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Gelsemium is a small genus of flowering plants from the family Loganiaceae comprising five species, three of which, Gelsemium sempervirens (L.) J. St.‐Hil., G. elegans Benth and G. rankinii Small, are particularly popular. Compared with other alkaloids from G. elegans, gelsemine, gelsevirine and koumine exhibit equally potent anxiolytic effects and low toxicity. Although the pharmacological activities and metabolism of koumine and gelsemine have been reported in previous studies, the species differences of gelsevirine metabolism have not been well studied. In this study, the metabolism of gelsevirine was investigated by using liver microsomes of humans, pigs, goats and rats by means of HPLC‐QqTOF/MS. The results indicated that the metabolism of gelsevirine in liver microsomes had qualitative and quantitative species differences. Based on the results, the possible metabolic pathways of gelsevirine in liver microsomes were proposed. Investigation of the metabolism of gelsevirine will provide a basis for further studies of the in vivo metabolism of this drug. [ABSTRACT FROM AUTHOR]

Rationale: Gelsemium elegans Benth. belongs to the family Loganiaceae and is widely distributed in northern America, east Asia, and southeast Asia. It has attracted wide attention for its diverse biological effects and complex architectures. Gelsevirine is one of the major components in G. elegans. Compared with other alkaloids from G. elegans, gelsevirine exhibits equally potent anxiolytic effects but with less toxicity. However, the metabolism of gelsevirine has not been clearly elucidated. Methods: The metabolism of gelsevirine was investigated using liver S9 fractions derived from rat liver homogenates by centrifugation at 9000 g. A rapid and accurate high‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (HPLC/QqTOF‐MS) method was applied to characterize the gelsevirine metabolites. Results: We discovered a total number of four metabolites of gelsevirine. The metabolic pathways of gelsevirine consisted of hydrogenation, N‐demethylenation and oxidation in rat liver S9. Conclusions: This is the first study on the metabolism of gelsevirine. We proposed possible metabolic pathways of gelsevirine. These findings may warrant future studies of the in vivo metabolism of gelsemine in animals. [ABSTRACT FROM AUTHOR]

Sanguinarine (SA) and chelerythrine (CHE) are the main active components of the phytogenic livestock feed additive, Sangrovit®. However, little information is available on the pharmacokinetics of Sangrovit® in poultry. The goal of this work was to study the pharmacokinetics of SA, CHE, and their metabolites, dihydrosanguinarine (DHSA) and dihydrochelerythrine (DHCHE), in 10 healthy female broiler chickens following oral (p.o.) administration of Sangrovit® and intravenous (i.v.) administration of a mixture of SA and CHE. The plasma samples were processed using two different simple protein precipitation methods because the parent drugs and metabolites are stable under different pH conditions. The absorption and metabolism of SA following p.o. administration were fast, with half‐life (t1/2) values of 1.05 ± 0.18 hr and 0.83 ± 0.10 hr for SA and DHSA, respectively. The maximum concentration (Cmax) of DHSA (2.49 ± 1.4 μg/L) was higher that of SA (1.89 ± 0.8 μg/L). The area under the concentration vs. time curve (AUC) values for SA and DHSA were 9.92 ± 5.4 and 6.08 ± 3.49 ng/ml hr, respectively. Following i.v. administration, the clearance (CL) of SA was 6.79 ± 0.63 (L·h−1·kg−1) with a t1/2 of 0.34 ± 0.13 hr. The AUC values for DHSA and DHCHE were 7.48 ± 1.05 and 0.52 ± 0.09 (ng/ml hr), respectively. These data suggested that Sangrovit® had low absorption and bioavailability in broiler chickens. The work reported here provides useful information on the pharmacokinetic behavior of Sangrovit® after p.o. and i.v. administration in broiler chickens, which is important for the evaluation of its use in poultry. [ABSTRACT FROM AUTHOR]

