Hesperetin-7-O-glucoside/ß-cyclodextrin Inclusion Complex Induces Acute Vasodilator Effect to Inhibit the Cold Sensation Response during Localized Cold-Stimulate Stress in Healthy Human Subjects: A Randomized, Double-Blind, Crossover, and Placebo-Controlled Study.

Hesperetin, a citrus flavonoid, exerts vasodilation and is expected to improve endothelial function and alleviate cold sensation by activating nervous system thermal transduction pathways. In this randomized, double-blind, crossover, and placebo-controlled study, the purpose was to assess the effect of an orally administered highly bioavailable soluble inclusion complex of hesperetine-7-O-glucoside with ß-cyclodextrin (HEPT7G/ßCD; SunActive® HES/HCD) on cold sensation response during localized cold-stimulated stress in healthy humans. A significant (p ≤ 0.05) dose-dependent increase in skin cutaneous blood flow following relatively small doses of HEPT7G/ßCD inclusion complex ingestion was confirmed, which led to a relatively effective recovery of peripheral skin temperature. The time delay of an increase in blood flow during rewarming varied significantly between low- and high-dose HEPT7G/ßCD inclusion complex consumption (e.g., 150 mg and 300 mg contain 19.5 mg and 39 mg of HEPT7G, respectively). In conclusion, the substantial alteration in peripheral skin blood flow observed during local cooling stress compared to placebo suggested that deconjugated hesperetin metabolites may have a distinct capacity for thermoregulatory control of human skin blood flow to maintain a constant body temperature during cold stress exposure via cutaneous vasodilation and vasoconstriction systems.

Increased bioavailability of diosmetin-7-glucoside-γ-cyclodextrin inclusion complex compared with diosmin in Sprague-Dawley rats.

Diosmin (DSN) is found mainly in citrus fruits, and has potent antioxidant effects. This study aimed to evaluate pharmacokinetics of diosmetin-7-glucoside-γ-cyclodextrin (DIOSG-CD) inclusion complex. The area under the curve values from AUC0-24 of DIOSG-CD, prepared by reacting DSN and naringinase with γ-CD, were approximately 800-fold higher than those of DSN following their administration in Sprague-Dawley rats.

Mutagenic, Acute, and Subchronic Toxicity Studies of the Hesperetin-7-Glucoside-ß-Cyclodextrin Inclusion Complex.

Hesperetin glucosides such as hesperidin and hesperetin-7-glucoside are abundantly present in citrus fruits and have various pharmacological properties. However, the potential toxicity of hesperetin glucosides remains unclear. An initial assessment of the safety of hesperetin-7-glucoside-ß-cyclodextrin inclusion complex (HPTGCD) as a functional food ingredient was undertaken to assess toxicity and mutagenic potential. A bacterial reverse mutation assay (Ames test) using Salmonella typhimurium (strains TA98, TA1535, TA100, and TA1537) and Escherichia coli (strain WP2 uvrA) with HPTGCD (up to 5000 µg/plate) in the absence and presence of metabolic activation was negative. In a single oral (gavage) toxicity study in male and female rats, HPTGCD at dose up to 2000 mg/kg did not produce mortality nor clinical signs of toxicity or change in body weight. In a subchronic oral (dietary admix) toxicity study in rats receiving 0, 1.5, 3, and 5% HPTGCD for 13 weeks, no adverse effects were noted and the no-observed-adverse-effect level (NOAEL) was 5% in the diet (equivalent to 3267.7 mg/kg/day for males and to 3652.4 mg/kg/day for females). These results provide initial evidence of the safety of HPTGCD.

Structural Investigation of Hesperetin-7-O-Glucoside Inclusion Complex with ß-Cyclodextrin: A Spectroscopic Assessment.

