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.

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.

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.

Gelsemine relieves the neuropathic pain by down-regulating DPP4 level in rats.

BACKGROUND: Based on the previous findings on the relieving role of gelsemine in neuropathic pain, this research aims to further investigate the relevant regulatory mechanism. METHODS: Targets of gelsemine were predicted using SwissTargetPrediction. The peripheral neuropathic pain rat model was established by ligating spinal nerves, and then gelsemine (10 µg for one day) or dipeptidyl peptidase 4 (DPP4) oligonucleotides (5 µg/day, for 7 days) was injected into intrathecal bolus of rats. The mechanical threshold (0, 1, 2, 4 h after the last injection) was examined to evaluate the mechanical allodynia of rats. After the mechanical threshold measurement, the rats were anesthetized with isoflurane and then sacrificed by cervical dislocation. IBA1- and DPP4-positive cells in the spinal dorsal horn of rats were determined using immunohistochemistry and immunofluorescence assays. The expressions of DPP4, IL-1ß and TNF-α in the spinal dorsal horn of rats were measured by Western blot and quantitative real-time PCR. RESULTS: DPP4 was one of the targets of gelsemine. Gelsemine could elevate the down-regulated mechanical threshold, and lessen the up-regulated IBA1- and DPP4-positive cells and expressions of DPP4, IL-1ß and TNF-α in the spinal dorsal horn of rats with neuropathic pain. DPP4 overexpression reversed the role of gelsemine in neuropathic pain. CONCLUSION: Gelsemine relieves neuropathic pain by down-regulating DPP4 level in rats, providing a novel drug candidate and biomarker for neuropathic pain treatment.

Gelsemine Exerts Neuroprotective Effects on Neonatal Mice with Hypoxic-Ischemic Brain Injury by Suppressing Inflammation and Oxidative Stress via Nrf2/HO-1 Pathway.

Given that the role of Gelsemine in neuroinflammation has been demonstrated, this research aimed to investigate the effect of Gelsemine on neonatal hypoxic-ischemic (HI) brain injury. An in vivo HI brain injury neonatal mouse model and an in vitro oxygen-glucose deprivation (OGD) cell model were established and pretreated with Gelsemine. The brain infarct volume, neuronal loss and apoptosis, as well as spatial learning and memory were examined by TTC staining, Nissl's staining, TUNEL staining and Morris water maze test. Immunohistochemical staining was applied to detect the microglia cells and astrocytes in the mouse brain tissue. The cell viability was analyzed by CCK-8 assay. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), TNF-α, IL-1ß, and IL-6 were determined via ELISA. The lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) level in OGD-treated cells were detected by colorimetry and DCFH-DA staining. Nrf2, HO-1, and inflammation-related factors were analyzed by immunofluorescence, qRT-PCR, or western blot. Gelsemine reduced the infarct volume and neuronal loss and apoptosis, yet improved spatial learning and memory impairment of HI-injured mice. Gelsemine inhibited the elevated MDA, TNF-α, IL-1ß, IL-6, LDH and ROS levels, promoted the reduced SOD level and viability, and strengthened the up-regulation of HO-1 and Nrf2 in brain tissues and OGD-treated cells. However, Nrf2 silencing reversed the effects of Gelsemine on the Nrf2/HO-1 pathway, inflammation, and oxidative stress in OGD-treated cells. Gelsemine produces neuroprotective effects on neonatal mice with HI brain injury by suppressing inflammation and oxidative stress via Nrf2/HO-1 pathway.

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.

Modulation of GABAA receptors and of GABAergic synapses by the natural alkaloid gelsemine.

The Gelsemium elegans plant preparations have shown beneficial activity against common diseases, including chronic pain and anxiety. Nevertheless, their clinical uses are limited by their toxicity. Gelsemine, one of the most abundant alkaloids in the Gelsemium plants, have replicated these therapeutic and toxic actions in experimental behavioral models. However, the molecular targets underlying these biological effects remain unclear. The behavioral activity profile of gelsemine suggests the involvement of GABAA receptors (GABAARs), which are the main biological targets of benzodiazepines (BDZs), a group of drugs with anxiolytic, hypnotic, and analgesic properties. Here, we aim to define the modulation of GABAARs by gelsemine, with a special focus on the subtypes involved in the BDZ actions. The gelsemine actions were determined by electrophysiological recordings of recombinant GABAARs expressed in HEK293 cells, and of native receptors in cortical neurons. Gelsemine inhibited the agonist-evoked currents of recombinant and native receptors. The functional inhibition was not associated with the BDZ binding site. We determined in addition that gelsemine diminished the frequency of GABAergic synaptic events, likely through a presynaptic modulation. Our findings establish gelsemine as a negative modulator of GABAARs and of GABAergic synaptic function. These pharmacological features discard direct anxiolytic or analgesic actions of gelsemine through GABAARs but support a role of GABAARs on the alkaloid induced toxicity. On the other hand, the presynaptic effects of the alkaloid provide an additional mechanism to explain their beneficial effects. Collectively, our results contribute novel information to improve understanding of gelsemine actions in the mammalian nervous system.