Rationale: N‐Methylcanadine and N‐methylstylopine are two types of isoquinoline alkaloids which are considered to be the main medicinally active constituents of the genus Papaveraceae. However, to date, no metabolism studies of N‐methylcanadine and N‐methylstylopine have been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of these two alkaloids in rat liver S9. Methods: N‐Methylcanadine or N‐methylstylopine was incubated with rat liver S9 for 1 h, and then the incubation mixture was processed with 15% trichloroacetic acid. High‐performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry (HPLC/QqTOF‐MS) as a reliable analytical method was used. The structural characterization of these metabolites was performed by the combination of the accurate MS/MS spectra and the known elemental composition. Results: As a result, a total of four metabolites of N‐methylcanadine and five metabolites of N‐methylstylopine in rat liver S9 were tentatively identified. The cleavage of the methylenedioxy group of the drugs was the main metabolic pathway of N‐methylcanadine and N‐methylstylopine. Conclusions: The present study is the first in vitro metabolic investigation of N‐methylcanadine and N‐methylstylopine in rat liver S9 using a reliable HPLC/QqTOF‐MS method. The metabolic pathways of N‐methylcanadine and N‐methylstylopine are tentatively proposed. This work lays the foundation for the in vivo metabolism of the two compounds in animals. [ABSTRACT FROM AUTHOR]

Rationale: Gelsemine has been extensively studied because of its anti-tumor, immuno- modulatory, insecticidal itching and other significant effects. However, limited information on the pharmacokinetics and metabolism of gelsemine has been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of gelsemine in rat liver S9 by using rapid and accurate high-performance liquid chromatography/ quadrupole-time-of-flight mass spectrometry (HPLC/QqTOF-MS). Methods: The incubation mixture was processed with 15% trichloroacetic acid. Multiple scans of gelsemine metabolites and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only 30 min. The structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses and product ions with those of the parent drug. Results: Five metabolites of gelsemine were identified in rat liver S9. Of these, four metabolites of gelsemine were identified for the first time. The present results showed that the metabolic pathways of gelsemine are oxidation, demethylation, and dehydrogenation in rat liver S9. Conclusions: In this study, metabolites of gelsemine in liver S9 were identified and elucidated firstly using the HPLC/QqTOF-MS method. The proposed metabolic pathways of gelsemine in liver S9 will provide a basis for further studies of the in vivo metabolism of gelsemine in animals and humans. [ABSTRACT FROM AUTHOR]

RATIONALE: Koumine is one of the major components of total alkaloids from Gelsemium. Koumine possesses a variety of interesting pharmacological effects, including anti-tumor, anti-inflammatory, and anxiolytic activities. It might be a promising lead drug because of its pharmacological activities and mild toxicity. However, little information is available on the metabolism of koumine. METHODS: A rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight (HPLC/QqTOF) mass spectrometry method was applied to characterize koumine metabolites. Multiple scans of koumine metabolites, which were formed in rat liver S9, were automatically performed simultaneously through auto MS/MS mode acquisition in only a 30-min analysis. The structural elucidation of these metabolites was performed by comparing their changes in accurate molecular masses and product ions with those of the parent drug or metabolites. RESULTS: As a result, a total of eleven metabolites of koumine were identified, of which nine new metabolites were found. The present results showed that the N-demethylenation, hydrogenation and the oxidation were the three main metabolic pathways of koumine. CONCLUSIONS: This was the first investigation of in vitro metabolism of koumine in rat liver S9 using a sensitive and specific HPLC/QqTOF-MS method. The possible metabolic pathways of koumine were tentatively proposed based on the structural elucidations of these metabolites. This work may be useful in the in vivo metabolism of koumine in animals and humans. [ABSTRACT FROM AUTHOR]

