Understanding the toxicity mechanism of gelsemine in zebrafish.

Gelsemium elegans (GE), also known as Duanchangcao, is a plant associated with toxic symptoms related to the abdomen; however, the toxicity caused by GE remains unknown. Gelsemine (GEL) is an alkaloid extracted from GE and is one of the most toxic alkaloids. This study used zebrafish as an animal model and employed high-throughput gene sequencing to identify genes and signaling pathways related to GEL toxicity. Exposure to GEL negatively impacted heart rate, swim bladder development, and activity in zebrafish larvae. Transcriptomics data revealed the enrichment of inflammatory and phagocyte signaling pathways. RT-PCR analysis revealed a decrease in the expression of pancreas-related genes, including the pancreatic coagulation protease (Ctr) family, such as Ctrl, Ctrb 1, and Ctrc, due to GEL exposure. Furthermore, GEL exposure significantly reduced Ctrb1 protein expression while elevating trypsin and serum amylase activities in zebrafish larvae. GEL also resulted in a decrease in pancreas-associated fluorescence area and an increase in neutrophil-related fluorescence area in transgenic zebrafish. This study revealed that GEL toxicity in zebrafish larvae is related to acute pancreatic inflammation.

Molecular identification and functional characterization of a transcription factor GeRAV1 from Gelsemium elegans.

BACKGROUND: Gelsemium elegans is a traditional Chinese medicinal plant and temperature is one of the key factors affecting its growth. RAV (related to ABI3/VP1) transcription factor plays multiple roles in higher plants, including the regulation of plant growth, development, and stress response. However, RAV transcription factor in G. elegans has not been reported. RESULTS: In this study, three novel GeRAV genes (GeRAV1-GeRAV3) were identified from the transcriptome of G. elegans under low temperature stress. Phylogenetic analysis showed that GeRAV1-GeRAV3 proteins were clustered into groups II, IV, and V, respectively. RNA-sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR) analyses indicated that the expression of GeRAV1 and GeRAV2 was increased in response to cold stress. Furthermore, the GeRAV1 gene was successfully cloned from G. elegans leaf. It encoded a hydrophilic, unstable, and non-secretory protein that contained both AP2 and B3 domains. The amino acid sequence of GeRAV1 protein shared a high similarity of 81.97% with Camptotheca acuminata CaRAV. Subcellular localization and transcriptional self-activation experiments demonstrated that GeRAV1 was a nucleoprotein without self-activating activity. The GeRAV1 gene was constitutively expressed in the leaves, stems, and roots of the G. elegans, with the highest expression levels in roots. In addition, the expression of the GeRAV1 gene was rapidly up-regulated under abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) stresses, suggesting that it may be involved in hormonal signaling pathways. Moreover, GeRAV1 conferred improved cold and sodium chloride tolerance in Escherichia coli Rosetta cells. CONCLUSIONS: These findings provided a foundation for further understanding on the function and regulatory mechanism of the GeRAV1 gene in response to low-temperature stress in G. elegans.

Gelsemium elegans cyclic peptide induces human cervical carcinoma cells apoptosis through intrinsic and extrinsic pathways.

Four novel Gelsemium elegans cyclic peptides (GEPs) were isolated in an antihuman cervical carcinoma activity tracking method, and their amino acid sequences were identified. The GEP-1 cyclic-(Trp-Leu-His-Val)-peptide inhibited HeLa cell proliferation in a dose- and time-dependent manner. GEP-1 induced intracellular reactive oxygen species (ROS) overproduction and induced HeLa cells apoptosis in a caspase-dependent manner. GEP-1 also induced collapse of the mitochondrial membrane potential and promoted the mitochondrial release of cytochrome c (cyt c), apoptosis-inducing factor (AIF), and endonuclease G (Endo G) in HeLa cells. Furthermore, GEP-1 triggered the extrinsic death receptor-dependent pathway, which was characterized by activating Fas and FADD. Notably, GEP-1 is a potential antihuman cervical carcinoma peptide.

The metabolism of gelsevirine in human, pig, goat and rat liver microsomes.

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.

Monoterpenoid indole alkaloids from the fruits of Gelsemium elegans and their anti-inflammatory activities.

Two novel monoterpenoid indole alkaloids (MIAs), gelsechizines A-B (1-2), along with four known ones (3-6) were isolated from the fruits of Gelsemium elegans. Compound 1 features a new carbon skeleton with two additional carbon atoms forming a 4-methylpyridine unit. Their structures with absolute configurations were elucidated by NMR, MS, X-ray diffraction and electronic circular dichroism (ECD) calculations. Compounds 1-3 showed significant anti-inflammatory effects in vivo and in vitro, which may be related to the inhibition of the trecruitment of neutrophils and macrophages as well as the secretion of TNF-α and IL-6. Preliminary structure-activity relationship analysis revealed that the ß-N-acrylate moiety plays an important role in the anti-inflammatory effect.

Gelsemine and koumine, principal active ingredients of Gelsemium, exhibit mechanical antiallodynia via spinal glycine receptor activation-induced allopregnanolone biosynthesis.

Gelsemine, the principal active alkaloid from Gelsemium sempervirens Ait., and koumine, the most dominant alkaloids from Gelsemium elegans Benth., produced antinociception in a variety of rodent models of painful hypersensitivity. The present study explored the molecular mechanisms underlying gelsemine- and koumine-induced mechanical antiallodynia in neuropathic pain. The radioligand binding and displacement assays indicated that gelsemine and koumine, like glycine, were reversible and orthosteric agonists of glycine receptors with full efficacy and probably acted on same binding site as the glycine receptor antagonist strychnine. Treatment with gelsemine, koumine and glycine in primary cultures of spinal neurons (but not microglia or astrocytes) concentration dependently increased 3α-hydroxysteroid oxidoreductase (3α-HSOR) mRNA expression, which was inhibited by pretreatment with strychnine but not the glial inhibitor minocycline. Intrathecal injection of gelsemine, koumine and glycine stimulated 3α-HSOR mRNA expression in the spinal cords of neuropathic rats and produced mechanical antiallodynia. Their spinal mechanical antiallodynia was completely blocked by strychnine, the selective 3α-HSOR inhibitor medroxyprogesterone acetate (MPA), 3α-HSOR gene silencer siRNA/3α-HSOR and specific GABAA receptor antagonist isoallopregnanolone, but not minocycline. All the results taken together uncovered that gelsemine and koumine are orthosteric agonists of glycine receptors, and produce mechanical antiallodynia through neuronal glycine receptor/3α-HSOR/allopregnanolone/GABAA receptor pathway.

The study of the constituents and source of toxicants in poisonous honey.

A novel method for non-target screening of toxicants in poisonous honey was established in this study. Poisonous honey and nontoxic honey were contrastive detected using liquid chromatography quadrupole-time-of-flight mass spectrometry and analyzed by Mass Profiler Professional Software. 4 poisonous alkaloids were screened out and confirmed by comparison with reference compounds. In order to investigate the source of these poisonous alkaloids, 6 poisonous alkaloids, ubiquitous in Gelsemium elegan, from honey, honeybees, pollen in honeycomb and different organs of Gelsemium elegan, were quantified by liquid chromatography triple-quadrupole tandem mass spectrometry. The results showed that alkaloids composition characteristics in honey, honeybees, and pollen were similar to those in the flower and bud of Gelsemium elegan and significant different from those in leave, stem and root. This result demonstrated that poisonous alkaloids in honey were come from the gathering honey process. This strategy provided an efficient and rapid method for non-target screening of toxicants in food.

Gelsemium poisoning mediated by the non-toxic plant Cassytha filiformis parasitizing Gelsemium elegans.

INTRODUCTION: Gelsemium poisoning is caused by consumption of the deadly Gelsemium species such as Gelsemium elegans, leading to significant gastrointestinal, neurological and cardio-respiratory toxicities. In 2011 (Cluster 1) and 2012 (Cluster 2), the authors encountered two clusters of gelsemium poisoning after consumption of the non-toxic parasitic plant Cassytha filiformis. The current study aims to examine the mechanism of gelsemium poisoning mediated by a benign parasitic plant. METHODS: Qualitative analysis of toxic gelsemium alkaloids using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on the herbal and urine samples from both clusters to confirm exposure. Morphological examination, qualitative analysis of aporphine alkaloids using liquid chromatography-ion trap-time of flight mass spectrometry (LC-IT-TOF/MS) and Sanger sequencing were performed on the plant sample from Cluster 2 to confirm its identity. A field study was conducted in local countryside and C. filiformis was collected for histological, LC-MS/MS and LC-IT-TOF/MS analyses to study its interaction with G. elegans. RESULTS: Gelsemium alkaloids that are not naturally present in C. filiformis were detected in the patients' herbal and urine samples. Misidentification and contamination with G. elegans during the preparation process were excluded by morphological examination of the plant sample from Cluster 2. Its identity as C. filiformis was verified with LC-IT-TOF/MS and molecular analyses. Histological, LC-MS/MS and LC-IT-TOF/MS analyses of C. filiformis collected during the field study confirmed that its haustoria penetrated the vascular bundles of G. elegans and absorbed its gelsemium toxins. CONCLUSIONS: The non-toxic plant C. filiformis absorbed toxic gelsemium alkaloids from its host, G. elegans, and led to gelsemium poisoning in our patients. Our study provides new insights into the toxicology of such plants. Benign parasitic plants may lead to potentially life-threatening poisoning if it parasitizes toxic hosts and absorbs their phytotoxins. The public awareness of risks associated with the use of these medicinal parasitic plants should be raised.

Confirmation of gelsemium poisoning by targeted analysis of toxic gelsemium alkaloids in urine.

The gelsemium plants are highly poisonous but toxicological evaluation of suspected poisoning cases has been hampered by the chemical complexity of the gelsemium toxins involved. A novel liquid chromatography-tandem mass spectrometry protocol was optimized for the collective detection of gelsemine and related alkaloids from Gelsemium elegans. The screening protocol was applied to the clinical investigation of unexplained intoxications following the ingestion of seemingly nontoxic herbs. In three clusters of toxicological emergencies ranging from severe dizziness to respiratory failure, Gelsemium elegans mistaken for various look-alike therapeutic herbs was suspected to be the hidden cause of poisoning. Nine cases of gelsemium poisonings were thus ascertained by the diagnostic urine alkaloid profiles. Gelsemine was sustained as the main urinary marker of Gelsemium exposure.