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.

Xanthones from Hypericum chinense and their cytotoxicity evaluation.

A xanthonolignoid, 2-O-demethylkielcorin, and a phenylxanthone, chinexanthone A, were isolated from stems of Hypericum chinense together with four known xanthonolignoids and seven known xanthones. Their structures were established by spectroscopic analysis, as their optical properties and absolute stereochemistry determined. The cytotoxicities of the isolated xanthone derivatives as well as additional 32 xanthones against a panel of human cancer cell lines were also evaluated.

Acylphloroglucinol, biyouyanagiol, biyouyanagin B, and related spiro-lactones from Hypericum chinense.

Biyouyanagiol (1), an acylphloroglucinol-related compound having a unique cyclopenta-1,3-dione moiety, was isolated from a Japanese traditional medicinal plant, Hypericum chinense, together with three new spiro-lactone-related derivatives, biyouyanagin B (2), 5,6-dihydrohyperolactone D (3), and 4-hydroxyhyperolactone D (4). Their structures were established on the basis of spectroscopic evidence. In a cytotoxicity assay against human cancer cell lines including multidrug-resistant (MDR) cancer cell lines, several compounds demonstrated enhanced cytotoxicity against MDR KB cells in the presence of colchicine.

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.

Chromone and chromanone glucosides from Hypericum sikokumontanum and their anti-Helicobacter pylori activities.

Chromone glucosides, takanechromones A-C (1, 2 and 5) and chromanone glucosides, named takanechromanones A and B (3 and 4), were isolated from the methanolic extracts of Hypericumsikokumontanum together with 27 known compounds. Their structures were established based on spectroscopic evidence. The isolated compounds and some chromone derivatives were assayed for antimicrobial activity against Helicobacter pylori and cytotoxicity against human cancer cell lines.

Tomoeones A-H, cytotoxic phloroglucinol derivatives from Hypericum ascyron.

Phloroglucinol derivatives tomoeones A-H (1-8) and three known compounds were isolated from leaves of Hypericum ascyron. Their structures were established based on spectroscopic analyses. They are all acylphloroglucinol derivatives possessing a spiro skeleton with geminal isoprenyl groups and a monoterpene moiety, and they are stereoisomers to each other at C-4 and C-13. They appear to be a class of phloroglucinol derivatives. Cytotoxicities of the isolated phloroglucinol derivatives against human tumor cell lines, including multidrug-resistant (MDR) cancer cell lines, were evaluated. Tomoeone F (6) demonstrated significant cytotoxicity against KB cells with an IC50 value of 6.2 microM. Compound 6 was also cytotoxic against MDR cancer cell lines (KB-C2 and K562/Adr), which was more potent than doxorubicin.

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.

Sesquiterpene lactones from the roots of Ferula varia and their cytotoxic activity.

The ethyl acetate-soluble fraction from a MeOH extract of the roots of Ferula varia gave six new sesquiterpene lactones (1-6) and five known sesquiterpenes (7-11). Their structures were established on the basis of spectroscopic evidence. The cytotoxic activities of 1-11 were evaluated against selected human cancer cell lines. Compound 4 showed significant selective cytotoxicity against multidrug-resistant cancer cells (KB-C2). The cytotoxicities of compounds 1, 3, 5, 8, and 11 against KB-C2 cells were enhanced in the presence of nontoxic concentrations of colchicine.

Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells.

Four new triterpenes, together with 16 known triterpenes, were isolated from the floral spikes of Betula platyphylla var. japonica in a search for compounds capable of reversing multidrug resistance in cancer cells. The structures of the new triterpenes were elucidated as 3,4-seco-olean-4(23),13(18)-dien-3-oic acid (1), 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (2), 3-O-methylmalonylepiocotillol II (6), and 3-O-methylmalonylcabraleahydroxylactone (16) by spectroscopic examination. The cytotoxicity of the isolated triterpenes against human cancer cell lines as well as multidrug-resistant cancer cell lines was evaluated. Most of the isolated triterpenes showed very weak cellular toxicities. Although no discernible differences were found in the cytotoxicities for the tested compounds against sensitive and resistant cell lines, the cytotoxicities for several triterpenes against multidrug-resistant cancer cell lines (KB-C2 or K562/Adr) were enhanced in the presence of nontoxic concentrations of colchicine or doxorubicin. Compound 10 reversed the cytotoxicity of colchicine against KB-C2 cells at 8.1 microM and showed comparable potency to 5 microM verapamil.

