Effect of Cytochrome P450 3A4 on Tissue Distribution of Humantenmine, Koumine, and Gelsemine, three Alkaloids from the Toxic Plant Gelsemium.

Humantenmine, koumine, and gelsemine are three indole alkaloids found in the highly toxic plant Gelsemium. Humantenmine was the most toxic, followed by gelsemine and koumine. The aim of this study was to investigate and analyze the effects of these three substances on tissue distribution and toxicity in mice pretreated with the Cytochrome P450 3A4 (CYP3A4) inducer ketoconazole and the inhibitor rifampicin. The in vivo test results showed that the three alkaloids were absorbed rapidly and had the ability to penetrate the blood-brain barrier. At 5minutes after intraperitoneal injection, the three alkaloids were widely distributed in various tissues and organs, the spleen and pancreas were the most distributed, and the content of all tissues decreased significantly at 20minutes. Induction or inhibition of CYP3A4 in vivo can regulate the distribution and elimination effects of the three alkaloids in various tissues and organs. Additionally, induction of CYP3A4 can reduce the toxicity of humantenmine, and vice versa. Changes in CYP3A4 levels may account for the difference in toxicity of humantenmine. These findings provide a reliable and detailed dataset for drug interactions, tissue distribution, and toxicity studies of Gelsemium alkaloids.

Comparative Analysis of the Gelsemium Alkaloids Metabolism in Human, Pig, Goat, and Rat Liver Microsomes.

AIM: The aim of this study was to investigate the metabolism of Gelsemium elegans in human, pig, goat and rat liver microsomes and to elucidate the metabolic pathways and cleavage patterns of the Gelsemium alkaloids among different species. METHODS: A human, goat, pig and rat liver microparticles were incubated in vitro. After incubating at 37°C for 1 hour and centrifuging, the processed samples were detected by HPLC/Qq-TOFMS was used to detect alcohol extract of Gelsemium elegans and its metabolites. RESULTS: Forty-six natural products were characterized from alcohol extract of Gelsemium elegans and 13 metabolites were identified. These 13 metabolites belong to the gelsemine, koumine, gelsedine, humantenine, yohimbane, and sarpagine classes of alkaloids. The metabolic pathways included oxidation, demethylation and dehydrogenation. After preliminary identification, the metabolites detected in the four species were different. All 13 metabolites were detected in pig and rat microsomes, but no oxidative metabolites of Gelsedine-type alkaloids were detected in goat and human microsomes. CONCLUSION: In this study, Gelsemium elegans metabolic patterns in different species are clarified and the in vitro metabolism of Gelsemium elegans is investigated. It is of great significance for its clinical development and rational application.

An efficient method for the preparative isolation and purification of alkaloids from Gelsemium by using high speed counter-current chromatography and preparative HPLC.

We established an efficient method using high-speed countercurrent chromatography (HSCCC) combined with preparative high-performance liquid chromatography (prep-HPLC) for isolating and purifying Gelsemium elegans (G. elegans) alkaloids. First, the two-phase solvent system composed of 1% triethylamine aqueous solution/n-hexane/ethyl acetate/ethanol (volume ratio 4:2:3:2) was employed to separate the crude extract (350 mg) using HSCCC. Subsequently, the mixture that resulted from HSCCC was further separated by Prep-HPLC, resulting in seven pure compounds including: 14-hydroxygelsenicine (1, 12.1 mg), sempervirine (2, 20.8 mg), 19-(R)-hydroxydihydrogelelsevirine (3, 10.1 mg), koumine (4, 50.5 mg), gelsemine (5, 32.2 mg), gelselvirine (6, 50.5 mg), and 11-hydroxyhumanmantenine (7, 12.5 mg). The purity of these seven compounds were 97.4, 98.9, 98.5, 99, 99.5, 96.8, and 85.5%, as determined by HPLC. The chemical structures of the seven compounds were analyzed and confirmed by electrospray ionization mass spectrometry (ESI-MS), 1H-nuclear magnetic resonance (1H NMR), and 13 C-nuclear magnetic resonance (13 C NMR) spectra. The results indicate that the HSCCC-prep-HPLC method can effectively separate the major alkaloids from the purified G. elegans, holding promising prospects for potential applications in the separation and identification of other traditional Chinese medicines.

