Gut microbiota-mediated associations of green tea and catechin intakes with glucose metabolism in individuals without type 2 diabetes mellitus: a four-season observational study with mediation analysis.

This four-season observational study aimed to examine the mediating role of the gut microbiota in the associations between green tea and catechin intakes and glucose metabolism in individuals without type 2 diabetes mellitus (T2DM). In each of the 4 seasons, 85 individuals without T2DM (56 male [65.9%]; mean [standard deviation] age: 43.3 [9.4] years) provided blood samples, stool samples, 3-day weighed dietary records, and green tea samples. Catechin intake was estimated by analyzing the tea samples. Linear mixed-effects model analysis showed that green tea intake was negatively associated with fasting blood glucose and insulin levels, even after considering the seasonal variations. Of the gut microbial species associated with green tea intake, the mediation analysis revealed that Phocaeicola vulgatus mediated the association between green tea intake and fasting blood glucose levels. These findings indicate that green tea can improve glucose metabolism by decreasing the abundance of P. vulgatus that is associated with elevated blood glucose levels in individuals without T2DM.

Acute Dose-Response Effectiveness of Combined Catechins and Chlorogenic Acids on Postprandial Glycemic Responses in Healthy Men: Results from Two Randomized Studies.

Epidemiologic studies show that the risk of diabetes can be reduced by ingesting green tea or coffee. Previous studies have shown that simultaneously taking green tea catechins (GTC) and coffee chlorogenic acid (CCA) alters postprandial gastrointestinal hormones secretion and improves insulin sensitivity. However, there is no evidence on the acute effects of GTC and CCA on incretin and blood glucose, and on the respective dose of polyphenols. In this randomized, double-blind, placebo-controlled crossover study, we examined the effective dose of GTC and CCA on postprandial glucose, insulin, and incretin responses to a high-fat and high-carbohydrate cookie meal containing 75 g of glucose in healthy men. Study 1 (n = 18) evaluated two doses of GTC (270 or 540 mg) containing a fixed dose of CCA (270 mg) with 113 mg of caffeine and a placebo (0 mg GTC and 0 mg CCA) with 112 mg of caffeine. Study 2 (n = 18) evaluated two doses of CCA (150 or 300 mg) containing a fixed dose of GTC (540 mg) and a placebo with 99 mg of caffeine. The single combined ingestion of GTC and CCA significantly altered the incretin response and suppressed glucose and insulin levels. These findings suggest that the effective minimum dose is 540 mg of GTC and 150 mg of CCA.

Effects of Ingesting Both Catechins and Chlorogenic Acids on Glucose, Incretin, and Insulin Sensitivity in Healthy Men: A Randomized, Double-Blinded, Placebo-Controlled Crossover Trial.

Epidemiologic studies have revealed that consuming green tea or coffee reduces diabetes risk. We evaluated the effects of the combined consumption of green tea catechins and coffee chlorogenic acids (GTC+CCA) on postprandial glucose, the insulin incretin response, and insulin sensitivity. Eleven healthy men were recruited for this randomized, double-blinded, placebo-controlled crossover trial. The participants consumed a GTC+CCA-enriched beverage (620 mg GTC, 373 mg CCA, and 119 mg caffeine/day) for three weeks; the placebo beverages (PLA) contained no GTC or CCA (PLA: 0 mg GTC, 0 mg CCA, and 119 mg caffeine/day). Postprandial glucose, insulin, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) responses were measured at baseline and after treatments. GTC+CCA consumption for three weeks showed a significant treatment-by-time interaction on glucose changes after the ingestion of high-fat and high-carbohydrate meals, however, it did not affect fasting glucose levels. Insulin sensitivity was enhanced by GCT+CCA compared with PLA. GTC+CCA consumption resulted in a significant increase in postprandial GLP-1 and a decrease in GIP compared to PLA. Consuming a combination of GTC and CCA for three weeks significantly improved postprandial glycemic control, GLP-1 response, and postprandial insulin sensitivity in healthy individuals and may be effective in preventing diabetes.