Confirmation of Gelsemium elegans poisoning by UHPLC-MS/MS analysis of koumine, gelsemine, and gelsenicine in hair.

A sensitive, accurate, simple, and rapid analytical UHPLC-MS/MS method was developed for identification and quantification of koumine, gelsemine, and gelsenicine in human hair. Approximately 10 mg of hair was extracted with methanol by cryogenic grinding. The limits of detection (LODs) ranged from 1 to 5 pg/mg, and the limits of quantitation (LOQs) ranged from 2 to 10 pg/mg. The method was linear over a concentration range from the LOQs to 1000 pg/mg, and the linear correlation (R2) of the calibration curves was above 0.998 for all three analytes. The bias varied from -6.5-13.1%, while the intra- and inter-day precision relative standard deviation (RSD) values were 4.3-12.4% and 3.7-13.2%, respectively. Recoveries ranged from 79.3% to 103.5%, and matrix effects ranged from 74.3% to 105.5%. The described method was used for the quantitative determination of koumine, gelsemine, and gelsenicine in a human hair sample from a Gelsemium elegans poisoning case. The highest concentrations of koumine, gelsemine, and gelsenicine were 27.2, 18.1, and 4.2 pg/mg, respectively, and corresponded to the segment associated with the ingestion period. To our knowledge, this is the first study to describe hair analysis in a G. elegans poisoning case and to provide quantitative toxicological findings.

The detoxification effect of cytochrome P450 3A4 on gelsemine-induced toxicity.

Gelsemine (GA), the principal alkaloid in Gelsemium elegans Benth, exhibits potent and specific antinociception in chronic pain without the induction of apparent tolerance. However, GA also exerts neurotoxicity and hepatotoxicity when overdosed, and potential detoxification pathways are urgently needed. Cytochrome P450 enzymes (CYPs) are important phase I enzymes involved in the detoxification of xenobiotic compounds. The study aimed to investigate the role of CYPs-mediated metabolism in GA-induced toxicity. Microsomes, chemical special inhibitors and human recombinant CYPs indicated that GA was mainly metabolized by CYP3A4/5. The major metabolite of GA was isolated and identified as 4-N-demethyl-GA by high-resolution mass spectrometry and nuclear magnetic resonance technology. The CYP3A4 inhibitor ketoconazole significantly inhibited the metabolism of GA. This drastically increased GA toxicity which is caused by increasing the level of malondialdehyde and decreasing the level of the superoxide dismutase in mice. In contrast, the CYP3A4 inducer dexamethasone significantly increased GA metabolism and markedly decreased GA toxicity in mice. Notably, in CYP3A4-humanized mice, the toxicity of GA was significantly reduced compared to normal mice. These findings demonstrated that CYP3A4-mediated metabolism is a robust detoxification pathway for GA-induced toxicity.

LC-MS/MS Quantification Reveals Ample Gut Uptake and Metabolization of Dietary Phytochemicals in Honey Bees (Apis mellifera).

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6-30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4-31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

Gelsemine, a natural alkaloid extracted from Gelsemium elegans Benth. alleviates neuroinflammation and cognitive impairments in Aß oligomer-treated mice.

INTRODUCTION: Gelsemine is a natural alkaloid extracted from Gelsemium elegans Benth., a traditional Chinese medicinal herb. Gelsemine has been shown to penetrate the brain, and could produce neurological activities, such as anxiolytic and neuralgia-alleviating effects, suggesting that this natural compound might be used for treating nervous system diseases. RESULTS: In this study, we have found, for the first time, that gelsemine at low concentrations (5-10 µg/kg) significantly alleviated cognitive impairments induced by ß-amyloid (Aß) oligomer, a main neurotoxin of Alzheimer's disease (AD). In addition, gelsemine substantially prevented Aß oligomer-induced over-activation of microglia and astrocytes, indicating that gelsemine might reduce AD-related gliosis. Consistently, gelsemine inhibited the over-expression of pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), in the brain of mice. Moreover, gelsemine largely increased the expression of pSer9-glycogen synthase kinase-3ß (GSK3ß), and decreased the hyper-phosphorylation of tau protein as evidenced by Western blotting analysis. Furthermore, gelsemine prevented Aß oligomer-induced reduction of PSD-95, a representative post-synaptic protein. CONCLUSION: All these results directly demonstrated the anti-Aß oligomer neuroprotective properties of gelsemine, opening a novel perspective for the development of gelsemine-based therapeutics against Aß-associated neurodegeneration disorders, including AD in particular.

