Oral acute toxicity study and in vivo antimalarial activity of Strychnos lucida R. Br. tablet.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria eradication has been a major goal of the Indonesian government since 2020. Medicinal plants, such as Strychnos lucida R. Br., are empirically used to treat malaria through traditional preparation methods. However, the safety and efficacy of these plants have not yet been confirmed. Therefore, further investigations are necessary to confirm the safety and efficacy of S. lucida as an antimalarial agent. AIMS OF THE STUDY: To quantify the concentration of brucine in the S. lucida extract, determine the acute oral toxicity of the standardized extract, and evaluate the in vivo antimalarial potency of S. lucida tablet (SLT). MATERIALS AND METHODS: Acute oral toxicity of S.lucida extract was determined using the Organization for Economic Co-operation and Development 420 procedure, and the analytical method for brucine quantification was validated using high-performance liquid chromatography. In addition, antimalarial activity was determined using the Peter's four-day suppressive method. RESULTS: Acute toxicity analysis revealed S. lucida as a low-toxicity compound with a cut-off median lethal dose of 2000-5000 mg/kg body weight [BW], which was supported by the hematological and biochemical profiles of the kidneys, liver, and pancreas (p > 0.05). Extract standardization revealed that S. lucida contained 3.91 ± 0.074% w/w brucine, adhering to the limit specified in the Indonesian Herbal Pharmacopeia. Antimalarial test revealed that SLT inhibited the growth of Plasmodium berghei by 27.74-45.27%. Moreover, SLT improved the hemoglobin and hematocrit levels. White blood cell and lymphocyte counts were lower in the SLT-treated group than in the K (+) group (p < 0.05). CONCLUSION: Histopathological and biochemical evaluations revealed that S. lucida extract was safe at a dose of 2000 mg/kg BW with low toxicity. SLT inhibited Plasmodium growth and improved the hemoglobin, hematocrit, and red blood cell profiles. Additionally, SLT reduced the lymphocyte and WBC counts and increased the monocyte and thrombocyte counts as part of the immune system response against Plasmodium infection.

A Combination of In Silico ADMET Prediction, In Vivo Toxicity Evaluation, and Potential Mechanism Exploration of Brucine and Brucine N-oxide-A Comparative Study.

Brucine (BRU) and brucine N-oxide (BNO) are prominent, bioactive, and toxic alkaloids in crude and processed Semen Strychni. Studies have demonstrated that BRU and BNO possess comprehensive pharmacological activities, such as anti-inflammatory and analgesic. In this context, a comparative study of BRU and BNO was performed by combination analysis of in silico ADMET prediction, in vivo toxicity evaluation, and potential action mechanism exploration. ADMET prediction showed that BRU and BNO might induce liver injury, and BRU may have a stronger hepatoxic effect. The prediction was experimentally verified using the zebrafish model. The BRU-induced hepatotoxicity of zebrafish larvae had a dose-response relationship. The mechanism of BRU-induced hepatotoxicity might relate to phosphorylation, kinase activity, and signal transduction. By comparison, signal transduction and gap junctions might involve BNO-induced hepatotoxicity. Our results provided a better understanding of BRU- and BNO-induced hepatotoxicity. We also built a foundation to elucidate the material base of the hepatotoxicity of traditional Chinese medicine Semen Strychni.

Network toxicology and molecular docking analyses on strychnine indicate CHRM1 is a potential neurotoxic target.

BACKGROUND: Improper use of strychnine can cause death. The aim of this study was to identify and evaluate toxic mechanisms of action associated with active compounds in strychnine using a network toxicology approach, and explore potential pathogenic targets. METHODS: In the present study, strychnine target and central nervous system-related gene set were established using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and four disease gene databases (Genecards, OMIM, PharmGkb, TTD). An "ingredient-target" interactive active network map was constructed using Cytoscape software (version 3.8.0). Functional enrichment analysis was performed based on the hub genes. A protein-protein interaction network was constructed using STRING database. The pharmacokinetics (ADMET) properties of strychnine were evaluated using SwissADME tool. Molecular docking was performed using Autodock Vina to explore the interactions between the active compounds and the target protein. RESULTS: Five strychnine toxicity-related components and a gene set of 40 genes were obtained. GO and KEGG analyses showed that Strychnine acts on the central nervous system through G protein-coupled receptor signaling pathway. Analysis of "ADMET" related parameters showed a high gastrointestinal tract absorption of (S)-stylopine and isobrucine and the compounds could cross the blood brain barrier. CHRM1 was selected as a key gene in strychnine toxicity. Molecular docking results showed that the co-crystalized ligands did not form hydrogen bond with CHRM1. (S)-stylopine had the highest binding affinity (binding energy = - 8.5 kcal/mol) compared with the other two compounds. CONCLUSION: Network toxicology and molecular docking reveal the toxicity mechanisms of strychnine active compounds. The findings showed that CHRM1 is a potential neurotoxic target. (S)-stylopine showed stronger neurotoxic effect compared with the other ligands.