Concomitant use of tea catechins affects absorption and serum triglyceride-lowering effects of monoglucosyl hesperidin.

The present study investigated whether combined ingestion of green tea catechins (GTC) and monoglucosyl hesperidin (GHES) influences the pharmacokinetic parameters of polyphenols and serum triglycerides (TG). We conducted 2 randomized, controlled trials. Study 1: 8 healthy male subjects participated in a crossover study in which they ingested a test beverage containing GHES (0, 84, 168, or 336 mg GHES) with GTC, or 336 mg GHES without GTC. After ingestion, the pharmacokinetic changes in plasma hesperetin (HEP) and catechins were measured. Study 2: 36 healthy male and female subjects (mean age, 53 ± 2 years; mean BMI, 25.2 ± 0.5 kg m-2) were recruited for a double-blind, placebo-controlled study in which they ingested a test beverage containing 165 mg GHES with 387 mg GTC or a placebo beverage daily for 4 weeks. Fasting serum TG and other lipids and glucose metabolites were analyzed. Study 1 showed that the pharmacokinetics of HEP did not differ significantly between the 336 mg GHES without GTC treatment and the 168 mg GHES with GTC treatment. Study 2 showed that continuous ingestion of 165 mg GHES and 387 mg GTC for 4 weeks significantly decreased fasting serum TG levels compared with baseline values (change in TG, -30 ± 13 mg dl-1, P = 0.040) in the intention-to-treat analysis. In conclusion, our findings suggest that GTC affects the oral bioavailability of GHES, and combined ingestion of low doses of GHES with GTC effectively improves fasting TG levels.

Effects of Timing of Acute and Consecutive Catechin Ingestion on Postprandial Glucose Metabolism in Mice and Humans.

We examined the effects of the timing of acute and consecutive epigallocatechin gallate (EGCG) and catechin-rich green tea ingestion on postprandial glucose in mice and human adults. In mouse experiments, we compared the effects of EGCG administration early (morning) and late (evening) in the active period on postprandial glucose. In human experiments, participants were randomly assigned to the morning-placebo (MP, n = 10), morning-green tea (MGT, n = 10), evening-placebo (EP, n = 9), and evening-green tea (EGT, n = 9) groups, and consumed either catechin-rich green tea or a placebo beverage for 1 week. At baseline and after 1 week, participants consumed their designated beverages with breakfast (MP and MGT) or supper (EP and EGT). Venous blood samples were collected in the fasted state and 30, 60, 120, and 180 min after each meal. Consecutive administration of EGCG in the evening, but not in the morning, reduced postprandial glucose at 30 (p = 0.006) and 60 (p = 0.037) min in the evening trials in mice. In humans, ingestion of catechin-rich green tea in the evening decreased postprandial glucose (three-factor analysis of variance, p < 0.05). Thus, catechin intake in the evening more effectively suppressed elevation of postprandial glucose.

Effect of tea catechins with caffeine on energy expenditure in middle-aged men and women: a randomized, double-blind, placebo-controlled, crossover trial.

