Teruaki Akao

Intestinal bacterial hydrolysis is required for the appearance of compound K in rat plasma after oral administration of ginsenoside Rb1 from Panax ginseng.

Ginsenoside Rb1 from Panax ginseng root is transformed into compound K via ginsenosides Rd and F2 by intestinal bacterial flora. Among 31 defined intestinal strains from man, only Eubacterium sp. A‐44 transformed ginsenoside Rb1 into compound K via ginsenoside Rd. The ginsenoside Rb1‐hydrolysing enzyme isolated from Eubacterium sp. A‐44 was identical to a previously purified geniposide‐hydrolysing β‐D‐glucosidase.

Balicalin, the predominant flavone glucuronide of scutellariae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form.

When baicalin was orally administered to conventional rats, it was detected in their plasma for 24 h after administration, but baicalein, the aglycone of baicalin, was not detected. However, when baicalin was given to germ‐free rats, only a small amount of baicalin was detected in their plasma within 2 h after the administration, its AUC0‐lim (the area under the concentration‐time curve from 0 to last determination time) being 12.0% of that in conventional rats. Subsequently, a considerable amount (55.1 ± 6.2%) of baicalin was recovered from the gastrointestinal tract even 4 h after administration. When baicalein was orally administered to conventional rats, however, baicalin appeared rapidly in their plasma at an AUC0‐lim value similar to that obtained after oral administration of baicalin, despite the absence of baicalein in plasma. When intestinal absorption was evaluated by the rat jejunal loop method, baicalein was absorbed readily, but only traces of baicalin were absorbed. Moreover, in conventional rats a small amount (13.4 ± 3.1%) of baicalin and an appreciable amount (21.9 ± 3.4%) of baicalein were recovered from the gastrointestinal tract even 4 h after oral administration of baicalin, but only a small amount (3.93 ± 1.43%) of baicalein was detected in the intestinal tract 1 h after administration of baicalein.

Pharmacokinetics of Berberine and Its Main Metabolites in Conventional and Pseudo Germ-Free Rats Determined by Liquid Chromatography Ion Trap Mass Spectrometry

Berberine (Ber) and its main metabolites were identified and quantified using liquid chromatography/electrospray ionization/ion trap mass spectrometry. Rat plasma contained the main metabolites, berberrubine, thalifendine, demethyleneberberine, and jatrorrhizine, as free and glucuronide conjugates after p.o. Ber administration. Moreover, the original drug, the four main metabolites, and their glucuronide conjugates were all detected in liver tissues after 0.5 h and in bile samples 1 h after p.o. Ber administration. Therefore, the metabolic site seemed to be the liver, and the metabolites and conjugates were evidently excreted into the duodenum as bile. The pharmacokinetics of Ber and the four metabolites were determined in conventional and pseudo germ-free rats (treated with antibiotics) after p.o. administration with 40 mg/kg Ber. The AUC0-limt and mean transit time values of the metabolites significantly differed between conventional and pseudo germ-free rats. The amounts of metabolites were remarkably reduced in the pseudo germ-free rats, whereas levels of Ber did not obviously differ between the two groups. The intestinal flora did not exert significant metabolic activity against Ber and its metabolites, but it played a significant role in the enterohepatic circulation of metabolites. In this sense, the liver and intestinal bacteria participate in the metabolism and disposition of Ber in vivo.

BIOAVAILABILITY STUDY OF GLYCYRRHETIC ACID AFTER ORAL ADMINISTRATION OF GLYCYRRHIZIN IN RATS; RELEVANCE TO THE INTESTINAL BACTERIAL HYDROLYSIS

To clarify the metabolic fate of glycyrrhizin when orally ingested, we investigated the bioavailability of glycyrrhetic acid, the aglycone of glycyrrhizin, after intravenous or oral administration of glycyrrhetic acid (5.7 mg kg−1, equimolar to glycyrrhizin) or glycyrrhizin (10 mg kg−1) at a therapeutic dose in rat.

