Increasing evidence suggests a link between gut microbial dysbiosis and the pathogenesis of depression. Alpha-glycosyl isoquercitrin (AGIQ), consisting of isoquercitrin and its glycosylated quercetin, has beneficial effects on the gut microbiome and brain function. Here, we detected the potential antidepressant impact of a four-week administration of AGIQ and its underlying mechanisms using a mouse model of depression.

Male C57BL/6 mice were orally administered AGIQ (0.05 % or 0.5 % in drinking water) for 28 days; subchronic social defeat stress was performed in the last 10 days. Behavior tests were conducted to assess anxiety and depressive-like behaviors. Additionally, evaluations encompassed 5-hydroxytryptamine (5-HT) levels, the gut microbiota composition, lipopolysaccharide (LPS) concentrations, short-chain fatty acids levels, and intestinal barrier integrity changes.

AGIQ significantly alleviated depression-like behaviors and increased hippocampal 5-HT levels. Further, AGIQ mitigated stress-induced gut microbial abnormalities and reduced the levels of LPS in the serum, which affected the relative gene expression levels of 5-HT biosynthesis enzymes in vitro. Furthermore, AGIQ reversed the reduced butyrate levels in cecal contents and improved the impaired intestinal barrier by increasing the expression of colonic zonula occluden-1 (ZO-1) and occludin, thereby decreasing LPS leakage.

Our results suggest that AGIQ could improve stress-induced depression by regulating the gut microbiome, which inhibits LPS production and maintains the gut barrier. This is the first report on the potential effect of AGIQ on depression via the gut microbiota-brain axis, shedding new light on treatment options.

Polyinosinic-polycytidylic acid (PIC) provides a model of developmental neuropathy by inducing maternal immune activation. We investigated the effects of an antioxidant, alpha-glycosyl isoquercitrin (AGIQ), on PIC-induced developmental neuropathy in rats, focusing on postnatal hippocampal neurogenesis. On gestational day 15, PIC at 4 mg/kg body weight was administered to dams intravenously. AGIQ either at 0.25% or 0.5% was administered through the diet to dams from gestational day 10 until weaning on day 21 post-delivery and, thereafter, to offspring until postnatal day 77 (adult stage). At weaning, the numbers of TBR2⁺ cells and PCNA⁺ cells in the subgranular zone and reelin⁺ cells in the dentate gyrus hilus in offspring of dams treated with PIC only were decreased compared with untreated controls. In contrast, 0.5% AGIQ ameliorated these changes and increased the transcript levels of genes related to signaling of reelin (Reln and Vldlr), growth factors (Bdnf, Cntf, Igf1, and Igf1r), and Wnt/β-catenin (Wnt5a, Lrp6, Fzd1, and Fzd3). In adults, AGIQ increased the number of FOS⁺ granule cells at 0.25% and the transcript levels of NMDA-type glutamate receptor genes, Grin2a and Grin2b, at 0.25% and 0.5%, respectively. These results suggest that mid-gestation PIC treatment decreased the abundance of type-2b neural progenitor cells (NPCs) by reducing NPC proliferation in relation with suppression of reelin signaling at weaning. We suggest that AGIQ ameliorated the PIC-induced suppressed neurogenesis by enhancing reelin, growth factor, and Wnt/β-catenin signaling at weaning to rescue NPC proliferation and increased synaptic plasticity by enhancing glutamatergic signaling via NMDA-type receptors after maturation.

alpha-Glycosyl isoquercitrin (AGIQ) is a flavonoid that possesses antioxidant and tumor suppressive capabilities and is marketed as a food additive in Japan. The aim of this study was to assess the potential for oral chronic toxicity and carcinogenicity of AGIQ in male and female Sprague Dawley rats following up to 5.0% dietary exposure. In the chronic toxicity study, rats were exposed to AGIQ or vehicle for one year with a 6-month interim termination point; for the carcinogenicity study, rats were treated for 24 months. No signs of AGIQ-related toxicity clinically or histologically were observed for up to one year except for yellow discoloration of bone. In the carcinogenicity study, a statistically significant increase in the incidence of malignant glioma of the brain or spinal cord was observed in female rats exposed to 5.0% AGIQ compared to those exposed to control feed. A Scientific Advisory Panel of experienced neuropathologists reviewed the gliomas (routine stains and glial cell markers) and concluded that the gliomas were a rare, spontaneous, rat-specific neoplasm: malignant microglial tumor. The lesions could not definitively be attributed to AGIQ exposure and have limited implications with respect to predicting human cancer risk.

Quercetin and its glycosides, such as isoquercitrin or rutin, are among the most ubiquitous flavonoids present in plants. They possess numerous health-promoting properties, whose applicability is, however, limited by poor water solubility and absorption issues. Enzymatically modified isoquercitrin (EMIQ) is an isoquercitrin derivative obtained from rutin via enzymatic transformations that greatly enhance its bioavailability. Due to advantageous reports on its safety and bioactivity, EMIQ is currently gaining importance as a food additive and a constituent of dietary supplements. This review summarizes the thus-far-conducted investigations into the metabolism, toxicity, biological properties, and molecular mechanisms of EMIQ and presents a comprehensive characterization of this valuable substance, which might represent the future of flavonoid supplementation.

Flavonoids such as quercetin and its glucosides, especially isoquercitrin are well known as anti-inflammatory, anti-allergic, and anti-carcinogenic, etc. The safety of isoquercitrin formulations needs to be established prior to their use in functional food applications. The mutagenicity and genotoxicity of the IQC-γCD inclusion complex were assessed with three standard assays of the bacterial reverse mutation assay (Ames test) and using a combined in-vivo micronucleus and comet assay under the Organisation for Economic Co-operation and Development (OECD) guidelines. In combined rat bone marrow micronucleus and rat liver comet assay performed in male Sprague Dawley (SD) rats, the various doses of IQC-γCD inclusion complex (max. 2000 mg/kg bw) and positive controls ethyl methanesulfonate (EMS) and mitomycin C (MMC), respectively, and negative control (vehicle) were administrated. The results of the Salmonella typhimurium mutagenicity assay (strains TA100, TA1535, WP2uvrA, TA98, and TA1537) after exposure to the IQC-γCD inclusion complex with the absence and presence of the metabolic activation system (S9 fraction from rat liver) revealed a weakly positive response but with no biologically relevant mutagenicity at the conditions examined according to recommended regulatory guidelines. The combined micronucleus and comet assay results reveal that the IQC-γCD inclusion complex did not induce in-vivo genotoxic potential or indication of any oxidative DNA damage in rat liver tissues. Altogether, considering the results of the study, it is unlikely that the consumption of IQC-γCD inclusion complex as food or supplement would present any concern for humans regarding the mutagenicity and genotoxicity.

Flavonoids such as quercetin and its glycoside Isoquercitrin and are abundantly present in the diet and have various pharmacological effects. However, limited data about its potential toxicity is available. In this study, we aim to evaluate the subchronic toxicity of the isoquercitrin-γ-cyclodextrin (IQC-γCD) molecular inclusion complex (SunActive® QCD/EN) in Sprague-Dawley (SD) rats. The IQC-γCD was administrated orally to 40 male and 40 female SD rats at dietary doses up to 5.0 % for 13 consecutive weeks. During the experiment periods, the general clinical signs, mortality, hematological, urinalysis values, biochemical, and histopathological parameters were examined. All animals survived until the scheduled necropsy, and no statistically significant or clinical sign of toxicologically relevant differences including pathology parameters, and histopathological endpoints were observed in any of the IQC-γCD treatment groups, compared with the control group. However, certain observations were noted in the male rats treated with the highest concentration (5.0 %), but these were not seen in female rats. A slight inhibition of weight gain was observed, probably linked to a fall in red blood cells, and hematocrit index in female rats. Statistically significant changes were noted in some clinical measures, such as plasma bilirubin level, alkaline phosphatase total bile acid without evidence of systemic clinical toxicity. The results support no observed adverse effect level (NOAEL) of IQC-γCD of 5.0 % in the diet for males (3338.55 mg/kg/day), and 3.0 % in the diet for females (2177.33 mg/kg/day) SD rats. Therefore, in this 13 weeks repeated-dose SD rat study there were no treatment-related adverse clinical or pathological findings for IQC-γCD of 5.0 % in the diet for males, and 3.0 % in the diet for females SD rats. The results of the present study support the safe use of IQC-γCD as a functional food, food additive, and natural ingredient.