Flavonoids are biologically active natural products of great interest for their potential applications in functional foods and pharmaceuticals. A hesperetin-7-O-glucoside inclusion complex with ß-cyclodextrin (HEPT7G/ßCD; SunActive® HCD) was formulated via the controlled enzymatic hydrolysis of hesperidin with naringinase enzyme. The conversion rate was nearly 98%, estimated using high-performance liquid chromatography analysis. The objective of this study was to investigate the stability, solubility, and spectroscopic features of the HEPT7G/ßCD inclusion complex using Fourier-transform infrared (FTIR), Raman, ultraviolet-visible absorption (UV-vis), 1H- and 13C- nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), liquid chromatography/mass spectroscopy (LC-MS), scanning electron microscopy (SEM), and powdered X-ray diffraction (PXRD) spectroscopic techniques including zeta potential, Job's plot, and phase solubility measurements. The effects of complexation on the profiles of supramolecular interactions in analytic features, especially the chemical shifts of ß-CD protons in the presence of the HEPT7G moiety, were evaluated. The stoichiometric ratio, stability, and solubility constants (binding affinity) describe the extent of complexation of a soluble complex in 1:1 stoichiometry that exhibits a greater affinity and fits better into the ß-CD inner cavity. The NMR spectroscopy results identified two different configurations of the HEPT7G moiety and revealed that the HEPT7G/ßCD inclusion complex has both -2S and -2R stereoisomers of hesperetin-7-O-glucoside possibly in the -2S/-2R epimeric ratio of 1/1.43 (i.e., -2S: 41.1% and -2R: 58.9%). The study indicated that encapsulation of the HEPT7G moiety in ß-CD is complete inclusion, wherein both ends of HEPT7G are included in the ß-CD inner hydrophobic cavity. The results showed that the water solubility and thermal stability of HEPT7G were apparently increased in the inclusion complex with ß-CD. This could potentially lead to increased bioavailability of HEPT7G and enhanced health benefits of this flavonoid.

Genotoxicity and mutagenicity evaluation of isoquercitrin-γ-cyclodextrin molecular inclusion complex using Ames test and a combined micronucleus and comet assay in rats.

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.

Bioavailability comparison between a compound comprising hesperetin-7-glucoside with ß-cyclodextrin and a mixture of hesperidin and dextrin in healthy adult human males.

The pharmacokinetics of compounds comprising hesperetin-7-glucoside with ß-cyclodextrin and physically mixed hesperidin/dextrin was compared in 8 healthy adult male subjects in a nonrandomized, double-blind, cross-over, controlled study. For 0-24 h, the area under the curve of the total plasma hesperetin concentration after hesperetin-7-glucoside with ß-cyclodextrin consumption was >100-fold higher than that after hesperidin/dextrin consumption.

13-Weeks subchronic toxicity of isoquercitrin-γ-cyclodextrin (IQC-γCD) molecular inclusion complex in Sprague-Dawley rats.

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.

Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium uses iron from the environment via the vulnibactin-mediated-iron-uptake system. In this study, we constructed the deletion mutants of the genes encoding the proteins involved in the vulnibactin-mediated-iron-uptake system, isochorismate synthase (ICS), vulnibactin utilization protein (VuuB), periplasmic ferric-vulnibactin binding protein (FatB), and ferric-vulnibactin receptor protein (VuuA). The Δics and ΔvuuA mutants were unable to grow under low-iron concentration conditions compared with the isogenic wild-type, indicating that the involvement of ICS in the vulnibactin biosynthesis pathway and uptake of ferric-vulnibactin through the VuuA receptor protein are essential for V. vulnificus M2799 growth under low-iron concentration conditions. Similar growth impairment was also observed in ΔfatB, with growth recovery of this mutant observed 6 h after the beginning of the culture. These results indicate that there must be other periplasmic ferric-vulnibactin binding proteins in V. vulnificus M2799 that complement the defective fatB gene. Complementary growth studies confirmed that VatD protein, which functions as a periplasmic ferric-aerobactin binding protein, was found to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the expression of ics, vuuB, fatB, vuuA, and vatD genes was found to be regulated by iron and the ferric uptake regulator.

Enzymatically modified isoquercitrin, alpha-oligoglucosyl quercetin 3-O-glucoside, is absorbed more easily than other quercetin glycosides or aglycone after oral administration in rats.

Quercetin, a flavonol contained in various vegetables and herbal medicines, has various biological activities including anti-cancer, anti-allergic and anti-oxidative activities. However, low oral bioavailability of quercetin due to insolubility in water has limited its use as a food additive or dietary supplement. Since the water solubility is enhanced by glycosyl conjugation, in the present study, we evaluated the bioavailability of several quercetin glycosides with different sugar moieties in rats. Quercetin, quercetin-3-O-rutinoside (rutin), and quercetin-3-O-glucoside (isoquercitrin, IQC) in suspension, and quercetin-3-O-maltoside (Q3M), quercetin-3-O-gentiobioside (Q3G), alpha-monoglucosyl rutin (alphaMR), alpha-oligoglucosyl rutin (alphaOR), and enzymatically modified isoquercitrin (alpha-oligoglucosyl isoquercitrin, EMIQ) dissolved in water, were orally administered to rats under anesthesia. Bioavailability (F value) was calculated from the concentrations of total quercetin in plasma from 0 to 12 h after the administration. F value of quercetin was 2.0%, and those of IQC, Q3M and EMIQ were 12%, 30%, and 35%, respectively. Although Q3G, alphaMR and alphaOR have high water solubility, their F values were low (3.0%, 4.1%, 1.8%, respectively). In the in vitro study, the homogenate of rat intestinal epithelium rapidly hydrolyzed IQC, Q3M and EMIQ to quercetin, and alphaMR and alphaOR to rutin. However, it could not hydrolyze Q3G or rutin to quercetin. Elongation of alpha-linkage of glucose moiety in IQC enhances the bioavailability of quercetin, and intestinal epithelial enzymes such as lactase-phrolizin hydrolase or mucosal maltase-glucoamylase would play important roles in the hydrolysis and absorption of these flavonol glycosides.

Preventative effect of a flavonoid, enzymatically modified isoquercitrin on ocular symptoms of Japanese cedar pollinosis.

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

Effect of enzymatically modified isoquercitrin, a flavonoid, on symptoms of Japanese cedar pollinosis: a randomized double-blind placebo-controlled trial.

BACKGROUND: Flavonoids exert antiallergic and antioxidant effects. We investigated the efficacy of enzymatically modified isoquercitrin (EMIQ), a flavonoid, to relieve symptoms of pollinosis. METHODS: In a parallel-group, double-blind placebo-controlled study design, 20 subjects with Japanese cedar pollinosis took two capsules daily of 100 mg EMIQ or a placebo for 8 weeks during the pollen season. Subjective symptoms and activities of daily living (ADL) scores were recorded every day, and the quality of life (QOL) score was obtained every 4 weeks. Blood sampling was performed before and after the study to measure serum cytokines, chemokines, IgE, quercetin and oxidized biomarkers. RESULTS: During the entire study period, total ocular score and ocular itching score for the EMIQ group were significantly lower (p < 0.05) than for the placebo group. When limited to the individual periods, total symptom score for the EMIQ group was significantly lower (p < 0.05, week 4-5) than that for the placebo group while other scores for the EMIQ group, such as total nasal score (p = 0.06, week 4-5), nasal obstruction score (p = 0.08, week 4-5), lacrimation score (p = 0.06, week 5-6), ocular congestion score (p = 0.08, week 4-7) and ADL score (p = 0.08, week 4-7), all tended to be lower. The levels of serum cytokines such as interleukin (IL)-4, IL-5, IL-12, IL-13, interferon-gamma, and eotaxin and IgE were not significantly downregulated by the intake of EMIQ but the serum concentrations of oxidized low-density lipoprotein and thymus and activation-regulated chemokine were reduced. CONCLUSION: Intake of the quercetin glycoside EMIQ was safe and influenced ocular symptoms caused by pollinosis.

Atheroprotective and plaque-stabilizing effects of enzymatically modified isoquercitrin in atherogenic apoE-deficient mice.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: EMIQ has atheroprotective and plaque-stabilizing effects.

Effect of enzymatically modified isoquercitrin in spontaneously hypertensive rats.

