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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. [ABSTRACT FROM AUTHOR]

The aim of this study was to validate the preventive effects of koumine (KM), a monoterpene indole alkaloid, on gouty arthritis (GA) and to explore its possible mechanisms. C57BL/6 mice were intraperitoneally administered KM (0.8, 2.4 or 7.2 mg/kg), colchicine (3.0 mg/kg) or sterile saline. One hour later, a monosodium urate (MSU) suspension was injected into the right hind paws of the mice to establish an acute gout model. Inflammation symptoms were evaluated at 0, 3, 6, 12 and 24 h, and the mechanical withdrawal threshold was evaluated at 0, 6 and 24 h. After 24 h, the mice were euthanized, and the joint tissue, kidney and blood were collected for subsequent experiments. Histological examination and antioxidant enzyme, kidney index and serum uric acid (UA) measurements were taken. The expression levels of the signalling pathway components were determined. KM effectively alleviated the symptoms of redness, swelling and pain; counteracted inflammatory cell infiltration; and increased antioxidant enzyme levels, reduced kidney index and serum UA levels through regulating UA excretion in MSU‐induced mice. The expression of toll‐like receptor 4 (TLR4)/nuclear factor kappa‐B (NF‐κB)/nucleotide‐binding oligomerization domain, leucine‐rich repeat and pyrin domain‐containing 3 (NLRP3) signalling pathway proteins and mRNA were reduced in the KM group. These results suggest that KM may be effective in alleviating GA through the TLR4/NF‐κB/NLRP3 pathway. [ABSTRACT FROM AUTHOR]

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. • Cytochrome P450 3A4 crucially impacts the tissue distribution and elimination of the three Gelsemium alkaloids. • The three Gelsemium alkaloids were mainly distributed in the spleen and pancreas after administration. • The difference in cytochrome P450 3A4 content may account for the difference in humantenmine toxicity. [ABSTRACT FROM AUTHOR]

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 ED 50 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 (LD 50 = 0.185 mg/kg). The study also investigated the mechanism of action by analyzing the expression levels of GlyRα3 and Gephyrin. The PGE 2 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 PGE 2 -induced hyperalgesia by upregulating the expression of GlyRα3 and Gephyrin, which are key targets of the Gly receptor pathway. • This study explored the analgesic mechanism of gelsenicine for the first time. • Gelsenicine can significantly relieve the hypersensitivity pain caused by PGE 2. • Gelsenicine did not cause pathological damage to the mouse's spinal cord. • Gelsenicine has the best analgesic effect among the G.elegans alkaloids. [ABSTRACT FROM AUTHOR]

Koumine is one of the most abundant alkaloids found in Gelsemium elegans, and it has a wide range of pharmacological effects including antitumor, anti-inflammatory, analgesic treatment effects, and antianxiety. However, its high toxicity and unclear mechanism of action have greatly limited the medicinal development and use of koumine. We investigated the toxic effects of koumine on the developmental toxicity and behavioral neurotoxicity of zebrafish embryos and larvae. Embryos at 6 h postfertilization (hpf) were exposed to 12.5, 25, 50, 75, and 100 mg/L of koumine until 120 hpf. Koumine affected the hatching and heartbeats of the embryos. The morphological analysis also revealed many abnormalities, such as shortened bodies, yolk sac edemas, tail malformations, and pericardial edemas. To identify the neurotoxicity of koumine, the behavior of the larvae was measured. Koumine at 50 and 100 mg/L affect the escape response. The embryos exhibited uncoordinated muscle contractions along the body axis in response to touch at 36 hpf. More importantly, we found that the neurotoxicity of koumine is mainly caused by influencing the ACh content and the activity of AChE without impairing motor neuron development. A comprehensive analysis shows that a high concentration of koumine has obvious toxic effects on zebrafish, and the safe concentration of koumine for zebrafish should be less than 25 mg/L. These results will be valuable for better understanding the toxicity of koumine and provide new insights into the application of koumine. [ABSTRACT FROM AUTHOR]

Qing Hu,1,2,* Xiao-Ling Fu,1,* Yi-Yan Dong,1,2 Ju Ma,1 Jian Hua,1 Jia-Ting Li,1 Kai-Xin Liu,1 Jian Yang,1,2 Chang-Xi Yu1,2 1School of Pharmacy, Fujian Medical University, Fuzhou, People’s Republic of China; 2Fujian Key Laboratory of Drug Target Discovery and Structural and Functional Research, School of Pharmacy, Fujian Medical University, Fuzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chang-Xi Yu; Jian Yang, School of Pharmacy, Fujian Medical University, No. 1 Xuefu North Road, Fuzhou, 350122, People’s Republic of China, Tel +86-0591-22862016, Email changxiyu@mail.fjmu.edu.cn; fjmukent@mail.fjmu.edu.cnIntroduction: Koumine (KME) is the most abundant active ingredient separated from Gelsemium elegans Benth and exhibits a significant therapeutic effect on rheumatoid arthritis (RA). It is a lipophilic compound with poor aqueous solubility, and there is an urgent need to develop novel dosage forms of KME and promote its clinical application for the treatment of RA. The aim of this study was to design and develop KME-loaded microemulsions (KME-MEs) for the effective management of RA.Methods: The composition of the microemulsion was selected by carrying out a solubility study and generating pseudoternary phase diagrams, and further optimized by D-Optimal design. The optimized KME-MEs was evaluated for particle size, viscosity, drug release, storage stability, cytotoxicity, cellular uptake, Caco-2 cell transport and everted gut sac investigations. In vivo fluorescence imaging and the therapeutic effects of KME and KME-MEs on collagen-induced arthritis (CIA) rats were also evaluated.Results: The optimized microemulsion contained 8% oil, 32% Smix (surfactant/cosurfactant) and 60% water and was used for in vivo and in vitro studies. The optimal KME-MEs exhibited a small globule size of 18.5 ± 0.14 nm and good stability over 3 months, and the release kinetics followed a first-order model. These KME-MEs had no toxic effect on Caco-2 cells but were efficiently internalized into the cytoplasm. Compared to KME, the KME-MEs displayed significantly increased permeability and absorption in Caco-2 cell monolayer assay and ex vivo everted gut sac experiment. As expected, the KME-MEs attenuated the progression of RA in CIA rats and were more effective than free KME with a reduced frequency of administration.Conclusion: The KME-MEs improved the solubility and therapeutic efficacy of KME by employing formulation technology. These results provide a promising vehicle for the oral delivery of KME to treat RA and have attractive potential for clinical translation.Graphical Abstract: Keywords: rheumatoid arthritis, koumine, microemulsion, permeability, oral administration

Abstract The genus Gelsemium belongs to the family Loganiaceae, one of the traditional Chinese herbs. Gelsemium is traditionally used to treat rheumatoid and neuropathic pain. Its root extracts were found to protect against anxiety, especially the alkaloids koumine and gelsemine. Indeed, koumine and gelsemine can act as positive agonists of the glycine receptor (GlyR), which reduces neuronal excitability through chloride influx and can also increase neuroactive steroid content by enhancing 3alpha‐hydroxysteroid oxidoreductase (3α‐HSOR) expression. The latter can activate the excitation‐inhibitory response via the γ‐aminobutyric acid type A receptor (GABAAR), reduce the abnormal corticotropin‐releasing hormone (CRH) increase in the hypothalamus, inhibit adrenocorticotropic hormone (ACTH) secretion, and effectively inhibit the abnormal ACTH and corticosterone increases in the circulation. In addition, koumine and gelsemine inhibited the expression of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, inhibiting the release of inflammatory factors and regulating anxiety‐related neural circuits. Gelsemine also inhibited the overexpression of brain‐derived neurotrophic factor (BDNF) and cAMP response element‐binding protein (CREB) in the hypothalamus to maintain the plasticity of brain neurons and protect neurogenesis to achieve anxiety regulation. In general, this article reviews the recent studies on Gelsemium in the anxiety field, discusses its possible antianxiety mechanism, and confirms the potential of Gelsemium as a therapeutic drug for anxiety‐related diseases.