alpha-Glycosyl Isoquercitrin (AGIQ), is used in Japan as a food additive and was granted generally recognized as safe (GRAS) status in 2005 (FEMA) and 2007 (FDA). The safety and toxicity information for AGIQ is sparse and therefore, the carcinogenicity potential of AGIQ was examined in the CByB6F1-Tg(HRAS)2Jic (rasH2) model. One hundred female and male rasH2 mice, each, were allocated to one of four designated dose groups; 0 (control)%, 1.5%, 3.0% or 5.0% AGIQ. Animals were administered the diets for six months and an additional 10 females and 10 males, each, were administered a positive control, N-methyl-N-nitrosourea (MNU). Body weights and clinical observations were collected. A full screen necropsy, organ weights, clinical chemistry, urinalysis and histopathology were performed. The positive control animals elicited appropriate responses specific to this strain (rasH2) of mice. There were statistically significant sporadic non-dose-dependent changes in clinical chemistries without corresponding pathological correlation. No microscopic AGIQ-related findings were noted; the range of pathology observations were all considered background findings, either specific to rasH2 mice or common to inbred strains of mice. Therefore, under the study conditions, the no-observed-adverse-effect level (NOAEL) was determined to be more than 5.0% (7215.4 mg/kg BW/day in male mice and 14685.5 mg/kg/day in female mice).

Soy isoflavones are bioactive phytoestrogens with known health benefits. Soybean embryo extract (SEE) has been consumed as a source of isoflavones, mainly daidzein, glycitein, and genistein. While previous studies have reported the anti-obesity effects of SEE, this study investigates their molecular mechanisms and the synergistic effects of co-treatment with SEE and enzymatically modified isoquercitrin (EMIQ). SEE upregulated genes involved in lipolysis and brown adipocyte markers and increased mitochondrial content in differentiated C3H10T1/2 adipocytes in vitro. Next, we use a high-fat diet-induced obesity mouse model to determine the anti-obesity effect of SEE. Two weeks of single or combined treatment with SEE and EMIQ significantly reduced body weight gain and improved glucose tolerance. Mechanistically, SEE treatment increased mitochondrial content and upregulated genes involved in lipolysis in adipose tissue through the cAMP/PKA-dependent signaling pathway. These effects required a cytosolic lipase adipose triglyceride lipase (ATGL) expression, confirmed by an adipocyte-specific ATGL knockout mouse study. Collectively, this study demonstrates that SEE exerts anti-obesity effects through the activation of adipose tissue metabolism and exhibits a synergistic effect of co-treatment with EMIQ. These results improve our understanding of the mechanisms underlying the anti-obesity effects of SEE related to adipose tissue metabolism.

The synthesis and biotransformation of five flavones containing methoxy substituents in the B ring: 2'-, 3'-, 4'-methoxyflavones, 2',5'-dimethoxyflavone and 3',4',5'-trimethoxyflavone are described. Strains of entomopathogenic filamentous fungi were used as biocatalysts. Five strains of the species Beauveria bassiana (KCh J1.5, J2.1, J3.2, J1, BBT), two of the species Beauveria caledonica (KCh J3.3, J3.4), one of Isaria fumosorosea (KCh J2) and one of Isaria farinosa (KCh KW 1.1) were investigated. Both the number and the place of attachment of the methoxy groups in the flavonoid structure influenced the biotransformation rate and the amount of nascent products. Based on the structures of products and semi-products, it can be concluded that their formation is the result of a cascading process. As a result of enzymes produced in the cells of the tested strains, the test compounds undergo progressive demethylation and/or hydroxylation and 4-O-methylglucosylation. Thirteen novel flavonoid 4-O-methylglucosides and five hydroxy flavones were isolated and identified.