Characterization of absorbed and produced constituents in goat plasma urine and faeces from the herbal medicine Gelsemium elegans by using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. MATERIAL AND METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Material Basis Analysis of Toxicity Attenuation and Efficacy Reservation of Processing of Gelsemium elegans Rhizomes / 中国实验方剂学杂志

Objective::On the basis of previous research, to detect the changes of six main alkaloids in Gelsemium elegans rhizomes before and after being processed, so as to reveal its internal mechanism of processing. Method::The contents of gelsemine, humantenidine, koumine, gelsenicine, gelsevirine and humantenine in G. elegans rhizomes was determined simultaneously by HPLC, the content changes of these components before and after processing and its reasons were analyzed by cluster analysis and principal component analysis. The mobile phase was methanol (A)-0.1%formic acid aqueous solution (B) for gradient elution (0-10 min, 22%A; 10-20 min, 22%-30%A; 20-30 min, 30%-40%A). The flow rate was 1.0 mL·min-1. The detection wavelength was set at 254 nm, the injection volume was 10 μL, and the column temperature was 30 ℃. Result::Before processing, contents of the above six components in raw products were 1.444, 1.129, 3.590, 1.603, 2.376, 1.631 mg·g-1, after processing, the contents of these six components were 2.258, 0.343, 1.176, 0.115, 0.459, 0.281 mg·g-1, respectively. Gelsenicine, the most toxic ingredient of G. elegans rhizomes, decreased most significantly with a decreasing rate of 92.83%, while the less toxic ingredient, gelsemine, increased by 56.37%after processing. The contents of other four components in G. elegans rhizomes decreased to varying degrees after processing. The results of cluster analysis indicated that G. elegans rhizomes were clearly divided into two categories before and after processing. Principal component analysis showed that the first principal component before and after processing was changed from koumine to gelsemine. Conclusion::The degradation of toxic components and content changes of other components may be one of the intrinsic mechanism of toxicity attenuation and efficacy reservation of G. elegans rhizomes being processed.

The Metabolism and Disposition of Koumine, Gelsemine and Humantenmine from Gelsemium.

BACKGROUND: Gelsemium is a toxic flowering plant of the Gelsemiaceae family. It is used to treat skin diseases in China, and it is an important medicinal and homeopathic plant in North America. Up to now, more than 200 compounds have been isolated and reported from Gelsemium. More than 120 of these are indole alkaloids, including the main components, koumine, gelsemine and humantenmine which produce the pharmacological and toxicological effects of Gelsemium. However, their clinical application their limited by its narrow therapeutic window. Therefore, it is very important to study the metabolism and disposition of indole alkaloids from Gelsemium before their clinical application. This paper reviews all the reports on the metabolism and disposition of alkaloids isolated from Gelsemium at home and abroad. METHODS: The metabolism and disposition of alkaloids from Gelsemium were searched by the Web of Science, NCBI, PubMed and some Chinese literature databases. RESULTS: Only koumine, gelsemine and humantenmine have been reported, and few other alkaloids have been described. These studies indicated that the three indole alkaloids are absorbed rapidly, widely distributed in tissues, extensively metabolized and rapidly eliminated. There are species differences in the metabolism of these alkaloids, which is the reason for the differences in their toxicity in animals and humans. CONCLUSION: This review not only explains the pharmacokinetics of indole alkaloids from Gelsemium but also facilitates further study on their metabolism and mechanism of toxicity.

The Difference in Cytotoxic Activity between Two Optical Isomers of Gelsemine from Gelsemium elegans Benth. on PC12 Cells.

Two optical isomers, +/- gelsemine (1, 2), together with one known compound were isolated from the whole plant of G. elegans. The structures of the separated constituents were elucidated on 1D and 2D (1H-1H COSY, HMBC, HSQC) NMR spectroscopy and high-resolution mass spectrometry (HRMS). The isolated alkaloids were tested in vitro for cytotoxic potential against PC12 cells by the MTT assay. As a result, (+) gelsemine (compound 1) exhibited cytotoxic activity against PC12 cells with an IC50 value of 31.59 µM, while (-) gelsemine (compound 2) was not cytotoxic.

A novel two-dimensional liquid chromatography system for the simultaneous determination of three monoterpene indole alkaloids in biological matrices.

The present paper describes a novel two-dimensional liquid chromatography (2D-LC) system, which is comprised of a first-dimensional ion exchange chromatography (IEX1) column, trap column, and second-dimensional reversed-phase chromatography (RP2) column system. The biological sample is separated by the first-dimensional LC using an IEX column to remove interferences. The analytes are transferred to the trap column after heart-cutting. Then, the analytes are transferred to the second-dimensional LC using an RP2 column for further separation and ultraviolet detection. This 2D-LC system can offer a large injection volume to provide sufficient sensitivity and exhibits a strong capacity for removing interferences. Here, the determination of three monoterpene indole alkaloids (MIAs; gelsemine, koumine, and humantenmine) from Gelsemium in biological matrices (plasma, tissue, and urine) was used this 2D-LC system. After a rapid and easy sample preparation method based on protein precipitation, the sample was injected into the 2D-LC. The method was developed and validated in terms of the selectivity, LOD, LOQ, linearity, precision, accuracy, and stability. The sample preparation time for the three MIAs was 15 min. The LOD for these compounds was 10 ng/mL, which was lower than the developed HPLC methods. The results showed that this method had good quantitation performance and allowed the determination of gelsemine, koumine, and humantenmine in biological matrices. The method is rapid, exhibits high selectivity, has good sensitivity, and is low-cost, thus making it well-suited for application in the pharmaceutical and toxicological analysis of Gelsemium. Graphical abstract.

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.

Effects of gelsemine on oxidative stress and DNA damage responses of Tetrahymena thermophila.

Gelsemine is an important toxic substance extracted from Gelsemium elegans, which has a lot of biological functions in cells and organisms, but its toxicity has been rarely reported in Tetrahymena thermophila. In this study, we used the protozoan T. thermophila as an experimental model to investigate the potential toxicity-induced mechanism of gelsemine in the unicellular eukaryote. Our results clearly showed gelsemine inhibited T. thermophila growth in a dose-dependent manner. This exposure also resulted in oxidative stress on T. thermophila cells and antioxidant enzyme levels were significantly altered at high gelsemine levels (p < 0.05). Gelsemine produced a slight apoptotic effect at the highest (0.8 mg/mL) gelsemine level used here (p < 0.05). Furthermore, the toxin-induced DNA damage in a dose-dependent manner. The ultrastructural analysis also revealed mitophagic vacuoles at 0.4 and 0.8 mg/mL levels of gelsemine exposure. Moreover, expressions of oxidative stress-related and MAP kinase genes were significantly changed after exposure to 0.8 mg/mL level of gelsemine (p < 0.05). Altogether, our results clearly show that gelsemine from G. elegans can inhibit the growth via inducing oxidative stress and DNA damage in T. thermophila cells.

Simultaneous determination of gelsemine and koumine in rat plasma by UPLC-MS/MS and application to pharmacokinetic study after oral administration of Gelsemium elegans Benth extract.

A simple, rapid and sensitive method using UPLC-MS/MS was established and validated for simultaneous determination of gelsemine and koumine in rat plasma after oral administration of Gelsemium elegans Benth extract. Plasma was performed with methanol precipitation and berberine was chosen as the internal standard. Plasma samples were separated on an Acquity UPLC® BEH C18 column (3.0 × 50 mm, 1.7 µm) with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase at a flow rate of 0.4 mL/min. Multiple reaction monitoring mode in positive ion mode was utilized for detection. The calibration curves were linear over the range of 0.2-100 ng/mL for gelsemine and 0.1-50 ng/mL for koumine, with the lower limits of quantification 0.2 and 0.1 ng/mL, respectively. The intra- and inter-precision and accuracy were well within the acceptable ranges. The developed method was successfully applied to an in vivo pharmacokinetic study in rat after oral administration of 10 mg/kg Gelsemium elegans Benth extract.

Development and in-house validation of a sensitive LC-MS/MS method for simultaneous quantification of gelsemine, koumine and humantenmine in porcine plasma.

Three monomers of G. elegans indole alkaloids (gelsemine, koumine and humantenmine) were simultaneously detected in porcine plasma for the first time with the development and validation of a sensitive and reliable LC-ESI-MS/MS method. Using a gradient mobile phase at a constant flow rate of 0.2 mL/min via electrospray ionization (positive ion mode) in a multiple reaction monitoring (MRM) scan, gelsemine, koumine and humantenmine were eluted, separated and detected at an appropriate retention time. The porcine plasma was prepared using protein precipitation with 1% formic acid-acetonitrile: methanol (2:1, v/v). Using matrix-matched calibration curves and weighted least squares linear regression, a good linearity (r2 > 0.99) was achieved with a concentration range of 0.1-200 µg/L for gelsemine, koumine and humantenmine; estimated LOD and LOQ values were 0.10 µg/L and 0.2 µg/L, respectively. The mean of the recoveries was in the range of 82.68-100.35% of porcine plasma at four different levels, and the intra-day and inter-day precision (CV) were lower than 15% with a range of 2.46-8.76% and 2.73-10.83%, respectively. The proposed method has proved to be suitable for accurate, quantitative determination of gelsemine, koumine and humantenmine in porcine plasma.