Heptamer-type small guide RNA that can shift macrophages toward the M1 state.

Multiple myeloma is a refractory cancer of plasma cells. Although treatment strategies for multiple myeloma are getting improved year by year, in most cases patients relapse due to the emergence of drug-resistant mutations in the myeloma cells. The interplay between myeloma cells and tumor-associated macrophages (TAM) is important for the pathology. We thought that some heptamer-type sgRNAs for TRUE gene silencing would be able to transform TAM toward the M1 state and might become therapeutic drugs for myeloma. Here, we searched for heptamer-type sgRNAs that can shift macrophages toward the M1 state. We screened a heptamer-type sgRNA library for the ability to up-regulate IL-12b gene expression in human macrophage-like cell lines, and found three such sgRNAs. One of the sgRNAs, H12960, which also showed such ability in human fresh macrophages and mouse macrophage-like cell lines, efficiently suppressed human myeloma cell growth in SCID/NOD mice.

Large-Scale Isolation of Three O-Methyl Anthocyanins from Bilberry (Vaccinium myrtillus L.) Extract.

Three O-methyl anthocyanidin 3-O-ß-D-glucopyranosides were isolated from bilberry extract on a large-scale basis together with two non O-methyl analogues. Anthocyanidin 3-O-ß-D-galactopyranosides were removed from bilberry extract together with parts of anthocyanidin 3-O-α-L-arabinopyranosides after treatment with ß-galactosidase. The remaining arabinopyranosides were removed by applying acid catalytic hydrolysis. The amounts of anthocyanins recovered as flavylium trifluoroacetic acid salt were as follows: 630 mg for petunidin 3-O-ß-D-glucopyranoside, 423 mg for peonidin 3-O-ß-D-glucopyranoside, 588 mg for malvidin 3-O-ß-D-glucopyranoside, 877 mg for delphinidin 3-O-ß-D-glucopyranoside, and 742 mg for cyanidin 3-O-ß-D-glucopyranoside.

Evaluation of double heptamer-type sgRNA as a potential therapeutic agent against multiple myeloma.

Emergence of drug-resistant mutations in the course of myeloma cell evolution and subsequent relapse of myeloma appears to be currently inevitable in most patients. To remedy this situation, we are trying to develop therapeutic small guide RNAs (sgRNAs) based on tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing), an RNA-mediated gene expression control technology. We designed two sets of double heptamer-type sgRNA, which target the human BCL2 mRNA. Both sets of double heptamer-type sgRNA reduced viability of human myeloma cell lines, RPMI-8226 and KMM-1. We also performed a mouse xenograft experiment to examine how the double heptamer-type sgRNA DHa1(BCL2)/DHa2(BCL2) can reduce the growth of KMM-1 cells in vivo. Median survival periods of the sgRNA cohorts were greater than that of the control cohort by 11-43 days. Furthermore, we designed two sets of double heptamer-type sgRNA, which target the human CCND1 mRNA, and both sets synergistically reduced RPMI-8226 cell viability.

Boysenberry Polyphenols Suppressed Elevation of Plasma Triglyceride Levels in Rats.

Boysenberry, a hybrid Rubus berry, is mainly cultivated in New Zealand. We previously reported that consumption of boysenberry juice (BBJ) exhibited anti-obesity effects in high-fat feeding rats. In this study, we focused on the suppressive effect of BBJ and its fraction on triglyceride absorption from the gastrointestinal tract. BBJ effectively inhibited pancreatic lipase activity in vitro, and was separated into four fractions (Fr1, Fr2, Fr3 and Fr4) by HP-20 column chromatography. Among all the fractions, Fr3, the ellagic acid-rich fraction, showed the most potent inhibition against pancreatic lipase in vitro with Fr2, the anthocyanin-rich fraction, second. Authentic ellagic acid equivalent in Fr3 showed poor activity against pancreatic lipase. Then, each fraction was orally administered with corn oil to rats fitted with a jugular catheter to examine the effects of each fraction on plasma triglyceride levels. Both Fr2 and Fr3 effectively suppressed the plasma triglyceride level elevation at a dose of 1,000 mg/kg body weight. These findings demonstrated that BBJ contains chemical components which inhibit triglyceride absorption from the gastrointestinal tract.

Absorption and metabolism of piceatannol in rats.