This study evaluated the effects of koumine on growth performance, brain transcriptional response, expression of genes associated with circadian rhythms, and physiological stress indicators in carp (5.21 ± 0.05 g). Fish were fed four different koumine levels (0, 0.2, 2, and 20 mg/kg) of diet (crude protein 29.71%, crude lipid 3.68%) for 10 weeks in a RAS (26.0–28.0 ℃, pH 7.3–7.5, and DO 5.5–6.1 mg/L). Each type of feed was administered to three replicate tanks (50 L, 12 cycles per day), each containing 30 fish, with feeding occurring twice daily. The results showed that koumine supplementation improved the growth performance of carp. Significant enrichment of circadian rhythm, memory, and behavior-related pathways in the brain of the koumine-supplemented group of fish. The circadian rhythm positive feedback-related genes MKP5, ATP1A, ATP2A, and CRY2 were significantly upregulated (P

Background and Purpose: New remedies are required for the treatment of diabetic neuropathic pain (DNP) due to insufficient efficacy of available therapies. Here, we used chemogenetic approaches combined with in vivo pharmacology to elucidate the role of basolateral amygdala (BLA) astrocytes in DNP pathogenesis and provide new insights into therapeutic strategies for DNP. Experimental Approach: A streptozotocin‐induced DNP model was established. Designer receptors exclusively activated by designer drugs (DREADDs) were used to regulate astrocyte activity. Mechanical hyperalgesia was assessed using the electronic von Frey test. Anxiety‐like behaviours were detected using open field and elevated plus maze tests. Astrocytic activity was detected by immunofluorescence, and cytokine content was determined by ELISA. Key Results: BLA astrocytes were regulated by DREADDs, and inhibition of BLA astrocytes attenuated mechanical allodynia and pain‐related negative emotions in DNP rats. In contrast, temporary activation of BLA astrocytes induced allodynia without anxious behaviours in naive rats. In addition, koumine (KM) alleviated mechanical allodynia and anxiety‐like behaviours in DNP rats, inhibited the activation of BLA astrocytes and suppressed the inflammatory response. Furthermore, persistent activation of BLA astrocytes through chemogenetics mimicked chronic pain, and KM alleviated the pain hypersensitivity and anxiety‐like behaviours. Conclusion and Implications: DREADDs bidirectionally regulate the activity of BLA astrocytes, which proves for the first time the role of BLA astrocyte activation in the pathogenesis of DNP and represents a novel therapeutic strategy for DNP. KM ameliorates DNP, perhaps by inhibiting the activation of BLA astrocytes and reveal KM as a potential candidate for treating DNP. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) caused by sepsis is one of the most common critical diseases, which is difficult to treat and has a high fatality rate. Koumine is one of the main active components of Gelsemium plants and has been confirmed to have potential for drug development; however, its therapeutic effects on ALI have not yet been studied. This study established ALI due to sepsis using cecal ligation and puncture (CLP) and assessed the therapeutic effects of koumine by measuring mouse survival rates, lung tissue pathological damage, inflammatory factors, and oxidative stress levels. Additionally, network pharmacology was utilized to explore the underlying mechanisms. The results showed that koumine inhibited the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), thereby inhibiting inflammatory response, reducing lung injury score and lung wet to dry ratio. In addition, koumine reduced oxidative stress in mice by reducing myeloperoxidase (MPO) and malondialdehyde (MDA) and increasing superoxide dismutase (SOD) content. Network pharmacology analysis showed that 52 putative targets were relevant, and SLC6A4, HTR3A, JAK2 and JAK3 were the key targets. GO and KEGG pathway enrichment analysis showed that the related mechanisms involved neuroactive ligand-receptor interaction, calcium signaling pathway, serotonergic synapses, cholinergic synapses, etc. In summary, this study confirmed the potential therapeutic effect of koumine in sepsis induced ALI, suggesting its development prospect as a novel candidate drug for ALI, and providing data support. [Display omitted] • To study for the first time the effect of active components of Gelsemium plant on pulmonary diseases. • Analyzed the role and mechanism of koumine in acute lung injury through inflammation, oxidative stress, and network pharmacology. • Koumine is a potential treatment for sepsis-induced acute lung injury. [ABSTRACT FROM AUTHOR]

Koumine is one of the most abundant alkaloids found in Gelsemium elegans, and it has a wide range of pharmacological effects including antitumor, anti-inflammatory, analgesic treatment effects, and antianxiety. However, its high toxicity and unclear mechanism of action have greatly limited the medicinal development and use of koumine. We investigated the toxic effects of koumine on the developmental toxicity and behavioral neurotoxicity of zebrafish embryos and larvae. Embryos at 6 h postfertilization (hpf) were exposed to 12.5, 25, 50, 75, and 100 mg/L of koumine until 120 hpf. Koumine affected the hatching and heartbeats of the embryos. The morphological analysis also revealed many abnormalities, such as shortened bodies, yolk sac edemas, tail malformations, and pericardial edemas. To identify the neurotoxicity of koumine, the behavior of the larvae was measured. Koumine at 50 and 100 mg/L affect the escape response. The embryos exhibited uncoordinated muscle contractions along the body axis in response to touch at 36 hpf. More importantly, we found that the neurotoxicity of koumine is mainly caused by influencing the ACh content and the activity of AChE without impairing motor neuron development. A comprehensive analysis shows that a high concentration of koumine has obvious toxic effects on zebrafish, and the safe concentration of koumine for zebrafish should be less than 25 mg/L. These results will be valuable for better understanding the toxicity of koumine and provide new insights into the application of koumine.