Intestinal bacterial hydrolysis is indispensable to absorption of 18β‐glycyrrhetic acid after oral administration of glycyrrhizin in rats

Abstract— Gnotobiote rats were prepared by infecting germ‐free rats with Eubaclerium sp. strain GLH, a human intestinal bacterium capable of hydrolysing glycyrrhizin to 18β‐glycyrrhetic acid. Their faeces and caecal contents showed glycyrrhizin‐hydrolysing activities (31·7 and 31·3 pmol min−1 (mg protein)−1, respectively) similar to those (81·0 and 39·9 pmol min−1 (mg protein)−1, respectively) of conventional rats, although there was no detectable activity in germ‐free rats. When glycyrrhizin (100 mg kg−1) was orally administered to conventional, germ‐free and gnotobiote rats, no glycyrrhizin could be detected in plasma 4 or 17 h after the administration, using EIA and HPLC assays. Plasma 18β‐glycyrrhetic acid was not detected 4 or 17 h after the administration of glycyrrhizin to germ‐free rats nor could this compound be detected in caecal contents or in the faeces. However, 18β‐glycyrrhetic acid (0·6–2·6 nmol mL−1) was detected in plasma of the conventional and the gnotobiote rats 4 and 17 h after the administration, and the caecal contents after 4 h and the cumulative faeces up to 17 h of the conventional and the gnotobiote rats contained considerable amounts of 18β‐glycyrrhetic acid. These findings indicate that orally administered glycyrrhizin is poorly absorbed from the gut, but is hydrolysed to 18β‐glycyrrhetic acid by intestinal bacteria such as E. sp. strain GLH, and the resulting 18β‐glycyrrhetic acid is absorbed.

Efflux of baicalin, a flavone glucuronide of Scutellariae Radix, on Caco-2 cells through multidrug resistance-associated protein 2

Baicalin and its aglycone, baicalein, being are strong antioxidants and have various pharmacological actions. Baicalein has shown a unique metabolic fate in rat intestine, being excreted into the intestinal lumen from mucosal (epithelial) cells following glucuronidation of baicalein absorbed after oral administration. The purpose of this study was to examine the absorption and excretion of baicalin and baicalein in a Caco‐2 cell monolayer model to evaluate the disposition of baicalin and baicalein in the human intestine. When baicalein at 5μM was loaded on the apical side of the Caco‐2 cell monolayer, baicalein was not transferred to the basolateral side, but more baicalin was excreted onto the apical side than was being absorbed onto the basolateral side. The amount of baicalin recovered on both sides accounted for more than 90% of the baicalein absorbed from the apical surface. This was supported by the fact that Caco‐2 cell microsomes showed UDP‐glucuronate glucuronosyl‐transferase activity towards baicalein to form baicalin. On the other hand, when baicalein was loaded at higher concentrations, baicalin excretion became saturated, and then baicalein was transferred to the basolateral side. Furthermore, baicalin efflux was not inhibited by MDR1/P‐glycoprotein substrates such as ciclosporin and vinblastine, but significantly inhibited by multidrug resistance‐associated protein 2 (MRP2, ABCC2) substrates such as probenecid and genistein. MRP2 was also detected in Caco‐2 cells by Western blotting using specific antibodies. In addition, baicalin, but not baicalein, enhanced dose‐dependently the vanadate‐sensitive ATPase activity of human MRP2. These results indicated that, in Caco‐2 cells, any baicalein absorbed after loading at low concentrations of baicalein was not transferred to the basolateral side, but was first transformed into baicalin in the cells and excreted through the action of MRP2, mainly to the apical side.

Metabolism of Sennosides by Human Intestinal Bacteria

During the course of studies on the metabolism of sennosides by human intestinal bacteria, an enzyme which takes part in the reduction of sennosides and sennidins was originally isolated from

Enteric Excretion of Baicalein, a Flavone of Scutellariae Radix, via Glucuronidation in Rat: Involvement of Multidrug Resistance-Associated Protein 2

