Microstructure, Content and in vitro Release of Brucine and Strychnine in Strychnos Nux-Vomica Powder with Different Particle Sizes

To explore the effect of particle size on the quality uniformity and in vitro release performance of Strychnos nux-vomica powder, seven samples of Strychnos nux-vomica powder with different particle sizes were prepared.Microstructures and particle sizes were analyzed, and high performance liquid chromatography(HPLC) was used to test the contents and in vitro release performances of brucine and strychnine in the samples. Results showed that the contents and the in vitro release rates of brucine(or strychnine) in different samples were different since there are different proportions of endosperms to epidermal cells in Strychnos nux-vomica powder with different particle sizes. Brucine and strychnine in each sample were promptly released in the first ten minutes and their cumulative release rates were higher than 70% after ten minutes. Eighty minutes later, the cumulative release rate tended to be a constant. Considering the quality uniformity and safety of Strychnos nux-vomica powder used as traditional Chinese medicine, it would be better to control the particle size of Strychnos nux-vomica powder between 100 and 140 mesh in which the maximum cumulative release rate in vitro of brucine and strychnine can be relatively low within this range.

Study on the Interaction between Strychnine and Bovine Serum Albumin by Capillary Electrophoretic Frontal Analysis

The protein binding constant, binding sites of the Strychnos alkaloid-strychnine and bovine serum albumin （BSA） was determined by capillary electrophoretic frontal analysis （CE-FA） for the first time. The experiment was carried out in a polyacrylamide-coated fused silica capillary （48.4 cm×50 μm i.d., 38.1 cm effective length） with 20 mmol/L citrate/MES buffer （pH 6.0, ionic strength 0.17）. The applied voltage was 12 kV and detection wavelength was set at 257 nm. The plateau height of the peak was employed to determine the unbound concentration of drug in BSA equilibrated sample solution based on the external drug standard in the absence of protein. The present method provides a convenient, accurate technique for the early stage of drug screening.

The pharmacological and toxicological action of strychnine on central nervous system

Strychnine is the main bioactive constituent responsible for the pharmacological and toxic properties of strychnos nux-vomica L.(Loganiaceae),which is known for severe neurotoxicity.Although its toxic threshold is easy to be reached and it is liable to cause poisoning,the effect of strychnine on the central nervous system(CNS)is crucial for its application in clinic.Therefore,the impacts of pharmacology and toxicology of strychnine on CNS have generated increasing interests.In this short review,we summarize the pharmacological activities,neurotoxicology,underlying mechanisms of the strychnine poisoning,and discuss the pharmacokinetics properties of strychnine.These findings could provide beneficial information for facilitating future mechanism research of neurotoxicity of strychnine and developing drugs to remedy neurotoxicity in the clinic.

ANALYSIS OF STRYCHNINE ALKALOIDS IN BIOLOGICAL SAMPLES BY THIN-LAYER CHROMATOGRAPHIC DENSITOMETRY AND DISTRIBUTION STUDY OF STRYCHNINE IN INTOXICATED RATS

A sensitive and rapid analytical procedure was established to detrmine strychnie alkaloids in diverse biological specimens. Samples were treated with hydrochloric acid to precipitate protein and scanned in thin-layer chromatograpic scanner in zig-zag dual-wavelength reflection mode.Detection sensitivity for strychniue was about 0. 05 μg. Quantification recoveries were between89. 2% and 99. 5% with RSD between 2. 92% and 13. 21 %. The method was applied to investigate thedistribution or strychnine in intoxicated rats. The results showed that there were remarkable difference in organs or tissues with the aigaest concentration 4. 56±1. 41μg/g in liver. Diversification experiment proves the method also could be used to analyze other alkaloids in biological samples if theconditions slightly modified.

Toxic effects of strychnine and strychnine N-oxide on zebrafish embryos

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.

In vitro Metabolism of Strychnine by Human Cytochrome P450 and Its Interaction with Glycyrrhetic Acid

Objective To investigate the metabolism of strychnine(STN)and the metabolic interaction between STN and glycyrrhetic acid(GA)in vitro.Methods Human liver microsomes(HLM)and human recombinant cytochrome P450(CYP)isoforms were employed to study the metabolism of STN and the metabolic interaction of STN with GA in vitro.Results In HLM,the Km,Vmax,and clearance of STN were 88.50μmol/L,0.88 nmol/(mg·min),and 9.93 mL/(mg·min),respectively.STN was metabolized mainly by CYP3A4.However,STN noncompetitively inhibited CYP3A4-catalyzed testosterone 6β-hydroxylation with IC50 value of 5.9μmol/L and Ki value of 5.5 μmol/L.Moreover,GA competitively inhibited STN metabolism with IC50 value of 10.6μmol/L and Ki value of 17.7μmol/L.Conclusion Although STN is mainly metabolized by CYP3A4 in vitro,STN has noncompetitive inhibition on CYP3A4-catalyzed testosterone 6β-hydroxylation.Moreover,GA could competitively inhibit STN metabolism.The present work is helpful to elucidate the metabolic interaction between STN and GA.