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. [ABSTRACT FROM AUTHOR]

Koumine (KME) is an active alkaloid extracted from Gelsemium elegans, and its diverse bioactivities have been studied for decades. However, KME exhibits poor solubility and low oral bioavailability, which hampers its potential therapeutic exploitation. This work aimed to develop optimized inclusion complexes to improve the bioavailability of KME. The KME/hydroxypropyl-β-cyclodextrin (KME/HP-β-CD) inclusion complexes were prepared by the solvent evaporation method and later optimized using the Box-Behnken design. The optimal KME/HP-β-CD was characterized by scanning electron microscopy, Fourier transforms infrared spectroscopy, differential scanning calorimetry, and nuclear magnetic resonance spectroscopy. The physicochemical characterization results revealed that the crystalline state of KME was transformed into an amorphous form, forming KME/HP-β-CD inclusion complexes. Compared with KME, the solubility and in vitro release rate of KME/HP-β-CD was significantly enhanced by 52.34- and 1.3-fold, respectively. Further research was performed to investigate the intestinal absorption characteristics and in vivo bioavailability in rats. The optimal KME/HP-β-CD showed enhanced absorptive permeability and relative bioavailability increased more than two-fold compared to that of raw KME. These results indicate that the optimal KME/HP-β-CD can be used as an effective drug carrier to improve the solubility, intestinal absorption, and bioavailability of KME.

Koumine (KME) is an active alkaloid extracted from Gelsemium elegans, and its diverse bioactivities have been studied for decades. However, KME exhibits poor solubility and low oral bioavailability, which hampers its potential therapeutic exploitation. This work aimed to develop optimized inclusion complexes to improve the bioavailability of KME. The KME/hydroxypropyl-b-cyclodextrin (KME/HPb-CD) inclusion complexes were prepared by the solvent evaporation method and later optimized using the Box-Behnken design. The optimal KME/HP-b-CD was characterized by scanning electron microscopy, Fourier transforms infrared spectroscopy, differential scanning calorimetry, and nuclear magnetic resonance spectroscopy. The physicochemical characterization results revealed that the crystalline state of KME was transformed into an amorphous form, forming KME/HP-b-CD inclusion complexes. Compared with KME, the solubility and in vitro release rate of KME/HP-b-CD was significantly enhanced by 52.34- and 1.3-fold, respectively. Further research was performed to investigate the intestinal absorption characteristics and in vivo bioavailability in rats. The optimal KME/HP-b-CD showed enhanced absorptive permeability and relative bioavailability increased more than two-fold compared to that of raw KME. These results indicate that the optimal KME/HP-b-CD can be used as an effective drug carrier to improve the solubility, intestinal absorption, and bioavailability of KME. [ABSTRACT FROM AUTHOR]

Translocator protein 18 kDa (TSPO) is an evolutionarily conserved 5-transmembrane domain protein, and has been considered as an important therapeutic target for the treatment of pain. We have recently reported the in vitro and in vivo pharmacological characterization of koumine as a TSPO positive allosteric modulator (PAM), more precisely ago-PAM. However, the probe dependence in the allostery of koumine is an important question to resolve, and the possible analgesic mechanism of koumine remains to be clarified. Here, we report the in vivo evaluation of the allostery of koumine when orthosteric ligand PK11195 was used and preliminarily explore the possible analgesic mechanism of koumine associated with neurosteroids. We find that koumine is an ago-PAM of the PK11195-mediated analgesic effect at TSPO, and the analgesic mechanism of this TSPO ago-PAM may be associated with neurosteroids as the analgesic effects of koumine in the formalin-induced inflammatory pain model and chronic constriction injury-induced neuropathic pain model can be antagonized by neurosteroid synthesis inhibitor aminoglutethimide. Although our results cannot fully clarify the allosteric modulatory effect of koumine, it further prove the allostery in TSPO and provide a solid foundation for koumine to be used as a new clinical candidate drug to treat pain. [ABSTRACT FROM AUTHOR]

Translocator protein 18 kDa (TSPO) is an evolutionarily conserved 5-transmembrane domain protein, and has been considered as an important therapeutic target for the treatment of pain. We have recently reported the in vitro and in vivo pharmacological characterization of koumine as a TSPO positive allosteric modulator (PAM), more precisely ago-PAM. However, the probe dependence in the allostery of koumine is an important question to resolve, and the possible analgesic mechanism of koumine remains to be clarified. Here, we report the in vivo evaluation of the allostery of koumine when orthosteric ligand PK11195 was used and preliminarily explore the possible analgesic mechanism of koumine associated with neurosteroids. We find that koumine is an ago-PAM of the PK11195-mediated analgesic effect at TSPO, and the analgesic mechanism of this TSPO ago-PAM may be associated with neurosteroids as the analgesic effects of koumine in the formalin-induced inflammatory pain model and chronic constriction injury-induced neuropathic pain model can be antagonized by neurosteroid synthesis inhibitor aminoglutethimide. Although our results cannot fully clarify the allosteric modulatory effect of koumine, it further prove the allostery in TSPO and provide a solid foundation for koumine to be used as a new clinical candidate drug to treat pain.

Osteoarthritis (OA) is a degenerative joint disease, Increasingly, mitochondrial autophagy has been found to play an important regulatory role in the prevention and treatment of osteoarthritis. Koumine is a bioactive alkaloid extracted from the plant Gelsemium elegans. In previous research, Koumine was found to have potential in improving the progression of OA in rats. However, the specific mechanism of its action has not been fully explained. Therefore, the aim of this study was to investigate whether Koumine can alleviate OA in rats by influencing mitochondrial autophagy. In the in vitro study, rat chondrocytes (RCCS-1) were induced with IL-1β (10 ng/mL) to induce inflammation, and Koumine (50 μg/mL) was co-treated. In the in vivo study, a rat OA model was established by intra-articular injection of 2% papain, and Koumine was administered orally (1 mg/kg, once daily for two weeks). It was found that Koumine effectively reduced cartilage erosion in rats with osteoarthritis. Additionally, it decreased the levels of inflammatory factors such as IL-1β, IL-6, and extracellular matrix (ECM) components MMP13 and ADAMTS5 in chondrocytes and articular cartilage tissue, while increasing the level of Collagen II.Koumine inhibited the production of reactive oxygen species (ROS) in cartilage tissue and increased the number of autophagosomes in chondrocytes and articular cartilage tissue. Additionally, it upregulated the expression of mitochondrial autophagy proteins LC3Ⅱ/Ⅰ, PINK1, Parkin, and Drp1. The administration of Mdivi-1 (50 μM) reversed the enhanced effect of Koumine on mitochondrial autophagy, as well as its anti-inflammatory and anti-ECM degradation effects in rats with OA. These findings suggest that Koumine can alleviate chondrocyte inflammation and improve the progression of OA in rats by activating PINK1/Parkin-mediated mitochondrial autophagy. [Display omitted] • Koumine alleviates osteoarthritis by regulating inflammatory cytokines. • Koumine ameliorates cartilage damage by regulating the levels of extracellular matrix synthase and degradative enzymes. • Koumine alleviates osteoarthritis by regulating the activation of PINK1/Parkin-mediated mitochondrial autophagy. [ABSTRACT FROM AUTHOR]

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.