PURPOSE: It has been reported that tea catechins increase energy metabolism, but their effect on resting metabolic rate (RMR) remains under debate. This study aimed to examine the effect of repeated intake of tea catechins on energy metabolism in the resting state in middle-aged men and women. METHODS: A total of 30 middle-aged men and women [13 women; age (mean ± SD) 52 ± 4 years; BMI 21.9 ± 2.2 kg/m2] were recruited. A randomized, double-blind, crossover study was conducted using a tea catechin-enriched beverage (611 mg catechins, 88 mg caffeine) and a placebo beverage (0 mg catechins, 81 mg caffeine) as test beverages. After 2 weeks of continuous test beverage intake, fasting RMR and energy expenditure (EE) after the ingestion of test beverage were measured. Measurements of forehead temperature (proxy for core temperature) and skin temperature were also obtained simultaneously. RESULTS: Among participants who underwent measurements, 26 (10 women; mean age 52 ± 4 years; mean BMI 22.1 ± 2.1 kg/m2) were analyzed. The EE increased significantly after ingestion of the tea catechin beverage compared with the placebo beverage (placebo treatment: 5502 ± 757 kJ/day; catechin treatment: 5598 ± 800 kJ/day; P = 0.041). No between-treatment differences in fasting RMR or the respiratory quotient were detected. In addition, the forehead and skin temperature did not differ significantly between the placebo and catechin treatments. CONCLUSION: This study revealed that continuous intake of tea catechins with caffeine for 2 weeks significantly increased EE after ingestion of the tea catechin but not fasting RMR in middle-aged men and women. CLINICAL TRIAL REGISTRY NUMBER AND WEBSITE: This trial was registered at www.umin.ac.jp/ctr/ as UMIN000025810 and UMIN000025811.

Prevention of Acute Upper Respiratory Infections by Consumption of Catechins in Healthcare Workers: A Randomized, Placebo-Controlled Trial.

Catechins, phytochemicals contained mainly in green tea, exhibit antiviral activity against various acute infectious diseases experimentally. Clinical evidence supporting these effects, however, is not conclusive. We performed a placebo-controlled, single-blind, randomized control trial to evaluate the clinical effectiveness of consumption of catechins-containing beverage for preventing acute upper respiratory tract infections (URTIs). Two hundred and seventy healthcare workers were randomly allocated to high-catechin (three daily doses of 57 mg catechins and 100 mg xanthan gum), low-catechin (one daily dose of 57 mg catechins and 100 mg xanthan gum), or placebo (0 mg catechins and 100 mg xanthan gum) group. Subjects consumed a beverage with or without catechins for 12 weeks from December 2017 through February 2018. The primary endpoint was incidence of URTIs compared among groups using a time-to-event analysis. A total of 255 subjects were analyzed (placebo group n = 86, low-catechin group n = 85, high catechin group n = 84). The URTI incidence rate was 26.7% in the placebo group, 28.2% in the low-catechin group, and 13.1% in the high-catechin group (log rank test, p = 0.042). The hazard ratio (95% confidence interval (CI)) with reference to the placebo group was 1.09 (0.61-1.92) in the low-catechin group and 0.46 (0.23-0.95) in the high-catechin group. These findings suggest that catechins combined with xanthan gum protect against URTIs.

Effects of timing of acute catechin-rich green tea ingestion on postprandial glucose metabolism in healthy men.

Green tea polyphenols, particularly catechins, decrease fasting and postprandial glucose. However, no studies have compared the timing of green tea ingestion on glucose metabolism and changes in catechin concentrations. Here, we examined the effects of timing of acute catechin-rich green tea ingestion on postprandial glucose metabolism in young men. Seventeen healthy young men completed four trials involving blood collection in a fasting state and at 30, 60, 120, and 180 min after meal consumption in a random order: 1) morning placebo trial (09:00 h; MP trial), 2) evening placebo trial (17:00 h; EP trial), 3) morning catechin-rich green tea trial (09:00 h; MGT trial), and 4) evening catechin-rich green tea trial (17:00 h; EGT trial). The concentrations of glucose at 120 min (P=.031) and 180 min (P=.013) after meal intake were significantly higher in the MGT trials than in the MP trials. Additionally, the concentration of glucose was significantly lower in EGT trials than in the EP trials at 60 min (P=.014). Moreover, the concentrations of glucose-dependent insulinotropic polypeptide were significantly lower in the green tea trials than in the placebo trials at 30 min (morning: P=.010, evening: P=.006) and 60 min (morning: P=.001, evening: P=.006) after meal intake in both the morning and evening trials. Our study demonstrated that acute ingestion of catechin-rich green tea in the evening reduced postprandial plasma glucose concentrations.