The Anticonvulsant Effect of Hydroethanolic Leaf Extract of Calotropis procera (Ait) R. Br. (Apocynaceae).

A number of currently used drugs have been obtained from medicinal plants which are a major source of drugs. These drugs are either used in their pure form or modified to a semisynthetic drug. Drug discovery through natural product research has been fruitful over the years. Traditionally, Calotropis procera is used extensively in the management of epilepsy. This study is conducted to explore the anticonvulsant effect of a hydroethanolic leaf extract of Calotropis procera (CPE) in murine models. This effect was evaluated using picrotoxin-induced convulsions, strychnine-induced convulsions, and isoniazid- and pilocarpine-induced status epilepticus in mice of both sexes. The results showed that CPE (100-300 mg/kg) exhibited an anticonvulsant effect against strychnine-induced clonic seizures by significantly reducing the duration (p = 0.0068) and frequency (p = 0.0016) of convulsions. The extract (100-300 mg/kg) caused a profound dose-dependent delay in the onset of clonic convulsions induced by picrotoxin (p < 0.0001) and tonic convulsions (p < 0.0001) in mice. The duration of convulsions was reduced significantly also for both clonic and tonic (p < 0.0001) seizures as well. CPE (100-300 mg/kg), showed a profound anticonvulsant effect and reduced mortality in the pilocarpine-induced convulsions. ED50 (~0.1007) determined demonstrated that the extract was less potent than diazepam in reducing the duration and onset of convulsions but had comparable efficacies. Flumazenil-a GABAA receptor antagonist-did not reverse the onset or duration of convulsions produced by the extract in the picrotoxin-induced seizure model. In isoniazid-induced seizure, CPE (300 mg kg1, p.o.) significantly (p < 0.001) delayed the onset of seizure in mice and prolonged latency to death in animals. Overall, the hydroethanolic leaf extract of Calotropis procera possesses anticonvulsant properties.

Chronotoxicity of Semen Strychni is associated with circadian metabolism and transport in mice.

OBJECTIVES: We aimed to determine the circadian responses of mice to Semen Strychni and to investigate the role of pharmacokinetics in generating chronotoxicity. METHODS: Total extract of Semen Strychni was administered by oral gavage to wild-type (WT) and Bmal1-/- (a circadian clock-deficient model) mice at different circadian time points for toxicity (including survival) and pharmacokinetic characterization. Nephrotoxicity and neurotoxicity were evaluated by measuring plasma creatinine and creatine kinase BB (CK-BB), respectively. Drug metabolism and transport assays were performed using liver/intestine microsomes and everted gut sacs, respectively. KEY FINDINGS: Semen Strychni nephrotoxicity and neurotoxicity as well as animal survival displayed significant circadian rhythms (the highest level of toxicity was observed at ZT18 and the lowest level at ZT2 to ZT6). According to pharmacokinetic experiments, herb dosing at ZT18 generated higher plasma concentrations (and systemic exposure) of strychnine and brucine (two toxic constituents) compared with ZT6 dosing. This was accompanied by reduced formation of both dihydroxystrychnine and strychnine glucuronide (two strychnine metabolites) at ZT18. Bmal1 ablation sensitized mice to Semen Strychni-induced toxicity (with increased levels of plasma creatinine and CK-BB) and abolished the time dependency of toxicity. Metabolism of Semen Strychni (strychnine and brucine) in the liver and intestine microsomes of WT mice was more extensive at ZT6 than at ZT18. These time differences in hepatic and intestinal metabolism were lost in Bmal1-/- mice. Additionally, the intestinal efflux transport of Semen Strychni (strychnine and brucine) was more extensive at ZT6 than ZT18 in WT mice. However, the time-varying transport difference was abolished in Bmal1-/- mice. CONCLUSIONS: Circadian responses of mice to Semen Strychni are associated with time-varying efflux transport and metabolism regulated by the circadian clock (Bmal1). Our findings may have implications for optimizing phytotherapy with Semen Strychni via timed delivery.

Anticonvulsant effects of the aqueous and methanol extracts from the stem bark of Psychotria camptopus Verdc. (Rubiacaea) in rats.