Previous rat toxicity studies of alpha-glycosyl isoquercitrin (AGIQ), a water-soluble flavonol glycoside derived from rutin, revealed systemic yellow bone discoloration. This investigative study was conducted to determine the AGIQ metabolite(s) responsible for the discoloration. Female Sprague-Dawley rats were administered dietary AGIQ at doses of 0%, 1.5%, 3.0%, or 5.0% (0, 1735.0, 3480.8, and 5873.7 mg/kg/day, respectively) for 14 days, followed by a 14- or 28-day recovery period. Measurements of quercetin in urine and quercetin, quercetin 3-O-glucuronide, kaempferol, and 3-o-methylquercetin metabolites of AGIQ in bone (femur), white and brown fat, and cerebrum samples were conducted following the exposure period and each recovery period. Gross examination of the femur revealed yellow discoloration that increased in intensity with dose and was still present in a dose-related manner following both recovery periods. Quercetin, at levels correlating with AGIQ dose, was measured in the urine following the 14-day exposure period and, at lower concentrations, 14 or 28 days following cessation of AGIQ exposure. All four metabolites were present in a dose-dependent manner in the femur following 14 days of dietary exposure; only quercetin, quercetin 3-O-glucuronide, and 3-o-methylquercetin were present during the recovery periods. Quercetin, quercetin 3-O-glucuronide, and 3-o-methylquercetin were detected in white fat (along with kaempferol), brown fat (excluding quercetin due to analytical interference), and cerebrum samples, indicating systemic availability of the metabolites. Collectively, these data implicate quercetin, quercetin 3-O-glucuronide, or 3-o-methylquercetin (or a combination thereof) as the most likely metabolite of AGIQ causing the yellow discoloration of bone in rats administered dietary AGIQ.

Quercetin and its glycosides possess potential benefits to human health. Several flavonols are available to consumers as dietary supplements, promoted as anti-oxidants; however, incorporation of natural quercetin glycosides into food and beverage products has been limited by poor miscibility in water. Enzymatic conjugation of multiple glucose moieties to isoquercitrin to produce alpha-glycosyl isoquercitrin (AGIQ) enhances solubility and bioavailability. AGIQ is used in Japan as a food additive and has been granted generally recognized as safe (GRAS) status. However, although substantial genotoxicity data exist for quercetin, there is very little available data for AGIQ and isoquercitrin. To support expanded global marketing of food products containing AGIQ, comprehensive testing of genotoxic potential of AGIQ and isoquercitrin was conducted according to current regulatory test guidelines. Both chemicals tested positive in bacterial reverse mutation assays, and exposure to isoquercitrin resulted in chromosomal aberrations in CHO-WBL cells. All other in vitro mammalian micronucleus and chromosomal aberration assays, micronucleus and comet assays in male and female B6C3F1 mice and Sprague Dawley rats, and Muta™ Mouse mutation assays evaluating multiple potential target tissues, were negative for both chemicals. These results supplement existing toxicity data to further support the safe use of AGIQ in food and beverage products.

Dietary flavonoid intake shows a significant inverse association with mortality from coronary heart disease, incidence of myocardial infarction and stroke. Quercetin is one of the most common flavonoids in our diet and has several favorable biological activities. Quercetin glucosides, which are enzymatically trans-glycosylated isoquercitrin, have high water-solubility and bioavailability compared with quercetin. Here, we investigated the effects of quercetin glucosides on collateral development in a murine hindlimb ischemia model.

We induced hindlimb ischemia in 24- to 32-week-old male C3H/HeJ mice by resecting the right femoral artery. Then, 0.5% carboxymethyl cellulose (control) or quercetin glucosides (100mg/kg/day) were administered daily by gavage. Blood flow was monitored weekly by laser Doppler imaging.

Recovery of blood flow to the ischemic leg was significantly enhanced by quercetin glucosides (blood flow ratio at 4 weeks: control, 0.57 ± 0.11; quercetin glucosides, 0.95 ± 0.10, p<0.05). Furthermore, anti-CD31 immunostaining revealed that quercetin glucosides increased capillary density in the ischemic muscle (control, 200 ± 24/mm(2); quercetin glucosides, 364 ± 41/mm(2), p<0.01). Quercetin glucosides did not promote tumor growth. The beneficial effect of quercetin glucosides was abrogated in eNOS-deficient mice.

These results suggest that quercetin glucosides may have therapeutic potential to promote angiogenesis in ischemic tissue.