Radical formation in individual aqueous solutions of some unsaturated fatty acids and in their mixtures.

This study examines oxidizability in individual aqueous solutions of oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid and arachidonic acid, and in their mixtures. We used electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance (HPLC-ESR) and high performance liquid chromatography-electron spin resonance-mass spectrometries (HPLC-ESR-MS). We detected 4-carboxybutyl radical derived from γ-linolenic acid, ethyl and 7-carboxyheptyl radicals derived from α-linolenic acid, and pentyl and 7-carboxyheptyl radicals derived from linoleic acid. HPLC-ESR analyses for the individual aqueous solutions of linoleic acid, α-linolenic acid, γ-linolenic acid and arachidonic acid showed less radical form for polyunsaturated fatty acids with more double bonds. On the other hand, HPLC-ESR peak height of 4-carboxybutyl radical, which form through hydrogen atom abstraction at the carbon close to the carboxy end, increased for linoleic acid/γ-linolenic acid, α-linolenic acid/γ-linolenic acid, and γ-linolenic acid/oleic acid mixtures compared to before mixing. Conversely, HPLC-ESR peak heights of ethyl, 7-carboxyheptyl and pentyl radicals, which form through hydrogen atom abstraction at the carbons close to the methyl end, decreased for linoleic acid/α-linolenic acid, linoleic acid/γ-linolenic acid, linoleic acid/oleic acid, linoleic acid/arachidonic acid, α-linolenic acid/γ-linolenic acid, and α-linolenic acid/oleic acid mixtures compared to before mixing.

Actual Daily Intakes of Tea Catechins and Thier Estimation According to Four Season 3 Day Weighed Dietary Records and a Short Food Frequency Questionnaire among Japanese Men and Women

Background: Tea catechins are considered to be important preventive factors of cancer on several organs; however, the relationships of the actual daily intakes (ADIs) on the preventive effects have not been adequately addressed. We measured the ADIs of tea catechins as annual averages derived from every their ingested cups recorded by each subject, and the estimation models were established considering tea origin. Methods: Fifty-nine Japanese men and women completed four season 3 day weighed dietary records (WDRs) and a food frequency questionnaire (FFQ), and samples of green, oolong and black teas, ingested during a total 12 days were collected for the analysis. The ADIs of the total and composed catechins of all tea samples were measured by a high-performance liquid chromatography. The estimation models for the ADIs (R2: coefficient of determination) based on the WDRs and FFQ were established with multiple regression analysis using appropriate confounding factors. Results: The ADIs of total catechins and epigallocatechin gallate (EGCg) were 110 and 21.4 mg/day in men and 157 and 34.7 mg/day in women, respectively. The total catechins ADIs were positively associated with green tea consumption based on WDRs and FFQ (adjusted R2 =0.421 and 0.341 for men and 0.346 and 0.238 for women, p<0.05 for all, respectively). Likewise, the EGCg ADIs were associated with green tea intake derived from WDRs and FFQ, respectively. Conclusions: We revealed the ADIs of total catechins and EGCg as annual averages could establish their estimation models. These provide reference information to clarify their relationships with cancer risks.

A comparative study of the inhibitory effects by caffeic acid, catechins and their related compounds on the generation of radicals in the reaction mixture of linoleic acid with iron ions.