RATIONALE: Allocryptopine (AL) and protopine (PR) have been extensively studied because of their anti-parasitic, antiarrhythmic, anti-thrombotic, anti-inflammatory and anti-bacterial activity. However, limited information on the pharmacokinetics and metabolism of AL and PR has been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of AL and PR in rat liver S9 using a rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOFMS) method. METHODS: The incubation mixture was processed with 15% trichloroacetic acid (TCA). Multiple scans of AL and PR metabolites and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. The structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses and product ions with those of the precursor ion or metabolite. RESULTS: Eight and five metabolites of AL and PR were identified in rat liver S9, respectively. Among these metabolites, seven and two metabolites of AL and PR were identified in the first time, respectively. The demethylenation of the 2,3-methylenedioxy, the demethylation of the 9,10-vicinal methoxyl group and the 2,3-methylenedioxy group were the main metabolic pathways of AL and PR in liver S9, respectively. In addition, the cleavage of the methylenedioxy group of the drugs and subsequent methylation or O-demethylation were also the common metabolic pathways of drugs in liver S9. In addition, the hydroxylation reaction was also the metabolic pathway of AL. CONCLUSIONS: This was the first investigation of in vitro metabolism of AL and PR in rat liver S9. The detailed structural elucidations of AL and PR metabolites were performed using a rapid and accurate HPLC/QqTOFMS method. The metabolic pathways of AL and PR in rat were tentatively proposed based on these characterized metabolites and early reports. [ABSTRACT FROM AUTHOR]

Abstract: Olaquindox is a growth-promoting feed additive for food-producing animals. Its toxicities were reported to be closely related to the metabolism. To provide the interpretation of toxicities in animals, this study explored the metabolism of olaquindox in rats, chickens and pigs of different genders by qualitative metabolite profiling. Animals were fed olaquindox in an oral dose, and then their urine, plasma, feces, liver, kidney and muscle were collected. Liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry was used for structural investigation and identification of metabolites. The structures of metabolites were elucidated based on the accurate MS2 spectra and comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite. A total of 18, 18 and 16 metabolites of rats, chickens and pigs were identified, respectively. Among the identified metabolites, 8 known metabolites were confirmed as an early study had stated, and 15 metabolites were found for the first time in vivo. The major metabolic pathways of olaquindox were proposed to be N-O reduction and oxidation of hydroxyl to carboxylic acid followed by N-O reduction. The qualitative species difference on the metabolite profiles of olaquindox among the three species was observed. However, metabolite profiles of olaquindox appeared to be qualitatively similar between female and male for the same species. The proposed metabolic pathways of olaquindox in animals will provide comprehensive data to clarify the metabolism of olaquindox among different species, and will give scientific explanation for toxicities and residues of olaquindox. [Copyright &y& Elsevier]

Abstract: Olaquindox, N-(2-hydroxyethyl)-3-methyl-2-quinoxalinecarboxamide-1,4-di-N-oxide, is one of the quinoxaline-dioxides used widely as an antimicrobial growth promoter in pig production. Its toxicities were reported to be closely related to the formation of N-oxide reductive metabolites. The present study presents the metabolism and N-oxide reduction of olaquindox incubated with liver microsomes and liver cytosol of rats, pigs and chicken. Metabolites were identified and characterized with a novel LC/MS-ITTOF. Thirteen metabolites were found in liver microsomes of rats, three of which were identified to be novel. Seven metabolites were found in liver microsomes of pigs and chicken. The N-oxide reduction was the major metabolic pathway of olaquindox in liver microsomes of the three species. The N1-reduction of olaquindox to metabolite O2 was found in not only liver microsomes but also cytosol of the three species in the presence of NAD(P)H under hypoxic conditions. The N1-reduction could be inhibited by air and carbon monoxide, and be significantly stimulated by riboflavin under various conditions. The N1-reduction in the liver cytosol of rats and pigs could be enhanced by menadione, but the reduction in liver cytosol of chicken could not be. The N1-reduction activities in all animals were not abolished when liver microsomes and cytosol were boiled. These findings suggested that the N1-reduction of olaquindox could be mediated by non-enzymatic and enzymatic conditions. This N1-reduction of olaquindox could also be catalyzed by a quinone-dependent reducing system in liver cytosol of rats and pigs. Moreover, liver cytosol of rats and pigs had an ability of N4-reduction that catalyzed olaquindox to metabolite O1 in the presence of benzaldehyde under hypoxic conditions, but the liver cytosol of chicken did not. The N4-reduction could be inhibited markedly in the cytosol rats and pigs by menadione, chlorpromazine and promethazine. In addition, 7-hydroxycoumarin was also found to inhibit the formation of O1 in the cytosol of rats. The inhibitory results suggested that the N4-reduction might be catalyzed by aldehyde oxidase in the cytosol of pigs, and by aldehyde oxidase and xanthine oxidase in the cytosol of rats. In conclusion, the N1-reduction and N4-reduction of olaquindox are mediated by multiple mechanisms and significant species differences are involved in both reductions. This work is a contribution to the understanding of toxicities and the relativities between toxicities and metabolism of olaquindox. [Copyright &y& Elsevier]