China Medicinal Plants of the Ampelopsis grossedentata-A Review of Their Botanical Characteristics, Use, Phytochemistry, Active Pharmacological Components, and Toxicology.

Ampelopsis grossedentata (AG) is mainly distributed in Chinese provinces and areas south of the Yangtze River Basin. It is mostly concentrated or scattered in mountainous bushes or woods with high humidity. Approximately 57 chemical components of AG have been identified, including flavonoids, phenols, steroids and terpenoids, volatile components, and other chemical components. In vitro studies have shown that the flavone of AG has therapeutic properties such as anti-bacteria, anti-inflammation, anti-oxidation, enhancing immunity, regulating glucose and lipid metabolism, being hepatoprotective, and being anti-tumor with no toxicity. Through searching and combing the related literature, this paper comprehensively and systematically summarizes the research progress of AG, including morphology, traditional and modern uses, chemical composition and structure, and pharmacological and toxicological effects, with a view to providing references for AG-related research.

Antinociceptive effect of gelsenicine, principal toxic alkaloids of gelsemium, on prostaglandin E2-induced hyperalgesia in mice: Comparison with gelsemine and koumine.

Gelsemium elegans (G.elegans) is a plant of the Loganiaceae family, known for its indole alkaloids, including gelsemine, koumine, and gelsenicine. Gelsemine and koumine are well-studied active alkaloids with low toxicity, valued for their anti-anxiety and analgesic properties. However, gelsenicine, another important alkaloid, remains underexplored due to its high toxicity. This study focuses on evaluating the analgesic properties of gelsenicine and comparing them with gelsemine and koumine. The results indicate that all three alkaloids exhibit robust analgesic properties, with gelsemine, koumine, and gelsenicine showing ED50 values of 0.82 mg/kg, 0.60 mg/kg, and 8.43 µg/kg, respectively, as assessed by the hot plate method. Notably, the therapeutic dose of gelsenicine was significantly lower than its toxic dose (LD50 = 0.185 mg/kg). The study also investigated the mechanism of action by analyzing the expression levels of GlyRα3 and Gephyrin. The PGE2 model group showed decreased expression levels of GlyRα3 and Gephyrin, while groups treated with gelsemine, koumine, and gelsenicine were able to reverse this decrease. These results suggest that gelsenicine effectively alleviates PGE2-induced hyperalgesia by upregulating the expression of GlyRα3 and Gephyrin, which are key targets of the Gly receptor pathway.

Effect of Dietary Gelsemium elegans Benth. Extract on the Growth, Slaughter Performance, Meat Quality, Intestinal Morphology, and Microflora of Yellow-Feathered Chickens.

The plant species Gelsemium elegans Benth. (GEB) promotes pig and sheep growth; however, little is known about its effects in chickens. In this study, a GEB extract (GEBE) was prepared, and its effects on the growth, slaughter, antioxidant performance, meat quality, serum biochemical indices, intestinal morphology, and microflora of yellow-feathered chickens were evaluated. In total, 600 chickens aged 15 days were randomly divided into four groups with five replicates each and fed a basal diet containing 0% (control), 0.25% (0.25 GEBE), 0.75% (0.75 GEBE), or 1.25% (1.25 GEBE) GEBE until 49 days of age. Chickens were then killed, and their meat, organs, and serum and cecal contents were collected. GEBE reduced the feed conversion ratio, particularly in the 0.75 and 1.25 GEBE groups. Furthermore, the GEBE diet improved meat tenderness and reduced the meat expressible moisture content and liver malondialdehyde content, indicating high meat quality. Whereas the 0.25 GEBE diet increased the level of Lactobacillus acidophilus in the cecum, the 0.75 GEBE diet decreased the Escherichia coli level therein. These findings demonstrate that GEBE may improve the meat quality and cecal microbiota of yellow-feathered chickens, providing a basis for identifying candidate alternatives to conventional antibiotics as growth promoting feed additives.