Caffeic acid and (+)-catechin, which are abundantly contained in coffee and tea, are typical polyphenols. In order to know the relative magnitudes of antioxidant activity, effects by caffeic acid, (+)-catechin and their derivatives on the formation of 4-POBN/carbon-centered linoleic acid-derived radical adducts were examined in the control reaction mixture of linoleic acid with FeCl3 at 30°C for 168 h. In the presence of 1.0 mM of the polyphenols, peak to peak heights of the third ESR signal resulted in 7.7 ± 2.4% (n = 3) (caffeic acid), 145 ± 13% (n = 3) (quinic acid), 4.4 ± 0.0% (n = 3) (chlorogenic acid), 104 ± 4.4% (n = 3) (ferulic acid), 4.3 ± 0.0% (n = 3) (noradrenaline), 12.5 ± 10.9% (n = 3) (gallic acid), 38.1 ± 7.1% (n = 3) [(+)-catechin], 47.9 ± 11.7% (n = 3) [(-)-epicatechin], 56.5 ± 1.6% (n = 3) (epigallocatechin), 13.5 ± 1.7% (n = 3) (catechol) and 83.7 ± 7.8% (n = 3) (resorcinol) of the control reaction mixture. All the compounds with catechol moiety exerted potent inhibitory effects on the radical formation except for (+)-catechin, (-)-epicatechin and epigallocatechin. (+)-Catechin, (-)-epicatechin and epigallocatechin may not exert the inhibitory effect as much possibly because they are less stable compared with caffeic acid. The resorcinol moiety in these molecules may also weaken their antioxidant activity.

Effects of long-term self-massage at the musculotendinous junction on hamstring extensibility, stiffness, stretch tolerance, and structural indices: A randomized controlled trial.

OBJECTIVES: The purpose of this study was to examine the effect of long-term self-massage at the musculotendinous junction on hamstring extensibility, stiffness, stretch tolerance, and structural indices. DESIGN: Single-blind, randomized, controlled trial. SETTING: Laboratory. PARTICIPANTS: Thirty-seven healthy men. INTERVENTION: The right or left leg of each participant was randomly assigned to the massage group, and the other leg was assigned to the control group. The participants conducted self-massage at the musculotendinous junction for 3 min daily, five times per week, for 12 weeks. MAIN OUTCOME MEASURES: Hamstring extensibility, stiffness, stretch tolerance, and structural indices were measured by a blinded examiner prior to the massage intervention and after 6 and 12 weeks of intervention. RESULTS: The maximum hip flexion angle (HFA) and the maximum passive pressure after 6 and 12 weeks of intervention in the massage group were significantly higher than prior to intervention. The visual analog scale (for pain perception) at maximum HFA, the stiffness of the hamstring, and the structural indices did not differ in either group over the 12 week period. CONCLUSIONS: Our results suggest that long-term self-massage at the musculotendinous junction increases hamstring extensibility by improving stretch tolerance. However, this intervention does not change hamstring stiffness. CLINICAL TRIAL REGISTRATION NUMBER: University Hospital Medical Information Network registration number UMIN000011233.

Acute ingestion of catechin-rich green tea improves postprandial glucose status and increases serum thioredoxin concentrations in postmenopausal women.

Elevated postprandial hyperglycaemia and oxidative stress increase the risks of type 2 diabetes and CVD. Green tea catechin possesses antidiabetic properties and antioxidant capacity. In the present study, we examined the acute and continuous effects of ingestion of catechin-rich green tea on postprandial hyperglycaemia and oxidative stress in healthy postmenopausal women. Participants were randomly assigned into the placebo (P, n 11) or green tea (GT, n 11) group. The GT group consumed a catechin-rich green tea (catechins 615 mg/350 ml) beverage per d for 4 weeks. The P group consumed a placebo (catechins 92 mg/350 ml) beverage per d for 4 weeks. At baseline and after 4 weeks, participants of each group consumed their designated beverages with breakfast and consumed lunch 3 h after breakfast. Venous blood samples were collected in the fasted state (0 h) and at 2, 4 and 6 h after breakfast. Postprandial glucose concentrations were 3 % lower in the GT group than in the P group (three-factor ANOVA, group × time interaction, P< 0·05). Serum concentrations of the derivatives of reactive oxygen metabolites increased after meals (P< 0·05), but no effect of catechin-rich green tea intake was observed. Conversely, serum postprandial thioredoxin concentrations were 5 % higher in the GT group than in the P group (three-factor ANOVA, group × time interaction, P< 0·05). These findings indicate that an acute ingestion of catechin-rich green tea has beneficial effects on postprandial glucose and redox homeostasis in postmenopausal women.