Abstract: Mequindox (MEQ) is a novel synthetic quinoxaline 1,4-dioxides antibacterial agent and growth promoter in animal husbandry. This study was to investigate whether reactive oxygen species (ROS), the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway, suppressors of cytokine signaling (SOCS) and inflammatory cytokines were involved in toxicities of MEQ. Our data demonstrated that high dose of MEQ (275mg/kg) apparently led to tissue impairment combined with imbalance of redox in liver. In liver and spleen samples, hydroxylation metabolites and desoxymequindox were detected, directly confirming the potential link of N→O group reduction metabolism with its organ toxicity. Moreover, up-regulation of JAK/STAT, SOCS family, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) were also observed in the high-dose group. Meanwhile, significant changes of oxidative stress indices in liver were observed in the high-dose group. As for NADPH subunit, the mRNA levels of many subunits were significantly up-regulated at low doses but down-regulated in a dose-dependent manner in liver and spleen, suggesting an involvement of NADPH in MEQ metabolism and ROS generation. In conclusion, we reported the dose-dependent long-term toxicity as well as the discussion of the potential mechanism and pathways of MEQ, which raised further awareness of its toxicity following with the dose change. [Copyright &y& Elsevier]

• The HPLC/QqTOF-MS approach was utilized for the identification of the humantenirine metabolites. • The metabolic profile and tissue distribution of humantenirine in rats were characterized firstly. • A total of 8 metabolites were tentatively identified in vitro and in vivo. • Humantenirine was widely distributed in tissues. Humantenirine is an active oxindole alkaloid extracted from Gelsemium elegans Benth (G. elegans). In the present study, the metabolites of humantenirine in liver microsomes were first identified by HPLC/QqTOF-MS. Then, the metabolic profile and tissue distribution after oral administration in rats were further investigated. A total of seven metabolites were identified in vitro , and five metabolites in vitro were found in vivo. Moreover, a Ⅱ-phase metabolite was identified first in vivo. The results indicated that humantenirine could be metabolized widely. The parent drug and its metabolites were distributed widely in various tissues and highly in the liver and pancreas. However, the parent drug and its metabolites had low peak intensities in plasma. The elimination of humantenirine occurred rapidly as well, the most unconverted forms of which were found in the kidney. Metabolic pathways, including demethylation, dehydrogenation, oxidation and glucuronidation, were proposed. The present findings may provide a basis for the study of pharmacokinetic characteristics and will contribute to the evaluation of the pharmacology and toxicity of G. elegans. [ABSTRACT FROM AUTHOR]

Herbal medicine contains hundreds of natural products, and studying their absorption, metabolism, distribution, and elimination presents great challenges. Gelsemium elegans (G. elegans) is a flowering plants in the Loganiaceae family. The plant is known to be toxic and has been used for many years as a traditional Chinese herbal medicine for the treatment of rheumatoid arthritis, neuropathic pain, spasticity, skin ulcers and cancer. It was also used as veterinary drugs for deworming, promoting animal growth, and pesticides. At present, studies on the metabolism of G. elegans have primarily focused on only a few single available reference ingredients, such as koumine, gelsemine and gelsedine. The goal of this work is to elucidate the overall metabolism of whole G. elegans powder in goats using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS). Analyses of plasma, urine and fecal samples identified or tentatively characterized a total of 44 absorbed natural products and 27 related produced metabolites. Gelsedine-type, sarpagine-type and gelsemine-type alkaloids were the compounds with the highest metabolite formation. In the present study, most natural products identified in G. elegans were metabolized through glucuronidation and oxidation. Hydrogenation, dehydrogenation and demethylation also occurred. To our knowledge, this is the first report of the metabolite profiling of the G. elegans crude extract in goats, which is of great significance for a safer and more rational application of this herbal medicine. Image 1 [ABSTRACT FROM AUTHOR]