Protocol to characterize target components of Gelsemium using an in-house herb database and high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Currently, the identification of herb metabolites is challenging due to a lack of clear standards. Here, using Gelsemium as an example, we present a protocol for characterizing target components of herbs. This approach utilizes high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry guided by an in-house herb metabolite database based on reported studies and mass spectrometry. We describe steps for creating an in-house database, preparing and detecting samples, processing data, and characterizing compounds. This approach offers a reference for future research on the identification of herb metabolites. For complete details on the use and execution of this protocol, please refer to Liu et al. (2017).1.

A Novel Two-Dimensional Liquid Chromatography Combined with Ultraviolet Detection Method for Quantitative Determination of Pyridoxal 5'-Phosphate, 4-Pyridoxine Acid and Pyridoxal in Animal Plasma.

Vitamin B6 is an indispensable micronutrient in organisms and is widely distributed in blood, tissues, and organs. Changes in the content and ratio of vitamin B6 can affect the entire physiological condition of the body, so it becomes particularly important to reveal the relationship between changes in its content and disease by monitoring vitamin B6 levels in the organism. In this study, a two-dimensional liquid chromatography-UV detector (2D-LC-UV) was used to establish a method for the simultaneous detection of PLP, PA, and PL for the first time. First, PLP, PA, and PL were extracted with plasma: 0.6 M TCA: ultrapure water = 1:2:3 (v/v/v) and then derivatized. Enrichment and preliminary separation were performed on a one-dimensional column and automatically entered into a two-dimensional column for further separation. This method exhibited good selectivity, and the correlation coefficients for the analyte calibration curves were >0.99. The detection limits for PLP, PA, and PL were 0.1, 0.2, and 4 nmol/L, respectively. The results showed that the system has high loading capacity, excellent resolution, and a good peak shape. This method is expected to provide applicability for the determination of PLP, PA, and PL in pharmacological, pharmaceutical, and clinical research.

Network Pharmacology and Experimental Verification to Unveil the Mechanism of N-Methyl-D-Aspartic Acid Rescue Humantenirine-Induced Excitotoxicity.

Gelsemium is a medicinal plant that has been used to treat various diseases, but it is also well-known for its high toxicity. Complex alkaloids are considered the main poisonous components in Gelsemium. However, the toxic mechanism of Gelsemium remains ambiguous. In this work, network pharmacology and experimental verification were combined to systematically explore the specific mechanism of Gelsemium toxicity. The alkaloid compounds and candidate targets of Gelsemium, as well as related targets of excitotoxicity, were collected from public databases. The crucial targets were determined by constructing a protein-protein interaction (PPI) network. Subsequently, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to explore the bioprocesses and signaling pathways involved in the excitotoxicity corresponding to alkaloids in Gelsemium. Then, the binding affinity between the main poisonous alkaloids and key targets was verified by molecular docking. Finally, animal experiments were conducted to further evaluate the potential mechanisms of Gelsemium toxicity. A total of 85 alkaloids in Gelsemium associated with 214 excitotoxicity-related targets were predicted by network pharmacology. Functional analysis showed that the toxicity of Gelsemium was mainly related to the protein phosphorylation reaction and plasma membrane function. There were also 164 pathways involved in the toxic mechanism, such as the calcium signaling pathway and MAPK signaling pathway. Molecular docking showed that alkaloids have high affinity with core targets, including MAPK3, SRC, MAPK1, NMDAR2B and NMDAR2A. In addition, the difference of binding affinity may be the basis of toxicity differences among different alkaloids. Humantenirine showed significant sex differences, and the LD50 values of female and male mice were 0.071 mg·kg-1 and 0.149 mg·kg-1, respectively. Furthermore, we found that N-methyl-D-aspartic acid (NMDA), a specific NMDA receptor agonist, could significantly increase the survival rate of acute humantenirine-poisoned mice. The results also show that humantenirine could upregulate the phosphorylation level of MAPK3/1 and decrease ATP content and mitochondrial membrane potential in hippocampal tissue, while NMDA could rescue humantenirine-induced excitotoxicity by restoring the function of mitochondria. This study revealed the toxic components and potential toxic mechanism of Gelsemium. These findings provide a theoretical basis for further study of the toxic mechanism of Gelsemium and potential therapeutic strategies for Gelsemium poisoning.