Effect of Baizhu(Rhizoma Atractylodis Macrocephalae)extract on intestinal absorption of brucine and strychnine in vitro and in situ

OBJECTIVE:To investigate the antagonistic effect of the extract of Baizhu(Rhizoma Atractylodis Macrocephalae)(RAM)on the intestinal absorption of brucine and strychnine in Strychnos nux-vomica(NUX)and propose the mechanism of these effects.METHODS:The apparent permeability value(Papp)and absorption rate constant(Ka)were chosen as indices.The everted intestinal sac model and in situ single-pass intestinal perfusion model were used to study the effects of the RAM extract on the absorption of brucine and strychnine.To confirm the results,the brucine and strychnine concentrations in hepatic portal venous blood were determined.Western blotting was used to study P-glycoprotein(P-gp)expression in the Caco-2 cell line.RESULTS:Papp and Ka of brucine and strychnine were significantly increased in the presence of a P-gp inhibitor,but no significant increase was noted in the presence of a tight junction regulator.The RAM extract inhibited the absorption of brucine and strychnine and enhanced P-gp expression.CONCLUSION:The primary absorption mechanism for brucine and strychnine is passive transport,which is affected by P-gp.

Effect of strychnine hydrochloride on liver cytochrome P450 mRNA expression and monooxygenase activities in rat

Strychnos nux-vomica L.has been frequently used in traditional Chinese medicine but has high acute toxicity.It is commonly taken with Glycyrrhizae radix to decrease its toxicity but the mechanism of this interaction is unknown.In this work,the mRNA expression and the activity of four cytochrome P450(CYP)enzymes representative of four subfamilies(CYP1A,CYP3A,CYP2C and CYP2E)were determined ex vivo in rat livers from groups of Wistar rats orally administered strychnine hydrochloride(SH)at three doses(0.1,0.3 and 0.9 mg/kg/day)alone and,at the highest dose,in combination with glycyrrhetinic acid(GA,25 mg/kg/day)or liquiritin(LQ,20 mg/kg/day)once a day for 7 consecutive days.Compared to control,the mRNA expressions of CYP3A1,1A2 and 2E1 were higher in rats receiving the highest dose of SH but lower for CYP3A1 and CYP2E1 in rats receiving the SH+GA and SH+LQ combinations.CYP2E1 activity was higher and CYP2C,CYP3A and CYP1A2 activities were lower in rats receiving the highest dose of SH.In contrast CYP1A2 and CYP2C activities were higher and CYP2E1 and CYP3A activities lower in rats receiving the SH+GA combination.CYP2E1 and CYP3A activities were also lower in rats receiving the SH+LQ combination.The results show that treatment with SH for 7 days affects the expression and the activity of CYP enzymes and that coadministration of GA and LQ modulates these effects.This modulation may explain the role of Glycyrrhizae radix in reducing the acute toxicity of Strychnos nux-vomica L.CYPs enzymes.

Determination of strychnine and brucine in traditional Chinese medicine preparations by capillary zone electrophoresis with micelle to solvent stacking

A novel micelle to solvent stacking on-line sample preconcentration technique in capillary zone electrophoresis（MSS-CZE） has been developed to determine the strychnine and brucine in traditional Chinese medicine preparations.The optimal running buffer was 30 mmol/L H_3PO_4 containing 20%acetonitrile at pH 4.0.The sample matrix was 8 mmol/L H_3PO_4 containing 5 mmol/L sodium dodecyl sulfate（SDS） at pH 3.0.The established MSS-CZE method afforded more than 50-fold improvements in concentration sensitivity compared with typical CZE-UV analysis.The calibration curve was linear in the range from 0.2 to 15.0μg/ mL for both strychnine and brucine,with correlation coefficients of 0.9984 and 0.9976,respectively.The limits of detection（5/ N = 3：1） for strychnine and brucine were 0.02 and 0.05μg/mL,respectively.The MSS-CZE method has been successfully applied to the analysis of strychnine and brucine in Chinese medicinal preparations.

Antiepileptic activity of lobeline isolated from the leaf of Lobelia nicotianaefolia and its effect on brain GABA level in mice

Objective:To investigate the anticonvulsant activity of the lobeline isolated from the Lobelia nicotianaefolia in chemoconvulsant-induced seizures and its biochemical mechanism by investigating relationship between seizure activities and altered gamma amino butyric acid(GABA)in brain of mice in Pentylenetetrazol(PTZ) seizure models.Methods:The anticonvulsant activity of the isolated lobelinc(5,10,20 and 30 mg/kg.i.p.) was investigated in PTZ and strychnine induced seizures in mice and the effect of isolated lolieline on brain GABA level in seizures induced by PTZ.Diazepam was used as reference anticonvulsant drugs for comparison.Results:Isolated lobeliue(10,20 and 30 nig/kg.up.) significautly delayed and antagonized(P < 0.050-0.001)the onset of PTZ-induced seizures.It also antagonized strychnine induced seizures.The mortality was also prevented in the test group of animals.In biochemical evaluation,isolated lolieline(5,10and 20 mg/kg,i.p.) significantly increased the brain GABA level.And at dose of 30 mg/kg GABA level showed slight decrease in PTZ model.Conclusions:In our findings,isolated lolieline(20mg/kg) exhibited potent anticonvulsant activity against PTZ induced seizures.Also a biochemical evaluation suggested significant increase in harain GABA level at 20 nig/kg up.of isolated lolieline.Hence,we may propose that lolieline reduces epileptic seizures by enhancing the GABA release supporting the GABAergic mechanism.