To investigate the protective effect of enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and thioacetamide (TAA)-promoted rats. We examined the modifying effect of co-administration with EMIQ on the liver tissue environment including hepatic macrophages and lymphocytes and on the induction mechanism of preneoplastic cell apoptosis during early stages of hepatocellular tumor promotion. TAA increased the number and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of proliferating and apoptotic cells in randomly selected areas in liver sections. Co-administration with EMIQ suppressed these effects. TAA also increased the numbers of ED2(+), cyclooxygenase-2(+), and heme oxygenase-1(+) liver cells, as well as the number of CD3(+) lymphocytes. These effects were also suppressed by EMIQ. EMIQ increased liver levels of thiobarbituric acid-reactive substance and 8-hydroxydeoxyguanosine, and TUNEL(+) apoptotic cells, death receptor 5 (DR5)(+) cells and 4-hydroxy-2-nonenal(+) cells within GST-P(+) foci. Outside the GST-P(+) foci, EMIQ decreased the numbers of apoptotic cells and DR5(+) cells. These results suggest that TAA-induced tumor promotion involves activation of hepatic macrophages producing proinflammatory factors. EMIQ may suppress the TAA-induced tumor-promoting activity by an anti-inflammatory mechanism mediated by suppressing the activation of these macrophages. Furthermore, EMIQ may suppress tumor-promoting activity differentially between the inside and outside of GST-P(+) foci. Within GST-P(+) foci, EMIQ facilitates the apoptosis of preneoplastic cells through the upregulation of DR5. Outside the GST-P(+) foci, EMIQ suppresses apoptosis and the subsequent regeneration of non-transformed liver cells.

Flavonoids are nutrients that exert anti-allergic effects. We investigated the preventative effect of enzymatically modified isoquercitrin (EMIQ), a flavonoid, to relieve the symptoms of Japanese cedar pollinosis.

In a parallel-group, double-blind placebo-controlled study design, 24 subjects with Japanese cedar pollinosis took 100mg EMIQ or a placebo for 8 weeks, starting 4 weeks prior to the onset of pollen release. Subjective symptoms, ADL scores and the usage of drugs were recorded daily, and the QOL score was obtained every 4 weeks. Blood sampling was performed before and after the study to measure serum levels of IgE and flavonoids.

During the entire study period, ocular symptom + medication score for the EMIQ group was significantly lower (p < 0.05) than that of the placebo group. When limited to the period, ocular symptom scores (p < 0.05, weeks 5-6), and ocular congestion scores (p < 0.05, weeks 5-6) for the EMIQ group was significantly lower than that for the placebo group while other scores for the EMIQ group, such as ocular itching scores (p = 0.09, weeks 4-5), lacrimation scores (p = 0.07, weeks 5-6), and ocular congestion scores (p = 0.06, weeks 4-5), all tended to be lower. However no significant differences were found in nasal symptoms between the two groups. Serum concentrations of IgE were not significantly downregulated but the serum concentrations of quercetin and its derivatives were elevated significantly by the intake of EMIQ.

Intake of the quercetin glycoside EMIQ proved to be effective for the relief of ocular symptoms caused by Japanese cedar pollinosis.

Enzymatically modified isoquercitrin (EMIQ), isoquercitrin with malto-oligosaccharides, has been recognized as "generally recognized as safe" by the Flavor and Extracts Manufacturers Association in the United States since 2003. The long-term antiatherogenic effect of EMIQ was examined using apolipoprotein E (apoE)-deficient atherogenic mice.

Male apoE-deficient mice (6 wk old) were fed with a high-fat diet alone or a diet containing EMIQ for 14 wk. At 20 wk old, atherosclerotic lesions in the aorta and aortic sinus were measured by morphometry and histomorphometry.

In apoE-deficient mice, EMIQ did not significantly affect body weight, plasma total cholesterol, triacylglycerol, and high-density lipoprotein cholesterol throughout the experiment. EMIQ significantly suppressed the aortic atherosclerotic lesion area (control 8.8 +/- 3.5% versus EMIQ 4.4 +/- 1.5%, mean +/- SD, P = 0.022). Similarly, atherosclerotic plaque lesions in the aortic sinus were significantly reduced by EMIQ (control 37.7 +/- 3.6% versus EMIQ 30.2 +/- 2.0%, P = 0.010). Of note, the immunostained area for macrophage or 4-hydroxy-2-nonenal, a well-recognized marker of oxidative stress, at the plaque in the aortic sinus was markedly suppressed, whereas the area for collagen or smooth muscle cell were increased by EMIQ, suggesting a plaque-stabilizing effect of EMIQ.