Development of a gastric retentive system as a sustained-release formulation of pranlukast hydrate and its subsequent in vivo verification in human studies.

Pranlukast hydrate was demonstrated in a human site-of-absorption study to have extremely poor absorption properties in the lower gastrointestinal tract. The ratios of AUC0-24 in the distal small bowel and colon compared to stomach delivery were approximately 1/7 and 1/70, respectively. As a consequence, a gastroretentive double-layered tablet formulation (gastric swelling system; GSS), consisting of a swelling layer and a drug release layer, was developed for once-daily dosing. To study the gastric retention of the optimized GSS, an in vivo gamma scintigraphic study was carried out in nine healthy volunteers. The transit profiles demonstrated that the GSS was retained in the stomach for more than 10h. The plasma profile was prolonged, especially following administration after an evening meal. The human data validated the design concept and suggest that GSS could be a promising approach for the development of sustained-release formulation for drugs with a limited absorption window in the upper small bowel.

Discovery and characterization of D-phenylserine deaminase from Arthrobacter sp. TKS1.

We discovered a D-phenylserine deaminase that catalyzed the pyridoxal 5'-phosphate (PLP)-dependent deamination reaction from D-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55°C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50°C. The enzyme acted on the D-forms of ß-hydroxy-α-amino acids, such as D-threo-phenylserine (K(m), 19 mM), D-serine (K(m), 5.8 mM), and D-threonine (K(m), 102 mM). As L-threonine, D-allo-threonine, L-allo-threonine, and DL-erythro-phenylserine were inert, the enzyme could distinguish D-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO(4), CuSO(4), BaCl(2), and CoCl(2) and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and DL-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes.

Liquid chromatography-electrospray ionization-tandem mass spectrometry for simultaneous analysis of chlorogenic acids and their metabolites in human plasma.

A method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was developed for the simultaneous analysis of nine chlorogenic acids (CGAs), three isomers each of caffeoylquinic acids (CQAs), feruloylquinic acids (FQAs) and dicaffeoylquinic acids (dCQAs), and their two metabolites, caffeic acid (CA) and ferulic acid (FA), in human plasma. In simultaneous multiple reaction monitoring (MRM) measurements using ESI-MS/MS with a negative ion mode, a deprotonated molecular ion derived from each of the 11 molecules was used as a precursor ion while three diagnostic product ions characteristic for each were selected for the qualitative analysis. To obtain maximal intensities for all diagnostic product ions, the collision energy was optimized for each one. LC separation was achieved under conditions of a reversed-phase Inertsil ODS-2 column combined with a gradient elution system using 50mM acetic acid with 3% acetonitrile aqueous solution and 50 mM acetic acid with 100% acetonitrile. In the quantitative analysis, one of the three diagnostic product ions for each of the 11 molecules was selected. Application of simultaneous LC-ESI-MS/MS MRM measurements to analyze the 11 standards spiked into blank human plasma indicated that all diagnostic product ions were detected without any interference, and that the sensitivity, linearity and recovery of this method were acceptable. When using this method to analyze those 11 molecules in the plasma after oral ingestion of 250 ml of a drink containing a green coffee bean extract (300 mg CGAs), all 11 molecules were identified and CQAs, FQAs and FA were quantified. CQAs, FQAs and dCQAs in human plasma were detected for the first time. This method should be useful to understand the biological and pharmacological effects of CGAs, such as improvement of human hypertension.

Determination of green tea catechins in human plasma using liquid chromatography-electrospray ionization mass spectrometry.