Koumine is an alkaloid that displays notable activity against inflammatory and neuropathic pain, but its therapeutic target and molecular mechanism still need further study. Translocator protein 18 kDa (TSPO) is a vital therapeutic target for pain treatment, and recent research implies that there may be allostery in TSPO. Our previous competitive binding assay hint that koumine may function as a TSPO positive allosteric modulator (PAM). Here, for the first time, we report the pharmacological characterization of koumine as a TSPO PAM. The results imply that koumine might be a high-affinity ligand of TSPO and that it likely acts as a PAM since it could delay the dissociation of 3H-PK11195 from TSPO. Importantly, the allostery was retained in vivo, as koumine augmented Ro5-4864-mediated analgesic and anti-inflammatory effects in several acute and chronic inflammatory and neuropathic pain models. Moreover, the positive allosteric modulatory effect of koumine on TSPO was further demonstrated in cell proliferation assays in T98G human glioblastoma cells. In summary, we have identified and characterized koumine as a TSPO PAM for the treatment of inflammatory and neuropathic pain. Our data lay a solid foundation for the use of the clinical candidate koumine to treat inflammatory and neuropathic pain, further demonstrate the allostery in TSPO, and provide the first proof of principle that TSPO PAM may be a novel avenue for the discovery of analgesics.

Koumine (KM), the most abundant alkaloid in Gelsemium elegans, has anti-neuropathic, anti-inflammatory, and analgesic activities; thus, it has the potential to be developed as a broad-spectrum analgesic drug. However, factors determining the relationship between analgesic efficacy and the corresponding plasma KM concentration are largely unclear. The pharmacokinetics and pharmacodynamics of KM and their optimization in the context of neuropathic pain have not been reported. We investigated the pharmacokinetics and pharmacodynamics of KM after oral administration in a streptozotocin-induced rat model of diabetic neuropathic pain (DNP) using a population approach. A first-order absorption and elimination pharmacokinetics model best described the plasma KM concentration. This pharmacokinetic model was then linked to a linear pharmacodynamic model with an effect compartment based on the measurement of the mechanical withdrawal threshold. KM was rapidly absorbed (time to maximum plasma concentration: 0.14–0.36 h) with similar values in both DNP and naïve rats, suggesting that DNP did not influence the KM absorption rate. However, the area under the curve (AUC0–∞) of KM in DNP rats was over 3-fold higher than that in naïve rats. The systemic clearance rate and volume of KM distribution were significantly lower in DNP rats than in naïve rats. Blood glucose value prior to KM treatment was a significant covariate for the systemic clearance rate of KM and baseline value of the threshold. Our results suggest that streptozotocin-induced hyperglycemia is an independent factor for decreased KM elimination and its anti-allodynic effects in a DNP rat model. To the best of our knowledge, this is the first study to investigate the role of DNP in the pharmacokinetics and pharmacokinetics-pharmacodynamics of KM in streptozotocin-induced diabetic rats.

Koumine is an alkaloid that displays notable activity against inflammatory and neuropathic pain, but its therapeutic target and molecular mechanism still need further study. Translocator protein 18 kDa (TSPO) is a vital therapeutic target for pain treatment, and recent research implies that there may be allostery in TSPO. Our previous competitive binding assay hint that koumine may function as a TSPO positive allosteric modulator (PAM). Here, for the first time, we report the pharmacological characterization of koumine as a TSPO PAM. The results imply that koumine might be a high-affinity ligand of TSPO and that it likely acts as a PAM since it could delay the dissociation of 3H-PK11195 from TSPO. Importantly, the allostery was retained in vivo , as koumine augmented Ro5-4864-mediated analgesic and anti-inflammatory effects in several acute and chronic inflammatory and neuropathic pain models. Moreover, the positive allosteric modulatory effect of koumine on TSPO was further demonstrated in cell proliferation assays in T98G human glioblastoma cells. In summary, we have identified and characterized koumine as a TSPO PAM for the treatment of inflammatory and neuropathic pain. Our data lay a solid foundation for the use of the clinical candidate koumine to treat inflammatory and neuropathic pain, further demonstrate the allostery in TSPO, and provide the first proof of principle that TSPO PAM may be a novel avenue for the discovery of analgesics. [ABSTRACT FROM AUTHOR]

We report here a concise, collective, and asymmetric total synthesis of sarpagine alkaloids and biogenetically related koumine alkaloids, which structurally feature a rigid cage scaffold, with L‐tryptophan as the starting material. Two key bridged skeleton‐forming reactions, namely tandem sequential oxidative cyclopropanol ring‐opening cyclization and ketone α‐allenylation, ensure concurrent assembly of the caged sarpagine scaffold and installation of requisite derivative handles. With a common caged intermediate as the branch point, by taking advantage of ketone and allene groups therein, total synthesis of five sarpagine alkaloids (affinisine, normacusine B, trinervine, Na‐methyl‐16‐epipericyclivine, and vellosimine) with various substituents and three koumine alkaloids (koumine, koumimine, and N‐demethylkoumine) with more complex cage scaffolds has been accomplished. [ABSTRACT FROM AUTHOR]

Koumine (KM), one of the primary constituents of Gelsemium elegans, has been used for the treatment of inflammatory diseases such as rheumatoid arthritis, but whether KM impacts the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome remains unknown. This study aimed to explore the inhibitory effect of KM on NLRP3 inflammasome activation and the underlying mechanisms both in vitro using macrophages stimulated with LPS plus ATP, nigericin or monosodium urate (MSU) crystals and in vivo using an MSU-induced peritonitis model. We found that KM dose-dependently inhibited IL-1β secretion in macrophages after NLRP3 inflammasome activators stimulation. Furthermore, KM treatment efficiently attenuated the infiltration of neutrophils and suppressed IL-1β production in mice with MSU-induced peritonitis. These results indicated that KM inhibited NLRP3 inflammasome activation, and consistent with this finding, KM effectively inhibited caspase-1 activation, mature IL-1β secretion, NLRP3 formation and pro-IL-1β expression in LPS-primed macrophages treated with ATP, nigericin or MSU. The mechanistic study showed that, KM exerted a potent inhibitory effect on the NLRP3 priming step, which decreased the phosphorylation of IκBα and p65, the nuclear localization of p65, and the secretion of TNF-α and IL-6. Moreover, the assembly of NLRP3 was also interrupted by KM. KM blocked apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and its oligomerization and hampered the NLRP3-ASC interaction. This suppression was attributed to the ability of KM to inhibit the production of reactive oxygen species (ROS). In support of this finding, the inhibitory effect of KM on ROS production was completely counteracted by H2O2, an ROS promoter. Our results provide the first indication that KM exerts an inhibitory effect on NLRP3 inflammasome activation associated with blocking the ROS/NF-κB/NLRP3 signal axis. KM might have potential clinical application in the treatment of NLRP3 inflammasome-related diseases.

In this study, a combination of network pharmacology, bioinformatics analysis, molecular docking and transcriptomics was used to investigate the active ingredient and potential target of Gelsemium elegans in the treatment of colorectal cancer. Koumine was screened as the active component by targeting PDK1 through network pharmacology and reverse docking. RNA-Seq, enrichment analysis and validation experiment were then further employed to reveal koumine might function in inhibiting Akt/mTOR/HK2 pathway to regulate cell glycolysis and detachment of HK2 from mitochondria and VDAC-1 to activate cell apoptosis both in vitro and in vivo. In the present study, we provide a systematical approach for the identification of effective ingredient and potential target of herbal medicine. Our results have important implication for the intensive study of koumine as novel anticancer agents for colorectal cancer and could be supportive in its further structural modification.