ETHNOPHARMACOLOGY RELEVANCE: The decoction from the stem bark of Psychotria camptopus (Rubiaceae) is used in the Cameroonian pharmacopoeia to treat neurological pathologies including epilepsy. AIM: The present work was undertaken to study the anticonvulsant properties of the aqueous (AE) and methanol (ME) extracts from the stem bark of P. camptopus in acute models of epileptic seizures in Wistar rats. METHOD: AE and ME were obtained by decoction and maceration of the stem bark powder in water and methanol, respectively. They were tested orally at the doses of 40, 80 and 120 mg/kg, on the latency of onset and duration of epileptic seizures induced by pentylene tetrazole (PTZ, 70 mg/kg, i.p.). The kinetic effect of both extracts at 120 mg/kg was evaluated. Their effects on diazepam (50 mg/kg) induced sleep and strychnine (STR, 2.5 mg/kg, i.p.) induced seizures were determined. ME was further tested on picrotoxin (PIC, 7.5 mg/kg, i.p.) and thiosemicarbazide (TSC, 50 mg/kg, i.p.) induced seizure models. The phytochemical composition of ME was assessed using LC-MS method, as well as its acute toxicity. RESULTS: AE and ME significantly (p < 0.001) reduced the duration of seizures in both PTZ and STR models. Their maximal effect was observed at 1 h after administration, though their effect at 120 mg/kg was maintained (p < 0.05) up to 24 h post-treatment. Both extracts significantly (p < 0.01) reduced sleep duration. ME significantly (p < 0.001) increased the latency of rat death on PIC-induced convulsions. In TSC rats, ME significantly (p < 0.001) delayed the latency to the first convulsion, and decreased the duration and frequency of convulsions. ME showed no acute toxicity while its phytochemical screening revealed the presence of two flavonoids (Rutin and Butin), two triterpenoid saponins (Psycotrianoside B and Bauerenone) and four alkaloids (10-Hydroxy-antirhine, 10-hydroxy-iso-deppeaninol, Emetine and Hodkinsine). In conclusion, AE and ME from the stem bark of P. camptopus have comparable anticonvulsant properties. The effect of ME is likely due to the presence of flavonoids and alkaloid and the activation of GABA pathway. These results further justify and support the use of P. camptopus in traditional medicine for the treatment of epilepsy.

Cyp3a11 metabolism-based chronotoxicity of brucine in mice.

Brucine is one of the main bioactive and toxic constituents of the herb drug Semen Strychni. Here we aimed to determine dosing time-dependent hepatotoxicity of brucine, and to investigate the role of metabolism in generation of brucine chronotoxicity. Brucine was administered to wild-type or Npas2-/- (a clock disrupted model) mice at different circadian time points for toxicity and pharmacokinetic characterization. The hepatotoxicity was evaluated by plasma alanine aminotransferase and aspartate aminotransferase measurements and histopathological analysis. The role of Cyp3a11 in brucine metabolism was determined by chemical inhibition assays and Cyp3a11-overexpressing HEK293 cells. Hepatic circadian Cyp3a11 mRNA and protein levels were determined by qPCR and Western blotting, respectively. The toxicity of brucine was more severe in the light phase [Zeitgeber time (ZT) 2 and ZT8] than in the dark phase (ZT14 and ZT20). Chemical inhibition and substrate metabolism assays suggested Cyp3a11 as a significant contributor to brucine metabolism. The Cyp3a11 mRNA, protein and activity in the livers of wild-type mice displayed significant circadian fluctuations. Npas2 ablation markedly down-regulated Cyp3a11 mRNA, protein and activity, and abrogated their circadian rhythms. The circadian time differences in brucine pharmacokinetics and liver distribution were lost in Npas2-/- mice, so were the time differences in brucine hepatotoxicity. In conclusion, chronotoxicity of brucine was determined by circadian variations in Cyp3a11 metabolism. The findings have implications in improving brucine (and possibly Semen Strychni) efficacy via dosing time optimization.

Neurochemical effects of motor cortex stimulation in the periaqueductal gray during neuropathic pain.

