This study aims to establish a rapid and sensitive UPLC-MS/MS method for simultaneously determining the content of strychnine and paeoniflorin in plasma and brain tissue of rats, and compare the pharmacokinetic behavior and brain tissue distribution of paeoniflorin combined with normal and toxic doses of strychnine in rats after percutaneous administration. Compared with those in the toxic-dose strychnine group, the AUC_(0-t), AUC_(0-∞), and C_(max) of strychnine decreased by 51.51%, 45.68%, and 46.03%, respectively(P<0.01), and the corresponding values of paeoniflorin increased by 91.41%, 102.31%, and 169.32%, respectively(P<0.01), in the compatibility group. Compared with the normal-dose strychnine group, the compatibility group showed insignificantly decreased C_(max), AUC_(0-t), and AUC_(0-∞) of strychnine, increased C_(max) and T_(max) of paeoniflorin(P<0.01), 66.88% increase in AUC_(0-t), and 70.55% increase in AUC_(0-∞) of paeoniflorin. In addition, the brain tissue concentration of strychnine decreased and that of paeoniflorin increased after compatibility. The combination of paeoniflorin with normal dose and toxic dose of strychnine can inhibit the percutaneous absorption of strychnine, and greatly promote the percutaneous penetration of paeoniflorin, whereas the interaction mechanism remains to be explored. The UPLC-MS/MS method established in this study is easy to operate and has good precision. It is suitable for in vivo study of pharmacokinetic behavior and brain tissue distribution of paeoniflorin and strychnine after percutaneous administration in rats, which provides reference for the safe and rational clinical use of strychnine and the combined use of drugs, and lays a solid foundation for the development of external preparations containing Strychni Semen.
Administration, Cutaneous ; Animals ; Brain ; Bridged-Ring Compounds/pharmacology* ; Chromatography, Liquid/methods* ; Glucosides ; Monoterpenes ; Rats ; Rats, Sprague-Dawley ; Strychnine ; Tandem Mass Spectrometry/methods* ; Tissue Distribution

Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.
Animals ; Bicuculline ; pharmacology ; Disease Models, Animal ; Glycine ; metabolism ; Hyperalgesia ; drug therapy ; etiology ; metabolism ; Imidazoles ; pharmacology ; Inhibitory Postsynaptic Potentials ; drug effects ; physiology ; Male ; Mice, Inbred C57BL ; Neurons ; drug effects ; metabolism ; Neurotransmitter Agents ; pharmacology ; Peripheral Nerve Injuries ; drug therapy ; metabolism ; Phenanthrolines ; pharmacology ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; metabolism ; Receptors, GABA-A ; metabolism ; Receptors, Glycine ; metabolism ; Strychnine ; pharmacology ; Synaptic Transmission ; drug effects ; physiology ; Tissue Culture Techniques ; Touch

Taurine has a number of beneficial pharmacological actions in the brain such as anxiolytic and neuroprotective actions. We explored to test whether taurine could be transported to the central nervous system through the intranasal route. Following intranasal administration of taurine in mice, elevated plus maze test, activity cage test and rota rod test were carried out to verify taurine’s effect on anxiety. For the characterization of potential mechanism of taurine’s anti-anxiety action, mouse convulsion tests with strychnine, picrotoxin, yohimbine, and isoniazid were employed. A significant increase in the time spent in the open arms was observed when taurine was administered through the nasal route in the elevated plus maze test. In addition, vertical and horizontal activities of mice treated with taurine via intranasal route were considerably diminished. These results support the hypothesis that taurine can be transported to the brain through intranasal route, thereby inducing anti-anxiety activity. Taurine’s anti-anxiety action may be mediated by the strychnine-sensitive glycine receptor as evidenced by the inhibition of strychnine-induced convulsion.
Administration, Intranasal ; Animals ; Anxiety ; Arm ; Brain ; Central Nervous System ; Isoniazid ; Mice ; Picrotoxin ; Receptors, Glycine ; Seizures ; Strychnine ; Taurine ; Yohimbine

OBJECTIVETo examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis.

METHODSThe osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL (50 ng/mL) and macrophage-colony stimulating factor (50 ng/mL) were added to this system, followed by treatment with brucine (0.02, 0.04 and 0.08 mmol/L), or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1 (TGF-β1), nuclear factor-kappa B (NF-κB) and Hes1 were determined by enzyme-linked immunosorbent assay.

RESULTSCompared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells (P<0.01). Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1 (P<0.05 or P<0.01).

CONCLUSIONBrucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.