Nasunin from eggplant consists of cis-trans isomers of delphinidin 3-[4-(p-coumaroyl)-L-rhamnosyl (1-->6)glucopyranoside]-5-glucopyranoside.

Two major anthocyanins were isolated from the acidified methanolic extract of eggplant (Solanum melongena L.) by column chromatography and preparative high-performance liquid chromatography. These anthocyanins were interconvertible under room light illumination condition. By means of tandem time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy, their structures were identified and elucidated as delphinidin 3-[4-(cis-p-coumaroyl)-l-rhamnosyl(1-->6)glucopyranoside]-5-glucopyranoside (compound 1) and delphinidin 3-[4-(trans-p-coumaroyl)-l-rhamnosyl-(1-->6)glucopyranoside]-5-glucopyranoside (compound 2), respectively. The results indicated that nasunin comprised cis and trans isomers of the p-coumaric acid moiety in its structure.

Effects of alkyl gallates on P-glycoprotein function.

In this study, we examined the effects of the food antioxidants, alkyl gallates, on the function of P-glycoprotein (P-gp) and elucidated the importance of alkyl chains and gallic acid moieties on the activity of P-gp. We examined the effects of three alkyl (n-butyl, n-octyl and n-dodecyl) gallates and their related compounds on the cellular accumulation and efflux of rhodamine 123 and daunorubicin in P-gp overexpressing KB-C2 cells. Alkyl gallates increased the cellular accumulation of these P-gp substrates dependent on their alkyl chain lengths by inhibiting the efflux of the substrates. n-Dodecylresorcinol also increased the accumulation, but its effect was less than that of n-dodecyl gallate. However, either lauric acid or n-dodecyl-beta-d-maltoside, which does not have a phenol group, did not increase the accumulation. The results indicated that both the gallic acid moiety and a long alkyl chain play important roles in the modification of P-gp function. The cytotoxicity of daunorubicin was recovered in the presence of alkyl gallates possibly due to their inhibition of P-gp function.

Protective role of Coptidis Rhizoma alkaloids against peroxynitrite-induced damage to renal tubular epithelial cells.

A study was conducted to elucidate and compare the protective activity of alkaloids from Coptidis Rhizoma (berberine, coptisine, palmatine, epiberberine, jatrorhizine, groenlandicine and magnoflorine) using an LLC-PK(1) cell under peroxynitrite (ONOO(-)) generation model. Treatment with 3-morpholinosydnonimine (SIN-1) led to an increase in cellular ONOO(-) generation in comparison with non-treated cells. However, Coptidis Rhizoma extract and its alkaloids, except for berberine, reduced the cellular ONOO(-) level. In addition, DNA fragmentation induced by SIN-1 was significantly decreased by the extract, and also by coptisine, epiberberine, jatrorhizine, groenlandicine and magnoflorine. Moreover, treatment with berberine, coptisine, palmatine and epiberberine exerted a protective effect against G(0)/G(1)phase arrest of cell cycle induced by SIN-1. The increase in cellular ONOO(-) generation, DNA damage and disturbance of the cell cycle by SIN-1 resulted in a decrease in cell viability. However, Coptidis Rhizoma extract, epiberberine, jatrorhizine, groenlandicine and magnoflorine significantly increased cell viability even at a concentration as low as 10 microg mL(-1). These findings demonstrate that Coptidis Rhizoma extract and its alkaloids can ameliorate the cell damage associated with ONOO(-) generation in renal tubular LLCPK(1) cells, and that the various alkaloids have distinctive mechanisms of action, such as ONOO(-) scavenging, protection from DNA damage and control of the cell cycle. Furthermore, the data suggest that among the Coptidis Rhizoma alkaloids, coptisine is the most effective for protection against SIN-1-induced cellular injury in terms of its potency and content.

