thalifendine and demethyleneberberine

To identify the structure of unknown metabolites of berberine (Ber) in human urine after oral administration.. Urine samples were obtained from 5 volunteers after they orally took Ber chloride 0.9 g per day for three days. Metabolites in urine samples were isolated and purified by polyporous resin column chromatography. The individual metabolites were identified mainly using electrospray ionization mass spectroscopy (ESI-MS) and proton nuclear magnetic resonance (1H NMR) spectroscopy.. Three unknown metabolites (M1, M2, and M3) were isolated. They were susceptible to arylsufatase. ESI-MS measurements of M1, M2, and M3 produced quasimolecular ions [M+H]+, m/z 17.9, 404.0, and 402.0 respectively. Especially, each of them produced a characteristic protonated ion [M-80+H]+, which can be ascribed as quasimolecular ions lost a SO3 fragment. 1H NMR spectra of the metabolites were also obtained and each of 1H signals was assigned.. Structures of M1, M2, and M3 were firmly identified as jatrorrhizine-3-sulfate, demethyleneberberine-2-sulfate, and thalifendine-10-sulfate, and the major metabolite was M2.

Topics: Adult; Antidiarrheals; Berberine; Humans; Magnetic Resonance Spectroscopy; Male; Spectrometry, Mass, Electrospray Ionization; Sulfates

Although metabolic study of individual active compounds isolated from herbal plants has been intensive, it cannot truly reflect the fate of herbs because the herbal extracts in use have many constituents. To address this problem, whole extracts of herbs should be investigated. Microsomes have been heavily used in the in vitro metabolic study of drugs, and various materials have been used to immobilize microsomes to develop highly effective and reusable bioreactors in this field. In this work, rat liver microsomes were immobilized on magnetic nanoparticles (LMMNPs) to develop a highly active and recoverable nanoparticle bioreactor. Using this bioreactor, we investigated the in vitro metabolism of Rhizoma coptidis extract. Incubation of berberine, a major active ingredient of R. coptidis, with LMMNPs for 20 min produced two metabolites, i.e., demethyleneberberine and thalifendine, at high levels. From a comparison of the time courses of thalifendine formation obtained by ultraperformance liquid chromatography-mass spectrometry analysis, it was found that LMMNPs had a higher biological activity than free liver microsomes in metabolizing berberine. Further, the activity of LMMNPs remained almost unchanged after six consecutive uses in the incubation tests. Metabolism of R. coptidis extracts by LMMNPs was studied. The same two metabolites of berberine, i.e., demethyleneberberine and thalifendine, were detected. After a thorough study seeking support for this observation, it was found that demethyleneberberine was the common metabolite of five protoberberine-type alkaloids present in R. coptidis extract, including palmatine, jatrorrhizine, columbanine, epiberberine, and berberine.

Topics: Animals; Berberine; Berberine Alkaloids; Bioreactors; Biotransformation; Chromatography, High Pressure Liquid; Coptis chinensis; Drugs, Chinese Herbal; Equipment Reuse; Kinetics; Magnetite Nanoparticles; Male; Microsomes, Liver; Rats; Rats, Sprague-Dawley

Berberine (BBR) is a drug with multiple effects on cellular energy metabolism. The present study explored answers to the question of which CYP450 (Cytochrome P450) isoenzymes execute the phase-I transformation for BBR, and what are the bioactivities of its metabolites on energy pathways.. BBR metabolites were detected using LC-MS/MS. Computer-assistant docking technology as well as bioassays with recombinant CYP450s were employed to identify CYP450 isoenzymes responsible for BBR phase-I transformation. Bioactivities of BBR metabolites in liver cells were examined with real time RT-PCR and kinase phosphorylation assay.. In rat experiments, 4 major metabolites of BBR, berberrubine (M1), thalifendine (M2), demethyleneberberine (M3) and jatrorrhizine (M4) were identified in rat's livers using LC-MS/MS (liquid chromatography-tandem mass spectrometry). In the cell-free transformation reactions, M2 and M3 were detectable after incubating BBR with rCYP450s or human liver microsomes; however, M1 and M4 were below detective level. CYP2D6 and CYP1A2 played a major role in transforming BBR into M2; CYP2D6, CYP1A2 and CYP3A4 were for M3 production. The hepatocyte culture showed that BBR was active in enhancing the expression of insulin receptor (InsR) and low-density-lipoprotein receptor (LDLR) mRNA, as well as in activating AMP-activated protein kinase (AMPK). BBR's metabolites, M1-M4, remained to be active in up-regulating InsR expression with a potency reduced by 50-70%; LDLR mRNA was increased only by M1 or M2 (but not M3 and M4) with an activity level 35% or 26% of that of BBR, respectively. Similarly, AMPK-α phosphorylation was enhanced by M1 and M2 only, with a degree less than that of BBR.. Four major BBR metabolites (M1-M4) were identified after phase-I transformation in rat liver. Cell-free reactions showed that CYP2D6, CYP1A2 and CYP3A4 seemed to be the dominant CYP450 isoenzymes transforming BBR into its metabolites M2 and M3. BBR's metabolites remained to be active on BBR's targets (InsR, LDLR, and AMPK) but with reduced potency.

Topics: AMP-Activated Protein Kinases; Animals; Berberine; Biotransformation; Cytochrome P-450 Enzyme System; Hep G2 Cells; Humans; Isoenzymes; Liver; Male; Metabolic Detoxication, Phase I; Rats; Rats, Wistar; Receptor, Insulin; Receptors, LDL

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.

Topics: Animals; Bacteria; Berberine; Bile; Chromatography, Liquid; Germ-Free Life; Intestinal Absorption; Intestine, Small; Liver; Male; Rats; Rats, Wistar; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry