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 5 min 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 20 min. 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.

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.

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.

Metabolism and Tissue Distribution of Chelerythrine and Effects of Macleaya Cordata Extracts on Liver NAD(P)H Quinone Oxidoreductase.

Background: Macleaya cordata (Willd.) (Papaveraceae) is listed as a feed additive in animal production by the European Food Authority. Methods: The metabolites of chelerythrine in rats were measured in vitro and in vivo by rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOF-MS). The structures of CHE metabolites were elucidated by comparing their changes in accurate molecular masses and fragment ions with those of parent ion or metabolite. The metabolic enzymes that were involved in chelerythrine reduction were investigated using an inhibition method. The tissue distribution of chelerythrine and the effects on NQO1 following intragastric administration with M. cordata extracts in rats were examined. Results: A total of twelve metabolites of chelerythrine were characterized by this approach in rat liver S9 and in vivo. The reduction of the iminium bond of chelerythrine and subsequent O-demethylation was the main metabolic pathway of chelerythrine in rat liver S9 while the reduction of the iminium bond of chelerythrine was the main metabolic pathway of chelerythrine in rats in vivo. After the rats were given intragastric administration, the low concentration residues of sanguinarine and chelerythrine in different rat tissues were found at 48 h after the last dose, suggesting that both compounds could be widely distributed in tissues. The results also indicated that XO, NQO1, NQO2, and carbonyl reductase are involved in chelerythrine reduction. Macleaya cordata extracts treated female and male rats, respectively, showed different responses, inhibiting NQO1 activity in males, but inducing NQO1 activity in females. Chelerythrine had a weak impact on NQO1 activity, but sanguinarine inhibited NQO1 activity Conclusion: Through studying the effects of cytosolic reductase inhibitors on chelerythrine reduction and the impact of chelerythrine and sanguinarine on the activity of NQO1 in vitro and in vivo, we clarified the potential drug interaction of Macleaya cordata extract in clinical application, so as to provide theoretical guidance for clinically safe medication. In addition, it provided a reference basis for the metabolic mechanism of chelerythrinein rats.

Integration of Metabolomics and Transcriptomicsto Comprehensively Evaluate the Metabolic Effects of Gelsemium elegans on Pigs.

Some naturalphytogenic feed additives, which contain several active compounds, have been shown to be effective alternatives to traditional antibiotics. Gelsemium elegans (G. elegans) is a whole grass in the family Loganiaceae. It is a known toxic plant widely distributed in China and has been used as a traditional Chinese herbal medicine for many years to treat neuropathic pain, rheumatoid pain, inflammation, skin ulcers, and cancer. However, G. elegans not only is nontoxic to animals such as pigs and sheep but also has an obvious growth-promoting effect. To our knowledge, the internal mechanism of the influence of G. elegans on the animal body is still unclear. The goal of this work is to evaluate the metabolic consequences of feeding piglets G. elegans for 45 days based on the combination of transcriptomics and metabolomics. According to growth measurement and evaluation, compared with piglets fed a complete diet, adding 20 g/kg G. elegans powder to the basal diet of piglets significantly reduced the feed conversion ratio. Results of the liver transcriptome suggest that glycine and cysteine-related regulatory pathways, including the MAPK signaling pathway and the mTOR signaling pathway, were extensively altered in G. elegans-induced piglets. Plasma metabolomics identified 21 and 18 differential metabolites (p < 0.05) in the plasma of piglets in the positive and negative ion modes, respectively, between G. elegans exposure and complete diet groups. The concentrations of glycine and its derivatives and N-acetylcysteine were higher in the G. elegans exposure group than in the complete diet group.This study demonstrated that G. elegans could be an alternative to antibiotics that improves the immune function of piglets, and the latent mechanism of G. elegans may be related to various signaling pathways, including the MAPK signaling pathway and the PPAR signaling pathway.

A comprehensive toxicity evaluation in rats after long-term oral Gelsemium elegans exposure.

BACKGROUND: Gelsemium elegans (G. elegans) is a flowering plant of the Loganiaceae family, which had been used in traditional Chinese herb medicine for many years for the treatment of rheumatoid pain, neuropathic pain, spasticity, skin ulcers, anxiety and cancer. Acute toxicity of the plant severely limits the application and development of G. elegans; however, long-term toxicity of exposure to G. elegans has not been illuminated. PURPOSE: This study is a comprehensive observation of the effects of long-term exposure (21 days at 70 mg/kg) to G. elegans in rats. METHODS AND RESULTS: The histopathological examination showed only a mild glial cell proliferation in the brain, and no lesions were observed in other organs. No abnormal changes in the biochemical parameters were observed that would have significant effects. The identification and analysis of absorbed natural ingredients showed that the active ingredients of the G. elegans could distribute to various tissues, and six compounds were identified in the brain, suggesting that they could cross the blood-brain barrier. Based on the intestinal content metabolomics, the tryptophan (Trp) biosynthesis, bile acid synthesis and bile secretion pathways have attracted our attention. Plasma metabolomic results showed that uric acid (UA) was significantly increased. The results of the brain metabolomic tests showed that the level of pyridoxal (PL) was decreased; considering the expression levels of the related enzymes, it was hypothesized that the level of pyridoxal 5'-phosphate (PLP) was decreased. PLP was important for the regulation of the neuronal γ-aminobutyric acid (GABA)/glutamate (Glu) interconversion and therefore neuronal excitability. The data of the study suggested that toxic reaction caused by G. elegans was due to a disruption of the balance of the neurotransmitter GABA/Glu transformation. CONCLUSIONS: Overall, G. elegans did not cause significant toxic reaction in the rats after long-term exposure. The results were significant for the future clinical applications of G. elegans and suggested that G. elegans could be potentially developed as a drug. The study provided a scientific basis for investigation of the mechanisms of toxicity and detoxification.

Pharmacokinetic Study of Multiple Components of Gelsemium elegans in Goats by Ultra-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry.

Gelsemium elegans (G. elegans) has been used in traditional Chinese medicine. This plant is highly toxic to humans, but can promote the growth of pigs and goats in the veterinary clinic. It is a very complex mixture containing tens or hundreds of different components. Therefore, multiple-component pharmacokinetic studies of G. elegans are a major challenge due to the lack of authentic standards of the components. The purpose of this study was to investigate the plasma pharmacokinetics of multiple components after a single oral dose of G. elegans in goat using a sensitive ultra-performance liquid chromatography coupled to tandem mass spectrometry method for the simultaneous semiquantification of multiple alkaloids without standards. The method was validated in terms of the specificity, LOD, LOQ, linearity, accuracy, precision and matrix effects. To validate the global pharmacokinetic characteristics, the results obtained from the semiquantitative analysis of three authentic compounds (gelsemine, koumine and humantenmine) were compared with the absolute quantification from our recently published method. The results showed that the two methods had similar analytical results, and the obtained values of Tmax, T1/2 and MRT0-t of the three alkaloids were similar between the two methods. In addition, the values of Cmax and AUC0-t of the three alkaloids after normalization were close to the real values, which indicated that this semiquantitative method could be used in the pharmacokinetic study of multiplecomponents. Then the pharmacokinetic parameters of 23 other G. elegans alkaloids in goats were obtained. The results suggested that the gelsedine-type alkaloids were the major active ingredients that predict and explain the efficacy and toxicity of G. elegans.

Gene expression profile analysis of ileum transcriptomes in pigs fed Gelsemium elegans plants.

Gelsemium elegans is a flowering plant in the Loganiaceae. Because it can promote the growth of pigs and sheep, it is widely used, including in veterinary clinics, but little information is available about its biological effects. Here, we used high-throughput sequencing to characterize the differentially expressed genes (DEGs) in the ileums of pigs between a control group and a group fed Gelsemium elegans for 45 days. We found that Gelsemium elegans affected many inflammatory and immune pathways, including biological processes such as defense responses, inflammation and immune responses. Moreover, this study identified several important genes related to the anti-inflammatory activity of Gelsemium elegans (e.g., CXCL-8, IL1A, and CSF2), which will be beneficial for further study of the pharmacological mechanisms and clinical applications of Gelsemium elegans.