EMIQ has atheroprotective and plaque-stabilizing effects.

To clarify the effects of purple corn color, enzymatically modified isoquercitrin (EMIQ), and isoquercitrin (IQ), registered as natural food additives in Japan, on liver carcinogenesis in vivo, a medium-term bioassay was employed. A total of 100 male F344 rats were divided into 5 groups; groups 1 to 4 were given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg b.w.) on day 1. From weeks 2 to 8, they were administered basal diet purple corn color, EMIQ, or IQ as containing test chemicals at doses of 1.0% (groups 1 and 5), 0.1% (group 2), 0.01% (group 3), or 0% (group 4) (experiments 1, 4, and 5). All rats were subjected to two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Purple corn color exerted no significant modifying effects on GST-P positive foci, preneoplastic foci, development in the liver. However, serum of rats treated with purple corn color provided evidence of antioxidant power significantly by potential antioxidant (PAO) test in vivo (experiment 2). And microarray analyses showed purple corn color to induce RNA expression such as P450 (cytochrome) oxidoreductase, phosphatidylinositol 3-kinase, and phospholipase A2 (experiment 3). Higher doses of EMIQ or IQ with strong antioxidant power in vivo by PAO test treated groups were correlated with smaller numbers of GST-P positive foci, with Spearman's rank correlation coefficients of P= 0.002 and P= 0.049, respectively (experiments 4 and 5). Therefore, the tested food additives may be effective as antioxidants in vivo and have chemopreventive potential against liver preneoplastic lesion development.

Renal histopathology in the most recent 2-year carcinogenicity bioassay of quercetin, in Fischer 344 rats, was re-evaluated in an attempt to determine a mode of action underlying a small increase in renal tubule tumors reported in the males (). The re-evaluation confirmed the reported increase in renal tumors in mid- and high-dose males, including a single carcinoma in a high-dose male, as well as an exacerbation of spontaneous, chronic progressive nephropathy (CPN) in male rats only. The re-evaluation also showed that there were no cellular alterations in the kidney indicative of chemical toxicity at 6 months, 15 months, or 2 years. The evidence linked the occurrence of the predominant basophilic adenomas and foci of atypical tubule hyperplasia (ATH) with the exacerbation of CPN to advanced grades of severity, supporting a mode of action involving quercetin interaction with CPN. This mode of action represents a secondary mechanism for renal tumor development, with no relevance for extrapolation to humans. In addition, the single carcinoma present in the high-dose males, along with 4 other lesions ranging from ATH to adenoma in male and female groups, were considered to have a unique phenotype associated previously with neoplasms of spontaneous and familial origin.

A subchronic toxicity study of a flavonoid morin was performed in both sexes of F344 rats with dietary administration at concentrations of 0%, 0.625%, 1.25%, 2.5% and 5% (w/w) for 13 weeks. No mortality or abnormal clinical signs were observed throughout the experimental period in any group. Although a slight tendency for increase in food intake was noted in both sexes of the 2.5% and 5.0% groups, slight non-significant body weight decrease was observed in 5.0% males. Significant increases in alanine transaminase (ALT; over 2.5%), alkali phosphatase (ALP; 1.25% and 5.0%) and relative liver weights (1.25% and 2.5%) in males and in gamma-glutamyl transpeptidase (gamma-GT), aspartate transaminase (AST), ALT, relative liver weights in the 2.5% and 5.0% females and ALP in 5.0% females were noted. Increased urea nitrogen and relative kidney weights at dose of 1.25% and above and creatinine at 5.0% were observed also in females. On histopathological observation, hepatocyte hypertrophy was detected in 3 of 10 5.0% females. Based on the above findings, the no-observed-adverse-effect level (NOAEL) for both sexes was estimated to be 0.625% (299 and 356 mg/kg b.w./day for males and females, respectively).