Enzymatically modified isoquercitrin (EMIQ) is a water-soluble glycoside of quercetin produced from rutin by enzymatic treatment. We investigated the anti-hypertensive effect of orally administered EMIQ in spontaneously hypertensive rats (SHR). The systolic blood pressure (SBP) in SHR administered EMIQ at a dose of 3 and 26 mg/kg/d was significantly lower than that in the control group on d 22, 36 and 50 of administration. The effect of EMIQ (26 mg/kg/d) was higher than equimolar administration of quercetin. Diltiazem administered as a positive control also suppressed the increase in SBP. and the effect was stronger than that of EMIQ. In the control group, the mean values of mean blood pressure (MBP) and diastolic blood pressure (DBP) were increased after the start of administration. Although diltiazem suppressed the increase in MBP, no significant changes were observed in the EMIQ groups. Compared with the control group, EMIQ groups showed the incidental changes of MBP and heart rate on day 22 of administration only. These results indicate that EMIQ suppressed the increase in SBP in SHR dose-dependently, and was more effective than the aglycone quercetin. It was also speculated that EMIQ showed higher anti-hypertensive effect than quercetin due to the high bioavailability, and the mechanism of SBP suppression is possibly through the improvement of endothelial NO production. In conclusion, our results suggest that EMIQ shows possibility as a naturally-derived safe food material which has an antihypertensive effect.

Effects of uptake of flavonoids on oxidative stress induced by hydrogen peroxide in human intestinal Caco-2 cells.

The relation between the uptake of flavonoids and the response of human colon adenocarcinoma Caco-2 cells exposed to oxidative stress induced by hydrogen peroxide (H(2)O(2)) was examined. Flavonoid aglycones were incorporated into Caco-2 cells in a concentration- and time-dependent manner, but neither glycosides nor unstable myricetin were incorporated into the cells. The incorporated flavonoids reduced the reactive oxygen species (ROS) induced by H(2)O(2) in the cells in proportion to the amount incorporated and the radical scavenging activity of flavonoids. But, flavonoids with high radical scavenging activity also generated H(2)O(2). The activity decreasing intracellular ROS was inversely related to the H(2)O(2) scavenging activity of flavonoids. Therefore, the decrease in the amount of intracellular ROS induced by H(2)O(2) was not directly due to the scavenging of H(2)O(2), but rather to the scavenging of ROS generated from H(2)O(2). These results suggest that strong antioxidative flavonoids have both a cytoprotective effect owing to the scavenging of ROS and cytotoxic effect caused by the generation of H(2)O(2).

Enzymatic production of highly soluble myricitrin glycosides using beta-galactosidase.

Myricitrin, a botanical flavonol glycoside, could be a useful ingredient of functional foods, cosmetics, and medicines because of its high anti-oxidative activity. However, due to its insolubility in water, it has a limited range of use. To improve this solubility, we glycosylated myricitrin by an enzymatic transglycosylate reaction. Myricitrin was galactosylated by beta-galactosidase from Bacillus circulans using lactose as a sugar donor. The reaction product was 480 times more soluble than myricitrin. Four myricitrin galactosides were isolated from the reaction products by column chromatography, and their molecular structures were identified by using ESI-MS, 1H-NMR, 13C-NMR, 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC analysis. The solubility of these four myricitrin galactosides was more than 3.9 x 10(3) fold that of myricitrin, and each had similar anti-oxidative activity to that of myricitrin.

Transepithelial permeability of myricitrin and its degradation by simulated digestion in human intestinal Caco-2 cell monolayer.

Myricitrin permeated the human intestinal Caco-2 cell monolayer via the paracellular pathway in a time- and concentration-dependent manner. Myricitrin was not conjugated by Caco-2 cells. Myricitrin was degraded by simulated intestinal digestion, but permeability did not change significantly.

Myricitrin degraded by simulated digestion inhibits oxidation of human low-density lipoprotein.

The inhibitory effects of myricitrin on the oxidation of human low-density lipoprotein were investigated before and after its degradation by simulated digestion. Myricitrin strongly inhibited the low-density lipoprotein oxidation induced by either 2,2'-azobis (2-amidinopropane) dihydrochloride or CuSO4 in a concentration-dependent manner. Myricitrin was very stable under an acidic condition (pH 1.8) corresponding to the gastric environment, but it was easily degraded under an alkaline condition (pH 8.5) corresponding to the intestinal environment. However, degraded myricitrin also had a strong inhibitory effect on the oxidative degradation of alpha-tocopherol, cholesterol and apolipoprotein B-100 in low-density lipoprotein. Our study revealed that myricitrin was degraded into many components under a mildly alkaline condition, but the degraded myricitrin still retained the free radical-scavenging and copper-chelating activities toward low-density lipoprotein.