Anti-HIV benzylisoquinoline alkaloids and flavonoids from the leaves of Nelumbo nucifera, and structure-activity correlations with related alkaloids.

(+)-1(R)-Coclaurine (1) and (-)-1(S)-norcoclaurine (3), together with quercetin 3-O-beta-D-glucuronide (4), were isolated from the leaves of Nelumbo nucifera (Nymphaceae), and identified as anti-HIV principles. Compounds 1 and 3 demonstrated potent anti-HIV activity with EC50 values of 0.8 and <0.8 microg/mL, respectively, and therapeutic index (TI) values of >125 and >25, respectively. Compound 4 was less potent (EC50 2 microg/mL). In a structure-activity relationship study, other benzylisoquinoline, aporphine, and bisbenzylisoquinoline alkaloids, including liensinine (14), negferine (15), and isoliensinine (16), which were previously isolated from the leaves and embryo of Nelumbo nucifera, were evaluated for anti-HIV activity. Compounds 14-16 showed potent anti-HIV activities with EC50 values of <0.8 microg/mL and TI values of >9.9, >8.6, and >6.5, respectively. Nuciferine (12), an aporphine alkaloid, had an EC50 value of 0.8 microg/mL and TI of 36. In addition, synthetic coclaurine analogs were also evaluated. Compounds 1, 3, 12, and 14-16 can serve as new leads for further development of anti-AIDS agents.

3-O-Glutaryl-dihydrobetulin and related monoacyl derivatives as potent anti-HIV agents.

3-O-Acyl-betulin and -dihydrobetulin derivatives were prepared and evaluated for anti-HIV activity. 3-O-Glutaryl-dihydrobetulin (17) demonstrated extremely potent anti-HIV activity with an EC(50) value of 2 x 10(-5) microM and a TI value of 1.12 x 10(6). 3-O-(3',3'-Dimethylsuccinyl)- and 3-O-(3',3'-dimethylglutaryl)-dihydrobetulins (15, 16) were also potent anti-HIV compounds with EC(50) values of 0.0017 and 0.0013 microM, respectively, and TI values of 16,160 and 19,530, respectively.

Coptidis Rhizoma: protective effects against peroxynitrite-induced oxidative damage and elucidation of its active components.

We have investigated the protective effects of Coptidis Rhizoma against peroxynitrite (ONOO(-))-induced oxidative damage and have elucidated the active components of this preparation. In an in-vitro system, Coptidis Rhizoma extract scavenged ONOO(-) and its precursors, nitric oxide (NO) and superoxide anion (O(2)(-)). This scavenging activity was more marked for ONOO(-) than its precursors. In addition, against 3-morpholinosydnonimine-induced cellular damage, this extract significantly reduced cellular ONOO(-) formation and increased cell viability. In an in-vivo lipopolysaccharide plus ischaemia-reperfusion system that generated ONOO(-), the administration of Coptidis Rhizoma extract at 50 and 100 mg kg(-1)/day for 30 days exerted greater inhibition of ONOO(-) than NO and O(2)(-). This suggested that it acted as a direct scavenger of ONOO(-) rather than as a scavenger of its precursors. Moreover, the suppression of the activities of the antioxidative enzymes superoxide dismutase, catalase and glutathione peroxidase was significantly attenuated by the administration of Coptidis Rhizoma extract. Furthermore, the extract ameliorated renal dysfunction judged by decreasing serum urea nitrogen and creatinine levels. To elucidate the active components of Coptidis Rhizoma extract, we evaluated and compared the effects of the phenol plus alkaloid and alkaloid fractions on ONOO-induced damage. We found that the alkaloid fraction consisting of berberine, palmatine and coptisine was the most effective at protecting against ONOO(-). We confirmed that berberine (10 and 20 mg kg(-1)/day for 10 days), the main and most active alkaloid in Coptidis Rhizoma extract, was also protective, exerting NO-, O(2)(-)- and ONOO(-)-scavenging activities. This study suggested that Coptidis Rhizoma could protect against ONOO(-)-induced oxidative damage and that this effect was mainly attributable to the constituent alkaloids, especially berberine. This study is the first to demonstrate an antioxidative effect of alkaloids, including berberine, against ONOO(-)-induced damage.