Two-Dimensional Liquid Chromatography Method for the Determination of Gelsemium Alkaloids in Honey.

Toxic Chinese medicine residues in honey pose a serious threat to consumer health. Gelsemium is one of the nine ancient poisons, making the whole plant virulent. The residue of Gelsemium alkaloid in honey causes poisoning from time to time. Therefore, it is very important to establish a method for the detection of Gelsemium alkaloids in honey. In this study, a method of solid phase extraction (SPE) with two-dimensional liquid chromatography (2D-LC) was developed for the first time for the simultaneous determination of Gelsemium alkaloids in honey, including gelsemine, koumine and humantenmine. First, the honey samples were purified by a PRS cation exchange column and extracted with 5% ammoniated methanol. Then, we verified the methodological indicators, which were in line with the Codex Guideline requirements. The verification results are as follows: matrix-matched calibrations indicated that the correlation coefficients were higher than 0.998. The recovery was in the range of 81%-94.2% with an intraday precision (RSD) of ≤5.0% and interday RSD of ≤3.8%. The limit of detection for the three alkaloids was 2 ng/g. The limits of quantification for gelsemine and koumine were 5 ng/g, and humantenmine was 20 ng/g. This method can be applied to the monitoring of Gelsemium alkaloids in honey.

Excretion, Metabolism, and Tissue Distribution of Gelsemium elegans (Gardn. & Champ.) Benth in Pigs.

Gelsemium elegans (Gardn. & Champ.) Benth is a toxic flowering plant in the family Loganiaceae used to treat skin diseases, neuralgia and acute pain. The high toxicity of G. elegans restricts its development and clinical applications, but in veterinary applications, G. elegans has been fed to pigs as a feed additive without poisoning. However, until now, the in vivo processes of the multiple components of G. elegans have not been studied. This study investigates the excretion, metabolism and tissue distribution of the multiple components of G. elegans after feeding it to pigs in medicated feed. Pigs were fed 2% G. elegans powder in feed for 45 days. The plasma, urine, bile, feces and tissues (heart, liver, lung, spleen, brain, spinal cord, adrenal gland, testis, thigh muscle, abdominal muscle and back muscle) were collected 6 h after the last feeding and analyzed using high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Five natural products in plasma, twelve natural products and five metabolites in urine, and three natural products in feces were characterized, suggesting that multiple components from G. elegans were excreted in the urine. However, ten natural products and four metabolites were detected in bile samples, which suggested that G. elegans is involved in enterohepatic circulation in pigs. A total of seven of these metabolites were characterized, and four metabolites were glucuronidated metabolites. Ten natural products and six metabolites were detected in the tissues, which indicates that G. elegans is widely distributed in tissues and can cross the blood-brain barrier. Among the characterized compounds, a highly toxic gelsedine-type alkaloid from G. elegans was the main compound detected in all biological samples. This is the first study of the excretion, metabolism and tissue distribution of multiple components from G. elegans in pigs. These data can provide an important reference to explain the efficacy and toxicity of G. elegans. Additionally, the results of the tissue distribution of G. elegans are of great value for further residue depletion studies and safety evaluations of products of animals fed G. elegans.

A high-resolution mass spectrometric approach to a qualitative and quantitative comparative metabolism of the humantenine-type alkaloid rankinidine.