Aging leads to changes in nearly all pharmacokinetic phases. Koumine (KM), an alkaloid derived from Gelsemium elegans Benth., is effective against age-associated chronic diseases, but its dose proportionality following oral administration in aged individuals remains unknown. Herein, we established and validated a simple method that requires low sample volumes to determine KM concentration in rats using ultra-performance liquid chromatography-tandem mass spectrometry. The maximum plasma concentration (Cmax) of 7 mg·kg−1 KM was ~12-fold and ~24-fold higher than that of 0.28 mg·kg−1 KM in adult and aged rats, respectively (P < 0.01). Time to reach Cmax (Tmax) for 7 mg·kg−1 KM was 4-fold longer in aged rats (P < 0.05). The area under the curve (AUC) of 7 mg·kg−1 KM was >17-fold and >43-fold higher than those of 0.28 mg·kg−1 KM in adult and aged rats, respectively (P < 0.01). The half-life (t1/2) of 7 mg·kg−1 KM was over 4-fold longer than that of 0.28 mg·kg−1 KM in adult rats (P < 0.01). The t1/2 of 1.4 and 7 mg·kg−1 KM were 1.5~2-fold longer, than that of 0.28 mg·kg−1 KM in aged rats (P < 0.05). The clearance rate of 7 mg·kg−1 KM was significantly lower in aged than in adult rats (P < 0.05). For 7.0 mg·kg−1 KM, the Cmax in aged rats was higher than in adult rats during the Tmax period (P < 0.05). In aged rats, the AUC for KM was >2.5-fold higher (P < 0.05) and the t1/2 was >60% longer than in adult rats (P < 0.05). These results help interpret the pharmacokinetics of KM in aging-associated diseases.

In this study, a combination of network pharmacology, bioinformatics analysis, molecular docking and transcriptomics was used to investigate the active ingredient and potential target of Gelsemium elegans in the treatment of colorectal cancer. Koumine was screened as the active component by targeting PDK1 through network pharmacology and reverse docking. RNA-Seq, enrichment analysis and validation experiment were then further employed to reveal koumine might function in inhibiting Akt/mTOR/HK2 pathway to regulate cell glycolysis and detachment of HK2 from mitochondria and VDAC-1 to activate cell apoptosis both in vitro and in vivo. In the present study, we provide a systematical approach for the identification of effective ingredient and potential target of herbal medicine. Our results have important implication for the intensive study of koumine as novel anticancer agents for colorectal cancer and could be supportive in its further structural modification. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a suitable drug-in-adhesive patch for transdermal delivery of koumine. Acrylic polymer Duro-Tak® 87-4287, which contains hydroxyl groups, may significantly enhance the skin permeation of koumine from transdermal patches containing 0.93–3.72% koumine. Among permeation enhancers, 10% azone showed the greatest potential and increased the flux of koumine to 1.48-fold that of the control. Therefore, an optimized patch formulation containing 3.72% koumine and 10% azone in Duro-Tak® 87-4287 that offers good physical properties was selected for an in vivo pharmacokinetic study using rats. The maximal plasma drug concentration (Cmax) of koumine after transdermal administration (4 mg/patch) was 25.80 ± 1.51 ng/mL, which was in the range of those after oral administration (3 mg/kg and 15 mg/kg). The time to the maximal concentration (Tmax) and the half-life (t1/2) of the drug with transdermal administration were 3.96 ± 0.46 h and 21.10 ± 1.36 h, respectively, which were longer than those with oral administration. Furthermore, the area under the concentration-time curve (AUC0–72 h) of 898.20 ± 45.57 ng·h/mL for the transdermal patch was much higher than that for oral administration (15 mg/kg). In conclusion, the drug-in-adhesive patch containing koumine provides a steady plasma koumine level and sustained release in vivo and can be an effective means of transdermal delivery for koumine. [ABSTRACT FROM AUTHOR]

Aging leads to changes in nearly all pharmacokinetic phases. Koumine (KM), an alkaloid derived from Gelsemium elegans Benth., is effective against age-associated chronic diseases, but its dose proportionality following oral administration in aged individuals remains unknown. Herein, we established and validated a simple method that requires low sample volumes to determine KM concentration in rats using ultra-performance liquid chromatography-tandem mass spectrometry. The maximum plasma concentration (Cmax) of 7 mg·kg−1 KM was ~12-fold and ~24-fold higher than that of 0.28 mg·kg−1 KM in adult and aged rats, respectively (P < 0.01). Time to reach Cmax (Tmax) for 7 mg·kg−1 KM was 4-fold longer in aged rats (P < 0.05). The area under the curve (AUC) of 7 mg·kg−1 KM was >17-fold and >43-fold higher than those of 0.28 mg·kg−1 KM in adult and aged rats, respectively (P < 0.01). The half-life (t1/2) of 7 mg·kg−1 KM was over 4-fold longer than that of 0.28 mg·kg−1 KM in adult rats (P < 0.01). The t1/2 of 1.4 and 7 mg·kg−1 KM were 1.5~2-fold longer, than that of 0.28 mg·kg−1 KM in aged rats (P < 0.05). The clearance rate of 7 mg·kg−1 KM was significantly lower in aged than in adult rats (P < 0.05). For 7.0 mg·kg−1 KM, the Cmax in aged rats was higher than in adult rats during the Tmax period (P < 0.05). In aged rats, the AUC for KM was >2.5-fold higher (P < 0.05) and the t1/2 was >60% longer than in adult rats (P < 0.05). These results help interpret the pharmacokinetics of KM in aging-associated diseases. [ABSTRACT FROM AUTHOR]

Gelsemium elegans Benth. (G. elegans), a traditional Chinese medicine, has great potential as an effective growth promoter in animals, however, the mechanism of its actin remains unclear. Here, we evaluated the protective effects of koumine extract from G. elegans against lipopolysaccharide (LPS)-induced intestinal barrier dysfunction in IPEC-J2 cells through alleviation of inflammation and oxidative stress. MTT and LDH assays revealed that koumine significantly reduced LPS cytotoxicity. Transepithelial electrical resistance (TEER) and cell monolayer permeability assays showed that koumine treatment attenuated the LPS-induced intestinal barrier dysfunction with no particularly different effects in tight junction proteins such as ZO-1, claudin-1, and occludin. LPS-triggered inflammatory response was also suppressed by koumine, as evidenced by the downregulated inflammatory factors, including TNF- α , IL-6, IL-1 β , NO, iNOS, and COX-2, which was closely connected with the inhibition of NF- κ B pathway for the decrease of phosphorylation of I κ B α and NF- κ B and nuclear translocation of p-p65. Amount of reactive oxygen species (ROS) and MDA induced by LPS was also reduced by koumine through activation of Nrf2 pathway, and increased in the levels of Nrf2 and HO-1 degradation of keap-1 to promote anti-oxidants, including superoxide dismutase (SOD) and catalase (CAT). To summarize, koumine-reduced the oxidative stress and inflammatory reaction triggered by LPS through regulation of the Nrf2/NF- κ B signaling pathway and preventing intestinal barrier dysfunction. [ABSTRACT FROM AUTHOR]

Koumine (KM) has anxiolytic, anti-inflammatory and growth-promoting effects in pigs and sheep. Based on the growth-promoting and immunological effects of koumine, the present study was conducted on Cyprinus carpio (C. carpio) with four KM concentrations: 0 mg/kg, 0.2 mg/kg, 2 mg/kg, and 20 mg/kg for 10 weeks, followed by a 1-week Aeromonas hydrophila (A. hydrophila) infection experiment. The effect of KM on the immunity of A. hydrophila infected carp was analyzed by histopathology, biochemical assay, and qRT-PCR to assess the feasibility of KM in aquaculture. The results showed that the presence of KM alleviated pathogen damage to carp tissues. At 2 mg/kg and 20 mg/kg concentrations of KM successively and significantly elevated (p < 0.05) the SOD activities in the intestinal tract, hepatopancreas and kidney of carp. The expression levels of hepatopancreatic antioxidant genes Nrf2 and IGF-1 were significantly up-regulated in the same group (p < 0.05), while the expression levels of immune genes IL-8 and IL-10 were down-regulated. In summary, KM at concentrations of 2 mg/kg and 20 mg/kg could regulate the expression of antioxidant and immune genes in various tissues in an orderly and rapid manner, and significantly improve the antioxidant and immune abilities of carp, which is conducive to the improvement of the resilience of carp. • The results showed that the presence of KM alleviated pathogen damage to carp tissues. • Medium and high concentrations of KM significantly increased the SOD activity in carp tissues (p < 0.05). • Medium and high concentrations of KM could regulate the expression of antioxidant and immune genes in various tissues. [ABSTRACT FROM AUTHOR]

Aims: Five series of novel koumine-like compounds were designed, semi-synthesized and systematically evaluated for antitumor activities. Methods: All compounds were evaluated for antiproliferative activity against four human cancer cell lines, including HT-29, HCT-116, HCT-15 and Caco-2. Results: Most compounds exhibited much higher antiproliferation activities (IC 50  <10 μM) than koumine. Two selected compounds A4 and C5 showed comparable antitumor effects to 5-FU in vivo , as well as better safety profiles. Further studies suggested that A4 and C5 could arrest HT-29 cell cycle in G2 phase and raise reactive oxygen species level, thus inducing cell apoptosis related to Erk MAPK and NF-κB signaling pathways inhibition. Conclusion: These results will greatly promote the druggability study of these koumine-like compounds.

Osteoporosis (OP) is a bone remodeling disease characterized by an imbalance between bone resorption and formation. Osteoclasts are the primary therapeutic targets for treating bone destruction. Koumine (KM), the most bioactive component in Gelsemium alkaloids, exhibits antitumor, immunosuppressive, anti-inflammatory, and analgesic properties. However, the effects of bone loss have not been well studied. This study conducted in vitro and in vivo verification experiments on KM. The results showed that KM inhibited bone resorption and tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts development by mature osteoclasts in a dose-dependent manner. Moreover, KM prevented OVX-induced OP in vivo and potentially inhibited ubiquitination, a process closely related to various biological activities, including protein interaction, transcription, and transmembrane signal transduction regulation, especially within the nuclear factor-κB (NF-κB) pathway. Previous studies have demonstrated that several proteins ubiquitination promotes osteoclastogenesis, our study indicated that KM inhibits early NF-κB activation and receptor activator of NF-κB ligand induced ubiquitination, a critical factor in osteoclast differentiation. In conclusion, our research suggests that KM holds potential as an effective therapeutic agent for OP., (© 2023 Wiley Periodicals LLC.)

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. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract 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 GABA A 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/GABA A receptor pathway. [ABSTRACT FROM AUTHOR]

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Gelsemium elegans Benth. is a toxic plant that has been used as an ancient Chinese herbal remedy for rheumatoid arthritis (RA) and nervous pain, spasticity, skin ulcers, and cancers. Koumine, one of its representative alkaloids, shows numerous promising pharmacological activities, including anti-inflammatory and analgesic activities. Here, we investigated the analgesic effect of koumine on the collagen-induced arthritis (CIA) rat model of RA and explored the potential pharmacological mechanisms underlying the analgesia. In the CIA rats, repeated koumine treatments significantly reduced pain compared to controls and attenuated the collagen-induced increase in levels of glial fibrillary acidic protein (GFAP) and the pro-inflammatory cytokines tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Cultured astrocytes showed reduced astrocyte reactivation and decreased production of both tested cytokines. Based on our results, koumine exerted both analgesic and anti-inflammatory effects on the CIA rat model that were apparently mediated by inhibiting astrocyte reactivation and pro-inflammatory cytokine production. [ABSTRACT FROM AUTHOR]

Gelsemium elegans (Gardner & Chapm.) Benth (G. elegans) has been widely used as a traditional folk medicine in China and Southeast Asia. As the most abundant alkaloid in G. elegans , Koumine (KM) has been revealed the effect of inflammatory attenuation modulating by macrophage activation and polarization. This study aimed to explore the effect of KM on modulation of microglia polarization that led to the suppression of neuroinflammation and further improved neurodegenerative behavior. Inflammatory mediators, microglia M1 and M2 phenotype markers and Nrf2/HO-1 pathway related protein were assessed in LPS-induced BV2 cells and LPS-treated mice by RT-PCR, immunohistochemistry, immunofluorescence and Western blotting. Moreover, the learning and memory abilities of mice were evaluated by Morris water maze test, and the neuronal damage was evaluated by the Nissl staining. KM attenuated LPS-induced viability and morphological changes in BV2 microglial cells. Our findings showed that KM activated the Nrf2/HO-1 signaling pathway to promote phenotypic switch from M1 to M2 phenotypes. This switch suppresses the release of inflammatory mediators in LPS-induced BV2 cells. Meanwhile, KM attenuated neuroinflammation through modulating microglia polarization and subsequently reversed the behavioral alterations in LPS-induced mice model of neuroinflammation. KM may alleviate neuroinflammation by regulating microglia polarization with the involvement of Nrf2/HO-1 pathway, resulting of the neuroprotective effect. [Display omitted] [ABSTRACT FROM AUTHOR]

Koumine, an indole alkaloid, is a major bioactive component of Gelsemium elegans. Previous studies have demonstrated that koumine has noticeable anti-inflammatory and analgesic effects in inflammatory and neuropathic pain (NP) models, but the mechanisms involved are not well understood. This study was designed to explore the analgesic effect of koumine on chronic constriction injury (CCI)-induced NP in rats and the underlying mechanisms, including astrocyte autophagy and apoptosis in the spinal cord. Rats with CCI-induced NP were used to evaluate the analgesic and anti-inflammatory effects of koumine. Lipopolysaccharide (LPS)-induced inflammation in rat primary astrocytes was also used to evaluate the anti-inflammatory effect of koumine. We found that repeated treatment with koumine significantly reduced and inhibited CCI-evoked astrocyte activation as well as the levels of pro-inflammatory cytokines. Meanwhile, we found that koumine promoted autophagy in the spinal cord of CCI rats, as reflected by decreases in the LC3-II/I ratio and P62 expression. Double immunofluorescence staining showed a high level of colocalization between LC3 and GFAP-positive glia cells, which could be decreased by koumine. Intrathecal injection of an autophagy inhibitor (chloroquine) reversed the analgesic effect of koumine, as well as the inhibitory effect of koumine on astrocyte activation in the spinal cord. In addition, TUNEL staining suggested that CCI-induced apoptosis was inhibited by koumine, and this inhibition could be abolished by chloroquine. Western blot analysis revealed that koumine significantly increased the level of Bcl-xl while inhibiting Bax expression and decreasing cleaved caspase-3. In addition, we found that koumine could decrease astrocyte-mediated neuroinflammation and enhance autophagy in primary cultured astrocytes. These results suggest that the analgesic effects of koumine on CCI-induced NP may involve inhibition of astrocyte activation and pro-inflammatory cytokine release, which may relate to the promotion of astrocyte autophagy and the inhibition for apoptosis in the spinal cord.

Koumine, an indole alkaloid, is a major bioactive component of Gelsemium elegans. Previous studies have demonstrated that koumine has noticeable anti-inflammatory and analgesic effects in inflammatory and neuropathic pain (NP) models, but the mechanisms involved are not well understood. This study was designed to explore the analgesic effect of koumine on chronic constriction injury (CCI)-induced NP in rats and the underlying mechanisms, including astrocyte autophagy and apoptosis in the spinal cord. Rats with CCI-induced NP were used to evaluate the analgesic and anti-inflammatory effects of koumine. Lipopolysaccharide (LPS)-induced inflammation in rat primary astrocytes was also used to evaluate the anti-inflammatory effect of koumine. We found that repeated treatment with koumine significantly reduced and inhibited CCI-evoked astrocyte activation as well as the levels of pro-inflammatory cytokines. Meanwhile, we found that koumine promoted autophagy in the spinal cord of CCI rats, as reflected by decreases in the LC3-II/I ratio and P62 expression. Double immunofluorescence staining showed a high level of colocalization between LC3 and GFAP-positive glia cells, which could be decreased by koumine. Intrathecal injection of an autophagy inhibitor (chloroquine) reversed the analgesic effect of koumine, as well as the inhibitory effect of koumine on astrocyte activation in the spinal cord. In addition, TUNEL staining suggested that CCI-induced apoptosis was inhibited by koumine, and this inhibition could be abolished by chloroquine. Western blot analysis revealed that koumine significantly increased the level of Bcl-xl while inhibiting Bax expression and decreasing cleaved caspase-3. In addition, we found that koumine could decrease astrocyte-mediated neuroinflammation and enhance autophagy in primary cultured astrocytes. These results suggest that the analgesic effects of koumine on CCI-induced NP may involve inhibition of astrocyte activation and pro-inflammatory cytokine release, which may relate to the promotion of astrocyte autophagy and the inhibition for apoptosis in the spinal cord. [ABSTRACT FROM AUTHOR]

Gelsemium elegans Benth. (G. elegans), which is a famous Chinese folk medicine, has been commonly used to treat certain types of skin ulcers and alleviate inflammation, headaches, and cancer pain. However, the extensive clinical use of G. elegans has been greatly hampered by its toxicity. As one of the most widely used herbal medicines, Glycyrrhiza uralensis Fisch, has a unique effect on detoxification of G. elegans. In the present study, a rapid and sensitive method using ultra-liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was established and validated for determination of koumine, the most abundant molecule among the alkaloids of G. elegans, in rat plasma, tissue, and liver microsome. The developed method was successfully applied to the pharmacokinetics, tissue distribution, and in vitro metabolism study in rat with or without pre-treated Glycyrrhiza uralensis Fisch extract. Meanwhile, the expression level of CYP3A1 mRNA was analyzed to explain the detoxification mechanism of Glycyrrhiza uralensis Fisch on G. elegans. As a result, our work demonstrated that Glycyrrhiza uralensis Fisch could significantly affect the pharmacokinetics and tissue distribution of koumine in rats. The detoxification mechanism of Glycyrrhiza uralensis Fisch on G. elegans may be its cytochrome enzyme up-regulation effect. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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 (r 2  > 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. [ABSTRACT FROM AUTHOR]

The imbalance between M1-and M2-polarized macrophages is one of the major pathophysiological changes in RA. Therefore, targeted macrophage polarization may be an effective therapy for RA. Koumine, an alkaloid monomer with the highest content and low toxicity in Gelsemium elegans Benth. , has the effect of treating RA by playing an immunomodulatory role by influencing various immune cells. However, whether koumine affects macrophage polarization in RA and the associated molecular mechanisms remain unknown. To investigate the mechanism of the anti-RA effect of koumine on macrophage polarization. The effect of koumine on macrophage polarization was investigated in vivo and in vitro. We first explored the effects of koumine on AIA rats and detected the levels of M1/M2 macrophage polarization markers in the spleen by western blotting. Then, we explored the regulatory effect of koumine on M1/M2 macrophage polarization and the effect on the PI3K/AKT signaling pathway in vitro. Finally, we verified the effects of koumine on macrophage polarization in CIA mice. We found that koumine alleviated symptoms, including relieving pain, reducing joint redness and swelling in AIA rats and restoring the M1/M2 macrophage balance in vivo. Interestingly, koumine had an inhibitory effect on both M1 and M2 macrophage polarization in vitro , but it had a stronger inhibitory effect on M1 macrophage. In a mixed polarization experiment, koumine mainly inhibited M1 macrophage polarization and had an inhibitory effect on the PI3K/AKT signaling pathway. Finally, we found that koumine had therapeutic effects on CIA mice, regulated macrophage polarization and inhibited the PI3K/AKT signaling pathway. Our results reveal that koumine regulates macrophage polarization through the PI3K/AKT signaling pathway. This may be one of the important mechanisms of its anti-RA effect, which provides a theoretical and scientific basis for the possible clinical application of koumine. [Display omitted] • This study provides insight into the effect of koumine on macrophages. • Koumine may have a stronger inhibitory effect on M1 macrophage polarization than M2. • Koumine can alleviate RA by regulating macrophage polarization via PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

In the past decades, hepatocellular carcinoma (HCC) has been receiving increased attention due to rising morbidity and mortality in both developing and developed countries. Koumine, one of the significant alkaloidal constituents of Gelsemium elegans Benth., has been regarded as a promising anti-inflammation, anxiolytic, and analgesic agent, as well as an anti-tumor agent. In the present study, we attempted to provide a novel mechanism by which koumine suppresses HCC cell proliferation. We demonstrated that koumine might suppress the proliferation of HCC cells and promote apoptosis in HCC cells dose-dependently. Under koumine treatment, the mitochondria membrane potential was significantly decreased while reactive oxygen species (ROS) production was increased in HCC cells; in the meantime, the phosphorylation of ERK, p38, p65, and IκBα could all be inhibited by koumine treatment dose-dependently. More importantly, the effects of koumine upon mitochondria membrane potential, ROS production, and the phosphorylation of ERK, p38, p65, and IκBα could be significantly reversed by ROS inhibitor, indicating that koumine affects HCC cell fate and ERK/p38 MAPK and NF-κB signaling activity through producing excess ROS. In conclusion, koumine could inhibit the proliferation of HCC cells and promote apoptosis in HCC cells; NF-κB and ERK/p38 MAPK pathways could contribute to koumine functions in a ROS-dependent manner.