Piceatannol (trans-3,3',4,5'-tetrahydroxystilbene), a natural analogue of resveratrol, has multiple biological functions. Nevertheless, piceatannol's biological fate is yet to be determined. In this study, we evaluated the absorption and metabolism of piceatannol in rats. Furthermore, the area under the plasma concentration curves (AUC) and metabolic pathway of piceatannol were compared with those of resveratrol. We determined the plasma concentrations of piceatannol, resveratrol, and their respective metabolites following their intragastric administration. Resveratrol metabolites were only conjugates, whereas piceatannol metabolites were piceatannol conjugates, O-methyl piceatannol, and its conjugates. The AUC for piceatannol, resveratrol, and their metabolites increased in a dose-dependent manner (90-360 µmol/kg). The AUC for total piceatannol was less than that for total resveratrol, whereas the AUC for piceatannol (8.6 µmol·h/L) after piceatannol and resveratrol coadministration was 2.1 times greater than that for resveratrol (4.1 µmol·h/L). The greater AUC for piceatannol was a result of its higher metabolic stability.

Comparison of (-)-epigallocatechin-3-O-gallate (EGCG) and O-methyl EGCG bioavailability in rats.

(-)-Epigallocatechin-3-O-(3-O-methyl)gallate (EGCG3″Me) and (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG4″Me) are O-methyl derivatives of (-)-epigallocatechin-3-O-gallate (EGCG) present in tea cultivars such as Benifuuki. Although O-methyl EGCGs have various bioactivities, their bioavailabilities have not been determined. In this study, we compared the bioavailability of EGCG and O-methyl EGCGs in rats, and clarified the pharmacokinetics of O-methyl EGCGs. Following oral administration (100 mg/kg), the areas under the concentration-time curves (AUCs) for EGCG, EGCG3″Me, and EGCG4″Me were 39.6 ± 14.2 µg·h/L, 317.2 ± 43.7 µg·h/L, and 51.9 ± 11.0 µg·h/L, respectively. The AUC after intravenous administration (10 mg/kg) was 2772 ± 480 µg·h/L for EGCG, 8209 ± 549 µg·h/L for EGCG3″Me, and 2465 ± 262 µg·h/L for EGCG4″Me. The bioavailability of EGCG3″Me (0.38%) was the highest (EGCG: 0.14% and EGCG4″Me: 0.21%). The distribution volume of EGCG3″Me (0.26 ± 0.02 L/kg) was the lowest (EGCG: 0.94 ± 0.16 L/kg and EGCG4″Me: 0.93 ± 0.14 L/kg). These results suggested that the higher AUC of EGCG3″Me after oral administration was related to its high bioavailability and low distribution volume. These findings supported the stronger bioactivity of EGCG3″Me in vivo.

Structural elucidation and biological fate of two glucuronyl metabolites of pelargonidin 3-O-ß-D-glucopyranoside in rats.

A high proportion of pelargonidin 3-O-ß-D-glucopyranoside (Pg3G) is metabolized to glucuronides and excreted in mammal urine after ingestion of strawberry fruit, suggesting that these metabolites play important functional roles in vivo. The aim of the present study was to elucidate the structures and determine the biological fate of the two dominant metabolites of Pg3G in rats to enable an accurate discussion of the biological properties of anthocyanins. Authentic Pg3G was orally administered to rats. One pelargonidin monoglucuronide and three Pg3G-monoglucuronides (glucuronides of the glucoside) were identified together with intact Pg3G in both blood plasma and urine samples. The structures of the two dominant metabolites were elucidated as pelargonidin 3-O-ß-D-glucuronide (Pg3GlcA) and pelargonidin 3-O-ß-D-glucuronyl-(1→2)-ß-D-glucoside by means of (1)H and (13)C nuclear magnetic resonance spectroscopy and heteronuclear multiple-bond connective spectroscopy. The bioavailability of Pg3G in its intact form was 0.31% of the orally administered dose, and 0.65% was absorbed in the Pg3GlcA form.

Suppression of lipopolysaccharide and galactosamine-induced hepatic inflammation by red grape pomace.

Grape pomace is generated in the production process of wine and grape juices and is an industrial waste. This study investigated whether an intake of grape pomace was able to suppress chronic inflammation induced by lipopolysaccharide (LPS) and galactosamine (GalN) in vivo. When Sprague-Dawley rats were orally given methanolic extracts from red and white grape pomace, the extracts inhibited the LPS/GalN-evoked activation of nuclear factor-κB (NF-κB) dose-dependently, and red grape pomace exerted a stronger effect than white grape one. Next, rats were fed an AIN93 M-based diet containing 5% red grape pomace for 7 days, followed by the intraperitoneal injection of LPS and GalN. The intake of the red grape pomace-supplemented diet was found to suppress the LPS/GalN-induced activation of NF-κB and expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins. These results suggest that red grape pomace may contain an abundance of effective compound(s) for anti-inflammatory action.