A method for the sensitive and specific determination of eight green tea catechins, consisting of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin-3-gallate (CG), epicatechin-3-gallate (ECG), gallocatechin-3-gallate (GCG) and epigallocatechin-3-gallate (EGCG), in human plasma was established. For optimization of conditions for LC-ESIMS, the separation of the eight catechins was achieved chromatographically using Inertsil ODS-2 column combined with a gradient elution system of 0.1M aqueous acetic acid and 0.1M acetic acid in acetonitrile. Detection using a mass spectrometer was performed with selected ion monitoring at m/z=289 for E and EC, 305 for GC and EGC, 441 for CG and ECG, and 457 for GCG and EGCG under negative ESI. A preparative procedure, consisting of the addition of perchloric acid and acetonitrile to the plasma for deproteinizing and the subsequent addition of potassium carbonate solution to remove excess acid, was developed. In six different plasma with the eight catechins spiked at two different concentrations, the average recoveries were in the range between 72.7 and 84.1%, which resulted from the matrix effect and preparative loss, with coefficients of variance being 8.2-19.8% among individuals. The levels of the catechins in prepared plasma solutions that were kept at 5 degrees C within 24h were stable, which allows us to simply analyze many prepared plasma solutions using an autosampler overnight. When using this method to analyze the eight catechins in human plasma after oral ingestion of a commercial green tea beverage, we detected all the catechins absorbed into human blood for the first time. This also suggested that extremely small amounts of the eight catechins orally ingested may be absorbed based on each absorptive property for the catechins. The method should enable pharmacokinetic studies of green tea catechins in humans.

Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systems.

To design an effective particulate drug delivery system having mucoadhesive function, several mucoadhesion tests for polymers and the resultant particulate systems were developed. Mucin particle method is a simple mucoadhesion test for polymers, in which the commercial mucin particles are used. By measuring the change in particle size or zeta potential of the mucin particle in a certain concentration of polymer solution, we could estimate the extent of their mucoadhesive property. BIACORE method is also a novel mucoadhesion test for polymers. On passing through the mucin suspension on the polymer-immobilized chip of BIACORE instrument, the interaction was quantitatively evaluated with the change in its response diagram. By using these mucoadhesion tests, we detected a strong mucoadhesive property of several types of chitosan and Carbopol. Evaluation of mucoadhesive property of polymer-coated particulate systems was demonstrated with the particle counting method developed by us. To detect the mucoadhesive phenomena in the intestinal tract, we observed the rat intestine with the confocal laser scanning microscope (CLSM) after oral administration of the particulate systems. The resultant photographs clearly showed a longer retention of submicron-sized chitosan-coated liposomes (ssCS-Lip) in the intestinal tract than other liposomal particles tested such as non-coated liposomes and chitosan-coated multilamellar one. These observations explained well the superiority of the ssCS-Lip as drug carrier in oral administration of calcitonin in rats than other liposomal particles.

Effectiveness of submicron-sized, chitosan-coated liposomes in oral administration of peptide drugs.

The mucoadhesive behavior of chitosan-coated liposomes in the intestinal tract of the rat was examined to elucidate their particle size effects on the absorption of an entrapped drug, calcitonin. The intestine was removed from rats after oral administration of liposomes containing a fluorescent dye, and its various parts were observed with confocal laser scanning microscopy. Penetration of submicron-sized liposomes (ssLip) or chitosan-coated ssLip (ssCS-Lip) into the mucosa was observed, while such behavior was not observed for the multilamellar liposomes, even when coated with chitosan (CS-Lip). The retentive property of ssCS-Lip was confirmed by measuring the amount of dye in each part of the intestine. The pharmacologic effects of calcitonin-loaded liposomes of different particle size were measured after oral administration in rats. The pharmacologic effect of oral administration of ssLip coated with chitosan was detected up to 120 h after administration. The extensive pharmacologic effect of ssCS-Lip was attributed to their prolonged retention in the intestinal mucosa, partly owing to their penetrative property into the intestinal mucosa. The chitosan-coated ssLip, with their higher retentive property in the intestinal tract, are much more effective than ssLip and CS-Lip in improving the enteral absorption of peptide drugs.