RATIONALE: Rankinidine belongs to the humantenine-type alkaloids isolated from Gelsemium. Currently, the mechanism behind the toxicity differences of rankinidine has not been explained. In this study, our purpose was to elucidate the major in vitro metabolic pathways of rankinidine and to compare the formation of metabolites of rankinidine in human (HLMs), rat (RLMs), goat (GLMs) and pig (PLMs) liver microsomes. METHODS: This is the first study to compare the in vitro metabolism of rankinidine with high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF). The MS/MS data and LC/MS peak area acquired in positive ion mode were used to analyze metabolite structures and compare metabolism. RESULTS: We identified 11 metabolites (M1-M11) in total and found five main metabolic pathways, consisting of demethylation (M1), reduction (M2), oxidation at different positions (M3-M5), oxidation and reduction (M6-M10) and demethylation and oxidation (M11). The metabolism of rankinidine has qualitative and quantitative species-specific differences in vitro. In PLMs and GLMs, the main metabolic pathway of rankinidine was oxidation. Notably, among the four species, the oxidation ability of rankinidine was highest in pigs and goats, and the demethylation and reduction abilities of rankinidine were highest in humans and rats. CONCLUSIONS: The interspecific metabolic differences of rankinidine in HLMs, PLMs, GLMs and RLMs were compared and studied for the first time using LC/QTOF. These findings will certainly support future studies of rankinidine metabolism in vivo and will contribute to elucidating the cause of species-specific differences behind Gelsemium toxicity.

Suppressive Effects of Gelsemine on Anxiety-like Behaviors Induced by Chronic Unpredictable Mild Stress in Mice.

Gelsemine is an active principle and a major alkaloid found in Gelsemium genus of plants belonging to the Loganiaceae family. The aim of the present study was to explore whether gelsemine exerts anxiolytic effects on a mouse model of chronic-unpredictable-mild-stress (CUMS)-induced anxiety-like behaviors. NOD-like receptor protein 3 (NLRP3) inflammasome, downregulated cAMP-response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) were also evaluated as potential mechanisms. First, gelsemine reversed a CUMS-induced decrease in body-weight gain in mice. Next, gelsemine alleviated CUMS-induced anxiety-like behaviors, as evidenced by the increased distance traveled in the central zone of the open-field test, both the increased percentage of time spent and distance traveled in the light compartment, the increased number of transitions between compartments in the light/dark-transition test, and the increased percentage of entries and time spent in the open arm of the elevated plus-maze. In addition, gelsemine decreased the levels of pro-inflammatory cytokines, including interleukin (IL)-1ß and IL-6, in the hypothalamus and hippocampus of CUMS mice. Interestingly, further investigations revealed that gelsemine inhibited the CUMS-induced activation of NLRP3-inflammasome pathways and downregulated CREB and BDNF overexpression in the hypothalamus. In summary, gelsemine alleviated anxiety-like behaviors in the CUMS-induced mouse model. Gelsemine exerted its anxiolytic effects by modulating the NLRP3 and CREB/BDNF pathways.

Toxicity assessment of gelsenicine and the search for effective antidotes.

BACKGROUND: Gelsenicine, one of the most toxic alkaloids of Gelsemium elegans Benth (G. elegans), causes severe respiratory depression. However, its toxicity mechanisms are yet to be elucidated and no effective antidotes are available. OBJECTIVE: This study aimed to analyse the toxicity characteristics of gelsenicine. METHODS: Both acute and sub-acute toxicities were evaluated. Gelsenicine distribution and elimination in the central nervous system (CNS) and blood were observed. Effective antidotes for gelsenicine poisoning were screened. RESULTS: In the acute toxicity study, gelsenicine was highly toxic, and female rats exhibited greater sensitivity to gelsenicine than male rats (LD50 0.520 mg/kg vs 0.996 mg/kg, respectively). Death was primarily caused by respiratory failure. However, in the sub-acute toxicity study, no significant organ damage was observed. Gelsenicine was easily absorbed from the gastrointestinal tract and penetrated the blood-brain barrier, reaching peak concentrations in the CNS within 15 min and rapidly decreasing thereafter. Flumazenil or diazepam combined with epinephrine reversed gelsenicine toxicity and significantly improved survival rate in mice. CONCLUSIONS: Gelsenicine is a highly toxic substance that affects nerve conduction without causing damage; the potential toxic mechanism is possibly associated with GABAA receptors. Our findings provide insights into the clinical treatment of gelsenicine-related poisoning and its toxicity mechanisms.