Absorption and metabolism of delphinidin 3-O-beta-D-glucopyranoside in rats.

The absorption and metabolism of delphinidin 3-O-beta-d-glucopyranoside (Dp3G), which is the most potent antioxidant among the blueberry anthocyanins, were studied in rats. Dp3G rapidly appeared in the blood plasma within 15 min of oral administration (100 mg/kg body wt). The plasma level of absorbed Dp3G showed two peaks at 15 and 60 min after ingestion and then decreased time-dependently. However, the plasma level was maintained at approximately 30 nmol/l even after 4 h. Besides the Dp3G peak, a single major metabolite peak was detected by HPLC in the blood plasma obtained at 15 min. MS and NMR spectroscopy clarified that the chemical structure of the metabolite was 4'-O-methyl delphinidin 3-O-beta-d-glucopyranoside (methylation of the 4'-OH on the delphinidin B-ring). The present finding of this unique metabolite in anthocyanin metabolism strongly suggests that methylation of the 4'-OH on the flavonoid B-ring is a common metabolic pathway for flavonoids that carry the pyrogallol structure on the B-ring, as the same type of metabolite has been reported for other flavonoids such as epigallocatechin, but not for flavonoids carrying the catechol structure.

Complete assignment of bilberry (Vaccinium myrtillus L.) anthocyanins separated by capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) mobilities of fifteen anthocyanins in bilberry extract were completely characterized. Four minor anthocyanins in bilberry extract (malvidin 3-O-alpha-L-arabinopyranoside (Mv 3-ara), peonidin 3-O-beta-D-galactopyranoside (Pn 3-gal), peonidin 3-O-alpha-L-arabinopyranoside (Pn 3-ara), and petunidin 3-O-alpha-L-arabinopyranoside (Pt 3-ara)) that remained unidentified in our previous CZE study were isolated from the bilberry extract, and the chemical structures were assigned by NMR and MS. Their CZE mobilities were then precisely examined together with those of other major anthocyanins in the extract. When the CZE mobilities of the fifteen anthocyanins assigned here were plotted against their molecular weight/numbers of free phenolic group, it was found that separation of anthocyanins by CZE is primarily determined by the type of conjugated sugar present, and secondly by the aglycon structure.

Absorption and metabolism of delphinidin 3-O-beta-D-glucoside in rats.

Anthocyanins, kind of flavonoids (FL) found in plants and vegetables, are known to have varieties of physiological functions. In the present study, we examined absorption and metabolism of delphinidin 3-O-beta-D-glucoside (Dp3G) in rats. Dp3G appeared in the plasma at 15 min after oral administration as an intact glucosidic form. The plasma level also showed another peak at 60 min. One metabolite peak was detected in the plasma and the structure was assigned as 4'-O-methyl Dp3G (MDp3G) by NMR and MS. The metabolite was also identified in several tissues as a major metabolite especially in the liver. No 3'-O-methyl Dp3G was detected in any tissues, therefore, 4'-OH methylation is the main path of Dp3G metabolism in rats. This finding generalized the metabolic formation of FL having pyrogallol B ring because it has been reported that FL having catechol structure produced 3'-O-methyl-derivatives, but FL having pyrogallol structure produced 4'-O-methyl-derivatives.