Stimulation of dendritic cell maturation and induction of apoptosis in leukemia cells by a heat-stable extract from azuki bean (Vigna angularis), a promising immunopotentiating food and dietary supplement for cancer prevention.

Non-toxic stimulation of dendritic cells (DCs), which are central immunomodulators, may aid the prevention of cancer. Furthermore, induction of apoptosis in cancer cells by anticancer agents contributes to the induction of DC maturation. We previously reported that extracts from Pinus parviflora Sieb. et Zucc pine cone and Mucuna seed induce differentiation of mouse bone marrow cells into mature dendritic cells and also induce apoptosis in various human cancer cell lines. In the present study, we screened 31 kinds of edible beans with biological activity similar to that of extracts from pine cone and Mucuna and found that the heat-stable extract from azuki bean (Vigna angula) stimulated differentiation of bone marrow cells into immature DCs with the greatest efficacy. The level of IL-6 produced by sequential treatment of DCs with azuki extract and lipopolysaccharide was the highest among the examined beans. Azuki extract also inhibited the growth of human leukemia U937 cells, leading to induction of apoptosis. These results suggest that azuki bean and its extract are immunopotentiating foods that can be used as a dietary supplement for cancer prevention and immunotherapy.

A heat-stable extract from Mucuna stimulates the differentiation of bone marrow cells into dendritic cells and induces apoptosis in cancer cells.

Pine cone extract is known to induce differentiation of human mononuclear cells into dendritic cells (DCs) and also to induce apoptosis in human cancer cells. In the present study, we screened edible plants that contain components with biological activities similar to or more potent than those of pine cone extract. We found that Mucuna (Mucuna pruviens var. utilis) contains a DC differentiation/maturation-inducing activity and a component that induces apoptosis in human cancer cell lines. Mucuna extract specifically stimulated differentiation of BM cells to immature DCs. Marked production of IL-6 was observed by sequential treatment with at least 10 µg/mL of Mucuna extract followed by LPS. The sequential treatment with Mucuna extract followed by LPS produced a much higher ratio of IL-12 to IL-6 and a lower ratio of TNF-α to IL-6 than that obtained by sequential treatment with a medicinal mushroom Phellinus linteus extract and then LPS. The DC differentiation/maturation activity and the component inducing apoptosis in cancer cells were separable by column chromatography.

The Chinese prescription Wen-Pi-Tang extract delays disease onset in amyotrophic lateral sclerosis model mice while attenuating the activation of glial cells in the spinal cord.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the selective loss of motor neurons. There is no effective treatment or drug against ALS, and the precise mechanisms leading to the selective loss of motor neurons are still unknown. We investigated the effect of a Chinese prescription, Wen-Pi-Tang, on the ALS model mouse SOD1(G93A). Although the oral administration of Wen-Pi-Tang extract to SOD1(G93A) mice had no significant effect on body weight loss and survival time, Wen-Pi-Tang delayed disease onset. Therefore, we evaluated immunohistological changes in the spinal cord of SOD1(G93A) mice during the early disease period, and found that Wen-Pi-Tang extract inhibited neuronal loss in the lumbar segment of the spinal cord of mice. Furthermore, increased astrocytes and microglial cells, which increase prior to neuronal loss, in spinal cords were significantly reduced in the Wen-Pi-Tang treated group. Since oxidative markers, heme oxygenase-1 and inducible nitric oxide synthase, in the spinal cord were also reduced as well as the change in microglia, the administration of Wen-Pi-Tang was thought to delay disease onset by inhibiting glial cell activation.

Structures and antioxidant activity of anthocyanins in many accessions of eggplant and its related species.