A Proteomics Study of the Subacute Toxicity of Rat Brain after Long- Term Exposure of Gelsemium elegans.

BACKGROUND: Gelsemium elegans (G. elegans) has been shown to have strong pharmacological and pharmacodynamic effects in relevant studies both in China and USA. G. elegans has been used as a traditional medicine to treat a variety of diseases and even has the potential to be an alternative to laboratory synthesized drugs. However, its toxicity severely limited its application and development. At present, there is little attention paid to protein changes in toxicity. AIM: This study investigated the toxicity effects after long-term exposure of G. elegans of the rat brain through proteomic. METHODS: 11 differential abundance proteins were detected, among which 8 proteins were higher in the G. elegans- exposure group than in the control group, including Ig-like domain-containing protein (N/A), receptor-type tyrosine-protein phosphatase C (Ptprc), disheveled segment polarity protein 3 (Dvl3), trafficking protein particle complex 12 (Trappc12), seizure-related 6 homologlike (Sez6l), transmembrane 9 superfamily member 4 (Tm9sf4), DENN domain-containing protein 5A (Dennd5a) and Tle4, whereas the other 3 proteins do the opposite including Golgi to ER traffic protein 4 (Get4), vacuolar protein sorting 4 homolog B (Vps4b) and cadherin-related 23 (CDH23). Furthermore, we performed validation of WB analysis on the key protein CDH23. RESULTS: Finally, only fewer proteins and related metabolic pathways were affected, indicating that there was no accumulative toxicity of G. elegans. G. elegans has the potential to develop and utilize of its pharmacological activity. CHD23, however, is a protein associated with hearing. CONCLUSION: Whether the hearing impairment is a sequela after G. elegans exposure remains to be further studied.

Sex Differences in the In Vivo Exposure Process of Multiple Components of Gelsemium elegans in Rats.

Asian Gelsemium elegans (G. elegans) has a wide range of pharmacological activities. However, its strong toxicity limits its potential development and application. Interestingly, there are significant gender differences in G. elegans toxicity in rats. This work aimed to elucidate the overall absorption, distribution, metabolism, and excretion (ADME) of whole G. elegans crude extract in female and male rats using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS), which facilitates determining the reasons for the gender differences in toxicity. A total of 25 absorbed bioactive components and 3 related produced metabolites were tentatively identified in female rats, while only 17 absorbed bioactive components and 3 related produced metabolites were identified in male rats. By comparison of peak intensities, most compounds were found to be more active in absorption, distribution and excretion in female rats than in male rats, which showed that female rats were more sensitive to G. elegans. This study was the first to investigate the multicomponent in vivo process of G. elegans in rats and compare the differences between sexes. It was hypothesized that differences in the absorption of gelsedine-type alkaloids were one of the main reasons for the sex differences in G. elegans toxicity.

The multicomponent residue depletion of Gelsemium elegans in pig tissues, urine, and plasma.