AbstractPurpose. Baicalin (BG) and its aglycone, baicalein (B), are strong antioxidants and have various pharmacological actions. The purpose of this study was to evaluate efflux of BG from rat intestinal mucosal cell following glucuronidation of B absorbed after oral administration of B. Methods. The absorption and excretion of BG and B were evaluated in rats using the in situ jejunal loop technique and in vitro jejunal everted sac experiments. BG and B levels were determined by high-performance liquid chromatography with electro-chemical detection to ensure selectivity and high sensitivity. Results. A large amount (30.4% recovery) of BG, but no B, was detected in the intestinal lumens of germ-free rats 4 h after oral administration of B (12.1 mg/kg), in comparison with a substantial recovery (55.1%) of unabsorbed BG 4 h after its administration. During the in situ rat jejunal loop absorption experiment, B disappeared rapidly, and 8% of the lost B was excreted into the loop as BG 20 min after infusing 0.1 mM B. In an in vitro absorption experiment using everted rat jejunal sac, BG also appeared outside the sac, accompanied by the disappearance of B from the outer (mucosal) side. However, very little of B was transferred to the inner (serosal) side of the sac, and only a trace of BG was detected inside the sac. Thus, in both the loop and the everted sac systems, the efflux of BG from the mucosal surface was saturated with the concentration of B added. Moreover, the efflux rate of BG in the everted jejunal sac from Eisai hyperbilirubinemic rat (EHBR) was significantly lower by 56.4% than that from Sprague-Dawley rat. Conclusions. These results indicate that, in rat, a large proportion of any B absorbed is retained, transformed into BG within the intestinal mucosal cells, and coordinately excreted through multidrug resis- tance-associated protein 2 (MRP2) into the intestinal lumen.

Antispasmodic activity of licochalcone A, a species‐specific ingredient of Glycyrrhiza inflata roots

Licochalcone A, a species‐specific and characteristic retrochalcone ingredient of Glycyrrhiza inflata root, has been shown to possess multiple bioactive properties. However, its muscle relaxant activity has not been reported previously. Licochalcone A showed a concentration‐dependent relaxant effect on the contraction induced by carbachol (50% effective concentration (EC50) = 5.64 ± 1.61 μm). KCl (EC50 5.12 ± 1.68 μm), BaCl2 (EC50 1.97 ± 0.48 μm) and A23187 (EC50 2.63 ± 2.05 μm). Pretreatment with licochalcone A enhanced the relaxant effect of forskolin, an adenylyl cyclase activator, on the contraction in a similar manner to 3‐isobutyl‐1‐methylxanthine (IBMX), a phosphodiesterase (PDE) inhibitor. Furthermore, the IC50 (22.1 ± 10.9 μm) of licochalcone A against cAMP PDE was similar to that of IBMX (26.2 ± 7.4 μm). These results indicated that licochalcone A may have been responsible for the relaxant activity of G. inflata root and acted through the inhibition of cAMP PDE.

In Vitro Metabolism of Isoline, a Pyrrolizidine Alkaloid from Ligularia duciformis, by Rodent Liver Microsomal Esterase and Enhanced Hepatotoxicity by Esterase Inhibitors

Isoline, a major retronecine-type pyrrolizidine alkaloid (PA) from the Chinese medicinal herb Ligularia duciformis, was suggested to be the most toxic known PA. Its in vitro metabolism was thus examined in rat and mouse liver microsomes, and its toxicity was compared with that of clivorine and monocrotaline after i.p. injection in mice. Isoline was more rapidly metabolized by both microsomes than clivorine and monocrotaline and converted to two polar metabolites M1 and M2, which were spectroscopically determined to be bisline (a deacetylated metabolite of isoline) and bisline lactone, respectively. Both metabolites were formed in the presence or absence of an NADPH-generating system with liver microsomes but not cytosol. Their formation was completely inhibited by the esterase inhibitors, triorthocresyl phosphate (TOCP) and phenylmethylsulfonyl fluoride, but not at all or partially by cytochrome P450 (P450) inhibitors, α-naphthoflavone and proadifen (SKF 525A), respectively. These results demonstrated that both metabolites were produced by microsomal esterase(s) but not P450 isozymes. The esterase(s) involved showed not only quite different activities but also responses to different inhibitors in rat and mouse liver microsomes, suggesting that different key isozyme(s) or combinations might be responsible for the deacetylation of isoline. Isoline injected i.p. into mice induced liver-specific toxicity that was much greater than that with either clivorine or monocrotaline, as judged by histopathology as well as serum alanine aminotransferase and aspartate aminotransferase levels. Isoline-induced hepatotoxicity was remarkably enhanced by the esterase inhibitor TOCP but was reduced by the P450 inhibitor SKF 525A, indicating that rodent hepatic esterase(s) played a principal role in the detoxification of isoline via rapid deacetylation in vivo.