Anthocyanins were detected in extracts from the peels of 123 accessions of eggplant (Solanum melongena) and its related species. Their anthocyanin profiles were classified into four types, including known Japanese eggplant type (type 1) and non-Japanese eggplant type (type 2). Although most of the eggplant accessions had one of the two known profiles, one accession had a novel profile (type 3). Two accessions of related species showed another novel profile (type 4). The major anthocyanins were identified as delphinidin 3-(p-coumaroylrutinoside)-5-glucoside (nasunin) (type 1), delphinidin 3-rutinoside (type 2), delphinidin 3-glucoside (type 3), and petunidin 3-(p-coumaroylrutinoside)-5-glucoside (petunidin 3RGc5G) (type 4). Delphinidin 3-caffeoylrutinoside-5-glucoside (delphinidin 3RGcaf5G) was isolated from the hybrid (F1) plants of a type 1 cultivar and a type 3 germplasm. Among the five purified anthocyanins, delphinidin 3RGcaf5G showed the highest radical-scavenging activities toward both 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and linoleic acid radical, followed in order by nasunin and petunidin 3RGc5G.

Effects of anthocyanins on psychological stress-induced oxidative stress and neurotransmitter status.

There is strong evidence that oxidative stress participates in the etiology of neurodegenerative diseases such as Parkinson's, and Alzheimer's diseases. Moreover, emotional stress effects in the central nervous system play a vital role in homeostasis. The protective effect of anthocyanins on the cerebral oxidative stress was studied using the whiskers cut model. In mice, such treatment causes psychological or emotional distress leading to oxidative stress in tissues. To investigate the in vivo antioxidant activity of anthocyanins, an extract of Vaccinium myrtillis L., an anthocyanin mixture, was orally administered (100 mg/kg of body weight.) to mice for 7 days, and then psychological stress was assessed by cutting off their whiskers. Whisker removal increased both protein carbonyl formation and lipid peroxidation in the brain, heart, kidney, and liver. Further, the levels of oxidative markers showed regional differences in the brain. Concomitantly, dopamine neurotransmitter levels were altered in both the midbrain and the brain cortex. Orally administered anthocyanins were also active in the brain, suppressing stress-induced cerebral oxidative stress and dopamine abnormalities in distressed mice. These effects of anthocyanin treatment suggest their possible usefulness for the treatment of cerebral disorders related to oxidative stress.

Effect on both aglycone and sugar moiety towards Phase II metabolism of anthocyanins.

The effect of sugar moiety on anthocyanin metabolism was studied using anthocyanidin 3-rutinosides (cyanidin 3-O-rutinoside (Cy3R) and delphinidin 3-O-rutinoside (Dp3R)) and 3-O-glucosides (delphinidin 3-O-glucoside (Dp3G)). O-methylated Cy3R and Dp3R were detected in rat blood plasma after oral administration of Cy3R and Dp3R (100mg/kg body weight). On the basis of HPLC retention time and UV-visible spectra together with the data of our previous studies on the hydrophobic metabolites of anthocyanidin 3-O-glucosides, it was concluded that both 3'- and 4'-O-methyl Cy3R were metabolites of Cy3R. On the other hand, only 4'-O-methyl Dp3R was detected as hydrophobic metabolite of Dp3R. A group of hydrophilic metabolites was also detected in rat blood plasma after oral administration of anthocyanins (Dp3G, Cy3R and Dp3R) and their structures were determined to be extended glucuronides and their O-methyl analogues by tandem MS analysis. The amounts of extended glucuronides of Dp3G, Cy3R and Dp3R were less than those of cyanidin 3-O-glucoside (Cy3G) reported in our previous study. On the other hand, anthocyanidin-glucuronides (both cyanidin-glucuronide and delphinidin-glucuronide) were not detected after oral administration of Cy3R, Dp3R and Dp3G. These results indicated that both the type of sugar moiety and stability of aglycone largely affected phase II metabolism of anthocyanins, and also indicated that the type of sugar moiety did not affect the O-methylation metabolism but affected glucuronyl conjugation in both liver and small intestine.

Effect of triterpenoids isolated from the floral spikes of Betula platyphylla var. japonica on P-glycoprotein function.

One of the major causes of multidrug resistance (MDR) in cancer cells is over-expression of P-glycoprotein (P-gp). We studied the effects of 20 triterpenes isolated from the floral spikes of Betula platyphylla var. japonica ( B. platyphylla) on P-gp function based on our previous finding that some of them showed MDR reversing effects. We evaluated accumulations and effluxes of rhodamine 123 as a P-gp substrate with P-gp over-expressing KB-C2 cells. Among the 20 triterpenes, compounds 3, 4, 8, 9, 13, 15, and 20 increased rhodamine 123 accumulations in KB-C2 cells, and three ( 8, 13, and 20) of them also inhibited efflux of rhodamine 123 out of cells. In addition, compounds 13 and 20 showed a weak inhibitory activity of P-gp ATPase. These results suggested that MDR reversing effects of compounds 13 and 20 are partly involved in inhibition of P-gp ATPase.