Introduction: Gelsemium elegans (G. elegans) as a traditional medicinal plant used in livestock production. The use of G. elegans in veterinary clinics may pose safety risks to human health. Objectives: The aim of this study was to investigate tissue residue depletion in pigs fed G. elegans powder. Methods: A precise quantitation method and a simultaneous semi-quantitation method for multiple components independently of standards in pig tissues were developed for the first time. The two methods were validated in terms of specificity, LODs, LOQs, linearity, accuracy, precision, and matrix effects. They were then applied to a tissue residue depletion study after G. elegans powder at a dose of 2% per kg feed were fed to pigs. Results: Compared with precise quantitation, the method validation results indicated that the semi-quantitation method was reliable and acceptable for multicomponent quantification independent of standards. Many G. elegans alkaloids are widely distributed in most tissues of pigs. Tissue residue depletion studies indicated that 14-hydroxygelsenicine, 11-hydroxygelsenicine, and gelsemoxonine could be used as potential residue markers, and pancreas, small intestine, and lung tissues could be considered as potential residue target tissues of G. elegans. In addition, both urine and plasma could be used to predict 14-hydroxygelsenicine and gelsemoxonine residues in the liver, pancreas, and small intestinal tissues of pigs. Conclusion: The developed semi-quantification method can be applied to monitor the application and residue of G. elegans. The results provide scientific evidence for evaluating the safety of animal-derived food from G. elegans for consumers and will be helpful for its application and future development.

In vitro Metabolism of Humantenine in Liver Microsomes from Human, Pig, Goat and Rat.

BACKGROUND: Gelsemium elegans Benth (G. elegans) is a well-known toxic plant. Alkaloids are the main active components of G. elegans. Currently, the metabolism of several alkaloids, such as gelsenicine, koumine, and gelsemine, has been widely studied. However, as one of the most important alkaloids in G. elegans, the metabolism of humantenine has not been studied yet. METHODS: In order to elaborate on the in vitro metabolism of humantenine, a comparative analysis of its metabolic profile in human, pig, goat and rat liver microsomes was carried out using high performance chromatography/ quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS) for the first time. RESULTS: Totally, ten metabolites of humantenine were identified in liver microsomes from human (HLMs), pig (PLMs), goat (GLMs) and rat (RLMs) based on the accurate MS/MS spectra. Five metabolic pathways of humantenine, including demethylation, dehydrogenation, oxidation, dehydrogenation and oxidation, and demethylation and oxidation, were proposed in this study. There were qualitative and quantitative species differences in the metabolism of humantenine among the four species. CONCLUSION: The in vitro metabolism of humantenine in HLMs, PLMs, GLMs and RLMs was studied by a sensitive and specific detection method based on HPLC/QqTOF-MS. The results indicated that there were species-related differences in the metabolism of humantenine. This work might be of great significance for the further research and explanation of species differences in terms of toxicological effects of G. elegans.

Metabolic profile and tissue distribution of Humantenirine, an oxindole alkaloid from Gelsemium, after oral administration in rats.

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.

Phosphoproteomics reveals NMDA receptor-mediated excitotoxicity as a key signaling pathway in the toxicity of gelsenicine.

Gelsenicine is one of the most toxic compounds in the genus Gelsemium, but the mechanism of toxicity is not clear. In this paper, tandem mass tag quantitative phosphoproteomics was used to study the changes in protein phosphorylation in different brain regions at different time points after gelsenicine poisoning in mice. The correlation between neurotransmitter receptors and the toxicity of gelsenicine was analyzed by molecular docking and rescue experiments. Parallel reaction monitoring (PRM) was used to verify the related proteins. A total of 17877 unique phosphosites were quantified and mapped to 4170 brain proteins to understand the signaling pathways. Phosphoproteomics revealed gelsenicine poisoning mainly affected protein phosphorylation levels in the hippocampus, and through bioinformatics analysis, it was found gelsenicine poisoning significantly affected neurotransmitter synaptic pathway. The molecular docking results showed that gelsenicine could bind to the N-methyl-D-aspartic acid receptor (NMDAR). In addition, we found that NMDA was effective in improving the survival rate of the animals tested, and this effect was associated with reduced protein phosphorylation by PRM validation. The results revealed that gelsenicine affects neurotransmitter release and receptor function. This is the first demonstration that NMDA receptor-mediated excitotoxicity is a key signaling pathway in the toxicity of gelsenicine.