Animals ; Bone Neoplasms ; metabolism ; prevention & control ; secondary ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Humans ; Jagged-1 Protein ; metabolism ; Macrophages ; drug effects ; physiology ; Mice ; Osteoclasts ; drug effects ; physiology ; Receptor, Notch1 ; metabolism ; Signal Transduction ; drug effects ; Strychnine ; analogs & derivatives ; pharmacology ; therapeutic use

OBJECTIVES: To establish a LC-MS/MS method which is accurate and sensitive for determination of koumine, gelsemine, and gelsenicine in biological samples and to verify the method. METHODS: Strychnine was used as internal standard. Analytes in blood, urine and liver with 1% sodium hydroxide solution were extracted by ethyl acetate. Chromatographic separation was achieved on a ZORBAX SB-C₁₈ column （150 mm×2.1 mm, 5 μm）, and gradient elution was performed with the buffer solution of methanol-20 mmol/L ammonium acetate （including 0.1% formic acid and 5% acetonitrile） as mobile phase. Qualitative and quantitative analysis was performed in the multiple reaction monitoring mode coupled with an electrospray ionization source under positive ion mode（ESI⁺）. RESULTS: The linearity of koumine, gelsemine and gelsenicine in blood, urine and liver was good within corresponding linear limitation and the correlation coefficients （r）>0.995 0. The limits of detection were 0.1 ng/mL （0.1 ng/g）, 0.1 ng/mL （0.1 ng/g） and 0.01 ng/mL （0.01 ng/g）, respectively. The extraction recovery and accuracy of the alkaloids ranged from 61.9% to 114.6% and 92.4% to 114.3%, respectively. The relative standard deviations of the intra-day and inter-day precisions were not more than 11.0%. CONCLUSIONS The method is selective, sensitive and suitable for simultaneous determination of koumine, gelsemine and gelsenicine in body fluids and tissues, which offering technical support for clinical diagnosis and treatment and forensic toxicological analysis of Gelsemium elegans poisoning.
Alkaloids/urine* ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Forensic Toxicology ; Formates ; Humans ; Indole Alkaloids/urine* ; Liver ; Reproducibility of Results ; Strychnine ; Tandem Mass Spectrometry

To provide insights into the mechanism for the attenuate-synergistic effect of Zuota to Tibetan medicine Renqing Mangjue, a contrasted study was carried out on the pharmacokinetics of brucine and strychnine in mice plasm, which are active and toxicant ingredient in the Tibetan medicine Renqing Mangjue. LC-MS/MS was used to detect simultaneously the concentrations of brucine and strychnine in mice plasm at-different time intervals after administration parallelly and randomly, and the pharmacokinetic software Kinetica 5. 0 was selected to non-compartmental analysis (NCA) for data, and statistical analysis software SPSS 19. 0 was used for significance test on the pharmacokinetic parameters. A reliable LC-MS/MS method was established for the determination of brucine and strychnine in blood plasma, which are consistent with the requirements of the preclinical pharmacokinetic study confirmed by the methodology. The linear concentration ranges of brucine and strychnine were 0.301-104.4 µg · L(-1) (r = 0.999 5) and 0.305-106 µg · L(-1) (r = 0.999 7), respectively; The intra-day and inter-day variable coefficients were both less than 10.0% with good precision; The average extraction recoveries of brucine and strychnine were 116.23% and 112.82%, and RSD were 3.2% and 2.3% separately;The average matrix effects of brucine and strychnine were 122.48% and 116.36%, and RSD were 7.7% and 4.4%, respectively. The pharmacokinetic results showed that AUCtot of brucine and strychnine in Zuota group were both increased remarkably (P < 0.05), and the Cmax of brucine in Zuota group was about 5.25-fold higher than that of brucine in non-Zuota group (P < 0.05). The Tmax of brucine and strychnine reduced to one-eighth and one-quarter respectively compared with those in Non-Zuota group. In addition, the eliminations of brucine and strychnine in vivo were accelerated after the compatibility of Zuota. A significant difference (P < 0.05) occurred at the MRT0-t, of brucine, while the MRT0-∞ and Lz of strychnine were statistically significant upon the inspection level α = 0.1. It was found that the absorption degree of brucine and strychnine in Zuota group increased in the range of the safe dose (or concentration), while their elimination rates were accelerated, which may be one of the mechanisms for attenuate-synergistic effect of Zuota to Tibetan medicine Renqing Mangjue.
Animals ; Female ; Male ; Medicine, Tibetan Traditional ; Mice ; Strychnine ; analogs & derivatives ; pharmacokinetics