Enhanced absorption of anthocyanins after oral administration of phytic acid in rats and humans.

Many studies on the bioavailability of polyphenols have been reported. However, the relative urinary excretions of AC are also low, ranging from 0.004% to 0.1%. By contrast, other polyphenols show higher urinary excretion levels. Here, we studied the enhancing effects of phytic acid (IP6) on absorption of blackcurrant anthocyanins (BCAs) in rats and humans. In rats after oral administration of BCAs (as 241 mg of AC/kg body weight) in IP6 (0%, 0.25%, 0.5%, 1%, 2.5%) solution, the ACs recovery in urine was increased dependent on IP6 dose. These results suggest that the IP6 enhances gastrointestinal absorption of ACs. At the further analysis of IP6 enhancement effect in rat, whereas BCAs were normally passed through the stomach and duodenum within 2 h, in IP6 group, after 2-6 h post-administration, stomach and jejunum content's weights were specifically heavy, and large amounts of ACs were also detected in stomach, duodenum, and jejunum. These results suggested that the mixture of BCAs and IP6 reduced the gastrointestinal motility. Prolongation of ACs residue in gastrointestinal tract then caused the enhancing effects of IP6 on absorption of AC. In the human study, each subject was orally administrated a BCA beverage containing BCA concentrate (AC 4 mg/kg body weight), 1% of IP6, and 1% of sodium citrate as a pH stabilizer. Both the plasma level and the urinary excretion of AC were increased as compared to BCA administration without IP6. AC intake with IP6 may increase the bioavailability of AC to the comparative level as other polyphenols. Yet, phytic acid, being a strong chelator of important minerals, contributes to mineral deficiencies. An interference with iron uptake has been reported. Safety tests are therefore necessary before high dose IP6 can be used in foods.

Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins; structure-activity relationship and their synergism.

Antioxidant activities of 15 purified bilberry anthocyanins together with pelargonidin 3-O-beta-D-glucopyranoside and 4'-O-methyl delphinidin 3-O-beta-D-glucopyranoside (MDp 3-glc), the major metabolite of delphinidin 3-O-beta-D-glucopyranoside (Dp 3-glc), were evaluated in order to study the structure-antioxidant activity relationship and any synergism among them in the mixture. Both aglycone structure and the attached sugar moiety affected the O*2- and ONOO- -scavenging activities, although the effect of the attached sugar moiety was smaller than that of the aglycone structure. The potency of activity toward the superoxide radical was in the following order: delphinidin > petunidin > malvidin =approximately cyanidin>(+)-catechin > peonidin > pelargonidin. The activity toward ONOO- was: delphinidin > cyanidin =approximately petunidin > malvidin =approximately (+)-catechin > peonidin > pelargonidin. It was confirmed that methylation of 4'-OH markedly reduced the antioxidant activity of anthocyanin. Further, it was revealed that synergism occurred in both - and ONOO- -scavenging activities among the anthocyanins in the mixture.

Bioavailability and tissue distribution of anthocyanins in bilberry (Vaccinium myrtillus L.) extract in rats.

To clarify how structural diversity of anthocyanins relates to their in vivo function, bioavailability was precisely studied in rats using bilberry (Vaccinium myrtillus L.) extract (Bilberon 25) as an anthocyanin source that contains 15 different anthocyanins. The bilberry extract was orally or intravenously administered to rats, and the plasma levels of each anthocyanin were determined by high-performance liquid chromatography. As the result, all anthocyanins except peonidin 3-O-alpha-L-arabinoside were detectable in the blood plasma. The plasma concentration of anthocyanins as a whole reached the maximum level of 1.2 microM at 15 min after oral administration of 400 mg/kg bilberry extract (153.2 mg/kg as anthocyanins) and then decreased with time. Uptake and decay profiles of each anthocyanin in the plasma were almost the same for all anthocyanins except a few with their maximum after 30 min. Among the anthocyanins carrying the same aglycone, the plasma level after 15 min of oral administration was as follows: galactoside > glucoside > arabinoside. Plasma clearance of anthocyanins after intravenous administration clearly showed that arabinoside disappeared more rapidly than glucoside and galactoside. On the other hand, when anthocyanins carrying the same sugar moiety were compared, the half disappearance time of plasma anthocyanins was in the following order: delphinidin > cyanidin > petunidin = peonidin > malvidin. The bioavailability of anthocyanins was in the range of 0.61-1.82% and was 0.93% as the anthocyanin mixture. The bioavailability of anthocyanins carrying the same aglycone was in the following order: Galactoside showed the highest followed by glucoside and arabinoside for cyanidin and delphinidin, but arabinoside and galactoside showed a higher bioavailability than glucoside for petunidin and malvidin. Anthocyanins recovered in urine and bile during the first 4 h after intravenous administration were only 30.8 and 13.4%, respectively. Anthocyanin profiles in tissues were quite different from those in blood plasma. The major anthocyanins distributed in liver and kidney were the O-methyl anthocyanins such as peonidin, malvidin, and other O-methyl anthocyanins derived from delphinidin, cyanidin, and petunidin-glycosides.

