYT group (∆CFS, P = 0.015; ∆VAS-A, P = 0.014). Liver dysfunction occurred in one patient of the NYT group.ConclusionsFor treating preoperative anemia in women with gynecological conditions, NYT administration along with iron supplementation safely and efficiently improved the preoperative fatigue and anxiety in addition to the recovery from anemia.Trial registration: jRCT1051190012 (28/April/2019, retrospectively registered).

Ninjin'yoeito, a traditional Japanese medicine, attenuates age-related deficits of muscle performance, self-care motivation, and body temperature in C57BL/6 mice.

As both physiological and psychological factors influence age-associated declines in older people, the development of drug therapy with multifaceted effects is required. To investigate the utility of ninjin'yoeito (NYT) against geriatric syndromes, we evaluated the effects of NYT on age-related declines in old C57BL/6 mice (88-week-old) as a preclinical model of frailty progression. Here, we showed that NYT reversed the decline of rectal temperature in old mice and also improved forelimb grip strength compared with that in the old control group without affecting skeletal muscle loss. Moreover, NYT significantly increased the duration of grooming after a sucrose solution was sprayed, which reflected self-care motivation. Finally, we revealed the antioxidant effects of NYT using a cell-free assay. These results suggest that NYT can improve both physiological and psychological declines associated with aging, and the mechanism may include antioxidant effects. NYT may have potential utility for maintaining the health of older people.