This study was aimed to investigate the inducing-apoptosis effect of brucine on human monocytic leukemia cell line THP-1 cells and its possible mechanism. The inhibition effect of brucine on growth of THP-1 cells was measured by CCK-8 method. Morphological changes of THP-1 cells treated with brucine was detected by acridine orange/ethidium bromide (AO/EB)double staining. Annexin-V/PI double labeling method was used to assay the apoptosis rate of THP-1 cells. The effect of brucine on THP-1 cell cycle distribution was detected by PI single staining. RT-PCR was used to detect the expression of BCL-2 and BAX. The results showed that the brucine could inhibit the THP-1 cell growth in concentration and time-dependent manners at the range of 50 to 400 µg/ml. The cells stained with AO/EB revealed that the brucine induced the nuclear chromatin condensation. After the THP-1 cells were treated with brucine of 400µg/ml for 48 hours, most nucleic were stained as orange-red, and condensed, displaying the late apoptotic cell morphology. Annexin-V/PI detection showed that brucine could induce apoptosis of THP-1 cells in a concentration-dependent manner. Compared with the control group, more cells in brucine-treated group were arrested at G0/G1 phase in a concentration-dependent manner. RT-PCR detection revealed that the expression of BCL-2 was down-regulated strikingly and BAX was up-regulated. It is concluded that brucine can efficiently inhibit cell growth and block THP-1 cells in G0/G1 phase. The mechanism of THP-1 cell apoptosis induced by brucine may be related to the inhibition of BCL-2 and activation of BAX.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Strychnine ; analogs & derivatives ; pharmacology ; bcl-2-Associated X Protein ; metabolism

AIM: The application of strychnine (S) is limited due to its toxicity; strychnine N-oxide (SNO) is a derivative of strychnine. The aim was to employ zebrafish embryos to investigate and compare the developmental toxicity induced by S and SNO. METHODS: The toxicity of S and SNO was examined through the hatching rate and survival rate. Morphological changes of the zebrafish were observed with a dissecting microscope. Apoptosis was detected through acridine orange (AO) staining and flow cytometry. Apoptotic genes were measured by RT-PCR. RESULTS: Embryo malformation was observed in the embryos exposed to S at 200 μmol·L(-1). When SNO concentration was increased to 1 mmol·L(-1), scoliolosis, and pericardial edema could be seen in some embryos. Results from fluorescence microscopy and flow cytometry analysis showed that S at 200 μmol·L(-1) induced apoptosis, whereas the apoptotic rate in the SNO-treated group (200 μmol·L(-1)) was much lower than that in the S group. RT-PCR analysis showed that p53 mRNA expression and the ratio of Bax/Bcl-2 in the S group were significantly altered compared with the control group (*P < 0.05). Moreover, Bax mRNA expression in both S and SNO group were significantly different from that in the control group (**P < 0.01). CONCLUSION These results lead to the conclusion that SNO has significantly lower toxicity than S in zebrafish embryos.
Animals ; Apoptosis ; drug effects ; Cyclic N-Oxides ; toxicity ; Drugs, Chinese Herbal ; toxicity ; Female ; Male ; Oxidative Stress ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; Strychnine ; analogs & derivatives ; toxicity ; Strychnos ; adverse effects ; chemistry ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Zebrafish ; embryology ; genetics ; metabolism ; Zebrafish Proteins ; genetics ; metabolism

OBJECTIVE: To investigate function of glycine receptors (GlyRs) at the hippocampal CA1 pyramidal cells and to characterize the pharmacological properties of these receptors at early postnatal stage. METHODS: We used whole cell patch clamp recording to study the current response in the acutely prepared hippocampal slices from postnatal day 11-13 rats induced by glycine applied in the artificial cerebrospinal fluid. RESULTS: Application of glycine to the pyramidal cells elicited strychnine sensitive chloride currents. EC50 for GlyRs respond to glycine was 123. 23 μmol/L and Hill coefficient was 1.24. Picrotoxin could partly blocked the currents. CONCLUSION Strychnine sensitive glycine receptors are functionally expressed in CA1 pyramidal neurons in rat hippocampal CA1 area at early postnatal stage, and some of GlyRs are αβ heteromeric receptors.
Animals ; CA1 Region, Hippocampal ; cytology ; Glycine ; pharmacology ; Patch-Clamp Techniques ; Pyramidal Cells ; drug effects ; Rats ; Receptors, Glycine ; metabolism ; Strychnine ; pharmacology

The HPLC method for determining plasma concentration of brucine was optimized during the study on the effect of the extraction reagent, the extraction frequency and the volume of extraction solvent on the extraction recovery of brucine. The optimum sample treatment method was obtained in the study. Specifically, ammonia water was added, 4 mL extraction solvent (N-hexane-methylene chloride-isopropyl alcohol 65:30:5) were adopted to extract brucine for twice. The method to determine plasma concentration of brucine was applied in pharmacokinetic study to compare pharmacokinetic properties of intravenous injection (5 mg x kg(-1)) and transdermal administration (40 mg x kg(-1)) of brucine aqueous alkali. The results showed that both pharmacokinetic parameters of brucine after intravenous injection and transdermal administration were in conformity with the two-compartment model. After transdermal administration, the absolute bioavailability was calculated to be 18.72%. The optimized HPLC method can satisfy the demands of the pharmacokinetic study on brucine.
Animals ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; administration & dosage ; analysis ; pharmacokinetics ; Male ; Rats ; Rats, Sprague-Dawley ; Strychnine ; administration & dosage ; analogs & derivatives ; blood ; pharmacokinetics