OBJECTIVE: Motor cortex stimulation (MCS) is a neurosurgical technique used to treat patients with refractory neuropathic pain syndromes. MCS activates the periaqueductal gray (PAG) matter, which is one of the major centers of the descending pain inhibitory system. However, the neurochemical mechanisms in the PAG that underlie the analgesic effect of MCS have not yet been described. The main goal of this study was to investigate the neurochemical mechanisms involved in the analgesic effect induced by MCS in neuropathic pain. Specifically, we investigated the release of γ-aminobutyric acid (GABA), glycine, and glutamate in the PAG and performed pharmacological antagonism experiments to validate of our findings. METHODS: Male Wistar rats with surgically induced chronic constriction of the sciatic nerve, along with sham-operated rats and naive rats, were implanted with both unilateral transdural electrodes in the motor cortex and a microdialysis guide cannula in the PAG and subjected to MCS. The MCS was delivered in single 15-minute sessions. Neurotransmitter release was evaluated in the PAG before, during, and after MCS. Quantification of the neurotransmitters GABA, glycine, and glutamate was performed using a high-performance liquid chromatography system. The mechanical nociceptive threshold was evaluated initially, on the 14th day following the surgery, and during the MCS. In another group of neuropathic rats, once the analgesic effect after MCS was confirmed by the mechanical nociceptive test, rats were microinjected with saline or a glycine antagonist (strychnine), a GABA antagonist (bicuculline), or a combination of glycine and GABA antagonists (strychnine+bicuculline) and reevaluated for the mechanical nociceptive threshold during MCS. RESULTS: MCS reversed the hyperalgesia induced by peripheral neuropathy in the rats with chronic sciatic nerve constriction and induced a significant increase in the glycine and GABA levels in the PAG in comparison with the naive and sham-treated rats. The glutamate levels remained stable under all conditions. The antagonism of glycine, GABA, and the combination of glycine and GABA reversed the MCS-induced analgesia. CONCLUSIONS: These results suggest that the neurotransmitters glycine and GABA released in the PAG may be involved in the analgesia induced by cortical stimulation in animals with neuropathic pain. Further investigation of the mechanisms involved in MCS-induced analgesia may contribute to clinical improvements for the treatment of persistent neuropathic pain syndromes.

High-Throughput Electrophysiology Screen Revealed Cardiotoxicity of Strychnine by Selectively Targeting hERG Channel.

Although the efficacy and the health care advantages of Chinese herbal medicine (CHM) have become increasingly recognized worldwide, the potential side effects and toxicity still restrict its broader application. This study established and applied an integrated platform anchored on automatic patch clamp system to screen and evaluate a collection of CHM extracts, compositions and monomeric compounds for in vitro cardiac toxicity. Of 1036 CHM samples screened, 2.79% significantly inhibited hERG channel activity. Among them, Strychnine was identified for the first time as a potent hERG inhibitor with an IC 50 of 6.65±1.04µ M in comparison to that of Dofetilide at 1.80±0.24µ M and Quinidine at 7.42±0.54µ M. Langendorff-perfusion experiments confirmed that strychnine increased QT interphase from 71.69±5.34 ms to 98.61±5.54 ms and decreased heart rates from 227.65±5.40 bmp to 162.91±14.70 bmp in isolated rat hearts. The cardiac toxicity effect of strychnine appears to be specific to hERG channel since an in vitro multiplex imaging analysis showed that it did not affect cellular phenotypes such as cell vitality, nucleus area, mitochondria mass and function, nor intracellular calcium in rat primary myocytes. This integrated high-throughput hERG patch clamp and high-content multi-parameter imaging cardiac toxicity screen approach should be useful for large-scale preclinical evaluation of complex Chinese herbal medicine.

Prophylactic Neuroprotection of Total Glucosides of Paeoniae Radix Alba against Semen Strychni-Induced Neurotoxicity in Rats: Suppressing Oxidative Stress and Reducing the Absorption of Toxic Components.

Strychnos alkaloids (SAs) are the main toxic constituents in Semen Strychni, a traditional Chinese medicine, which is known for its fatal neurotoxicity. Hence, the present study was carried out to evaluate the neurotoxicity induced by SAs and the pre-protective effects of the total glucosides of Paeoniae Radix Alba (TGP). An SA brain damage model was firstly established. The neurotoxicity induced by SAs and the pre-protective effects of TGP were confirmed by physical and behavioral testing, biochemical assay, and histological examination. Then, a liquid chromatography-tandem mass spectrometry method was developed and validated to investigate the time-course change and distribution of strychnine and brucine (two main SAs) in the brain after oral SA administration with or without TGP pretreatment. Biochemical analysis results indicated that TGP could ameliorate the oxidative stress status caused by SAs. Time-course change and distribution studies demonstrated that strychnine and brucine were rapidly absorbed into the brain, peaked early at 0.5 h, and were mainly located in the hippocampus and cerebellum. TGP showed a pre-protective effect against neurotoxicity by reducing the absorption of toxic alkaloids into the brain. These findings could provide beneficial information in facilitating future studies of Semen Strychni neurotoxicity and developing herbal medicines to alleviate neurotoxicity in the clinic.