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.

1. Gelsemium elegans Benth (Loganiaceae)is a toxic plant that can be used for committing suicide besides alleviating pains. Its anti-inflammatory and analgesic effect mainly come from its active ingredient, namely koumine. Koumine, an indole alkaloid, possesses widely pharmacological effects especially inhibition of neuropathic pain. 2. This study aimed to investigate the metabolic profile of koumine using human liver microsomes (HLMs), selective chemical inhibitors and recombinant human CYP isoforms. Ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) was used to detect and identify metabolites. 3. Four major metabolites of koumine were found after incubation with HLMs or individual CYP isoforms. The metabolic pathways of koumine included demethylation, dehydrogenation, oxidation and demethyl-dehydrogenation. Chemical inhibition study showed that the inhibitor of CYP3A4/3A5 significantly decreased (93%) the formation of koumine metabolites. Further, CYP3A4/3A5 was shown as the most efficient isoform in biotransformation of koumine, among a series of CYP isoforms tested. 4. In conclusion, koumine was metabolized into four oxidative metabolites in HLMs. And CYP3A4/3A5 was probably the main contributor to the hepatic oxidative metabolism of koumine. [ABSTRACT FROM AUTHOR]

Koumine, an active alkaloid of neurotoxic plant Gelsemium, has been focused on its therapeutic uses, especially in central nervous system. Nevertheless, less is known about the neurological effects of koumine, which hampers its potential therapeutic exploitation. Moreover, as the anxiolytic potential of Gelsemium has raised many critical issues, its active principles on the anxiolytic and other neurological effects need to be further investigated. Here, we used functional observation battery (FOB) of mice to systematically measure the neurological effects of koumine at the effective doses, and then further confirmed its anxiolytic properties in open-field test (OFT) of mice and Vogel conflict test (VCT) of rats. Koumine exhibited anxiolytic-like activities but did not affect other autonomic, neurological and physical functions in FOB. Furthermore, koumine released anxiolytic responses and anti-punishment action in a manner similar to diazepam in OFT and VCT, respectively. The results constitutes solid set of fundamental data further demonstrating anxiolytic properties of koumine at the therapeutic doses without inducing adverse neurological effects, which supports the perspectives for the development of safe and effective koumine medicine against pathological anxiety. [ABSTRACT FROM AUTHOR]

Koumine, an indole alkaloid extracted from Gelsemium elegans Benth , exerts anti-inflammation and antioxidant activities. However, the effects of koumine on intestinal injury induced by H 2 O 2 and its potential molecular mechanisms need larger studies. Aim of the study: We established an IPEC-J2 cell damage model induced by H 2 O 2 to explore the protective mechanism of koumine on intestinal injury. In the experiment, cell damage models were made with hydrogen peroxide. To assess the protective effect of koumine on H 2 O 2 -induced IPEC-J2 cell injury, CCK-8, the release of LDH and ROS, transmission electron microscopy and Annexin V-FITC/PI were employed. Western Blot and Quantitative Real-time PCR were used to determine the potential alleviated mechanism of koumine on H 2 O 2 -trigged IPEC-J2 cell damage. The results of CCK-8 and LDH implied that koumine has a mitigative effect on H 2 O 2 -induced cell damage via upregulating cell viability and suppressing cell membrane fragmentation. Simultaneously, koumine notably inhibited the level of pro-inflammatory factors (IL-1β, IL-6, IL-8, TNF-α and TGF-β), the over-production of ROS along with decreasing the injury of mitochondrion, endoplasmic reticulum and lysosome induced by H 2 O 2. Moreover, koumine dramatically attenuated H 2 O 2 -triggered IPEC-J2 cell apoptosis and autophagy. Subsequently, Western blot analysis identified NF-ΚB, PI3K and ERS as possible pathway responsible for the protective effect of koumine on H 2 O 2 -stimulated IPEC-J2 cell inflammation. This in vitro experimental study suggests that koumine suppresses the H 2 O 2 -induced activation of inflammatory pathways, oxidative injury, ER stress, apoptosis and autophagy, which provide a rationale for therapeutically use in major intestinal diseases. [Display omitted] • Koumine alleviates H 2 O 2 -induced intestinal damage by attenuating inflammation, apoptosis and autophagy in vitro. • Koumine protects IPEC-J2 cells from inflammation caused by H 2 O 2 through inhibiting PI3K/AKT/mTOR/NF-κB pathway. • Inhibition of ER stress can relieve autophagy and apoptosis of IPEC-J2 cells induced by oxidative stress. [ABSTRACT FROM AUTHOR]

Koumine is an alkaloid with significant anti-anxiety, anticancer cell proliferation, and analgesic activities, and our previous studies have shown that koumine can be used as an immunostimulant in aquaculture, but the molecular mechanism of its effect remains unclear. We fed a basal diet with 0, 0.2, 2, and 20 mg/kg koumine to C. carpio for 10 weeks, and comprehensive studies of the histological and biochemical parameters and transcriptomes of the four groups were performed. Histological results indicated that the number of apoptotic cells in the liver increased with increasing koumine concentration. Compared with those of the control group, the malondialdehyde, superoxide dismutase, catalase, acid phosphatase, alkaline phosphatase, and lactate dehydrogenase levels of the treatment group increased to varying degrees. In total, 100.11 GB of clean data, 4774 DEGs, and 138 differentially expressed genes were obtained from the transcriptome data. Differentially expressed genes were classified into 187 signalling pathways, and the circadian rhythm signalling pathway, the JAK-STAT signalling pathway, the p53 signalling pathway and the PPAR signalling pathway were the top enriched pathways. The qRT-PCR results confirmed that the key genes ifnar1 , socs3l , epoa , ghra , cMyc , mcl-1 , shisa4 , and gtse1 involved in balancing cell proliferation and apoptosis were enriched in these pathways. We discovered that the JAK-STAT and p53 pathways are important targets of koumine. Such information contributes to a better understanding of the potential mechanism by which koumine regulates hepatic immunity as well as to lays the theoretical foundation for its application. [Display omitted] • Dietary koumine elevated the level of apoptosis in Cyprinus carpio liver cells. • Dietary koumine can effectively activate the antioxidant protective and the drug metabolic of the Cyprinus carpio liver. • The JAK-STAT and p53 pathways play an important role in the regulation of liver apoptosis by koumine subunits. [ABSTRACT FROM AUTHOR]