Gastrointestinal uptake of nasunin, acylated anthocyanin in eggplant.

We previously showed that nasunin, acylated anthocyanins in eggplant peel, comprises two isomers, cis-nasunin and trans-nasunin. In this study, gastrointestinal absorption of cis- and trans-nasunins was studied in rats. Orally administered nasunins were quickly absorbed in their original acylated forms and maximally appeared in blood plasma after 15 min. When the maximum plasma concentration and area under the plasma concentration curve were normalized by orally administered dose (micromoles per kilogram), there was no significant difference in the uptake efficiency between two isomers and both exhibited a plasma level almost identical to that of delphinidin 3-O-beta-D-glucopyranoside. However, metabolites such as 4'-O-methyl analogues and extended glucuronides which were observed for delphinidin 3-O-beta-D-glucopyranoside and cyanidin 3-O-beta-D-glucopyranoside metabolisms were not detected in urine or blood plasma. Moreover, deacylated and glycolytic products of nasunins such as delphinidin 3-O-beta-D-glucopyranoside or delphinidin (aglycone) were also not detected in blood plasma even after oral administration for 8 h. These results indicated that nasunins were absorbed in their original acylated forms and exhibit a bioavailability almost identical to that of nonacylated anthocyanins.

Ingested delphinidin-3-rutinoside is primarily excreted to urine as the intact form and to bile as the methylated form in rats.

Many reports have described the bioavailability of anthocyanins; however, most of these reports investigated only the amount of anthocyanins excreted in urine. In the present study, we calculated the pharmacokinetic bioavailability of anthocyanins in rats by measuring the plasma concentration of delphinidin-3-rutinoside that had been administered orally or intravenously. Delphinidin-3-rutinoside was primarily absorbed in the blood and excreted into urine as unmetabolized forms with a T(max) of 26.3 min and a C(max) of 0.285 +/- 0.071 micromol/L. We detected small amounts of the metabolite 4'-O-methyl-delphinidin-3-rutinoside in the plasma, but we detected neither anthocyanidin (aglycone) nor glucuro- or sulfoconjugates. For the 8 h period after intake, delphinidin-3-rutinoside and 4'-O-methyl-delphinidin-3-rutinoside were excreted to urine at 795 +/- 375 and 12.3 +/- 2.91 nmol, respectively. Relative to intravenous injection, oral administration of delphinidin-3-rutinoside resulted in complete bioavailability (0.49 +/- 0.06%). Analysis of delphinidin-3-rutinoside plasma concentrations in bile cannulated rats revealed that, for the 8-h period after intake, the intact delphinidin-3-rutinoside excretion ratio in bile was 11% of the excretion ratio of 4'-O-methyl-delphinidin-3-rutinoside, 1.91 +/- 0.35 nmol versus 17.4 +/- 8.67 nmol, respectively. Setting the bile duct cannulation in a Bollman-type cage, however, significantly increased the bioavailability of orally administered delphinidin-3-rutinoside (18.14 +/- 6.24%). This effect appears to stem immobilization stress by reducing gastrointestinal motility. The cumulative excretion of delphinidin-3-rutinoside and 4'-O-methyl-delphinidin-3-rutinoside in urine and bile was 2.67 +/- 1.24% (w/w) of the dose ingested. Studies report that several metabolites are formed after oral ingestion of anthocyanins. Examples include glucuronyl from cyanidin-3-glucoside and both glucuronyl and sulfate conjugates from pelargonidin-3-glucoside. Our results indicate that delphinidin-3-rutinoside might be metabolized differently from cyanidin-3-glucoside and pelargonidin-3-glucoside.