Erysothrine,An Alkaloid Extracted from Flowers of Erythrina Mulungu Mart
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Erysothrine,an alkaloid extracted from flowers of Erythrina mulungu Mart. ex Benth:Evaluating its anticonvulsant and anxiolytic potential Epppyilepsy & Behavior 23(2012)205-212 Presented by Pratthana Srichomphu 1st year master student Neurosciences Program Content Introduction & Rationale Objective MtMater ilials &Mthd& Methods Result & discussion Conclusions What is the seizure? Excitation Inhibition Inhibition Excitation Too much excitation Increased activation of NMDA receptors Increased inward Na+, Ca++ currents Too littl e in hibition Defective GABA A or GABA-B inhibition Defective activation of GABA neurons Defective intracellular buffering of calcium Defective inward CI-, outward K+ currents Classification of seizures Partial seizures Generalized seizures Standard Treatment of seizures More than 80% of patients with epilepsy can have their seizures controlled with medication Excitation Inhibitation Cl- Cl- Diazepam Chemical Convulsant ¾Pentylenetetrazol (PTZ) ¾Bicuculline GABA antagonist ¾Kainic acid Glutamate agonist ¾N-Methyl D-Aspartate (NMDA) Tonic – clonic seizure Chemical Convulsant Cl- Bicuculine Ca++ Ca++ Kainic acid NMDA PTZ Folk medicine AtiAnticonvu lsan t, ana lges ic, se dtidative, hypno tic Family: Fabaceae Genus:andErythrina hypotension properties Species: mulungu, cristi -galli Synonyms: Erythrina verna, Corallodendron mulungu Common Names: Mulungu, corticeira, murungu, muchocho, murungo, totocero, Lourenzi et al 1992,Rodrigues et al 2001 Hydroalcohoholic extract Analgesic Anticonvulsant Vasconcelos et al2007 The purification of hydroalcoholic extract of E. mulungu (+)-11-α-hydroxyerythravine(+)-erythravine anticonvulsant Chroma tograp hic iso la tion Erysothrine ???? Central effect of Erythrina mulungu (from flowers) Anticonvulsant ??? Anxiolytic activities ?? Modulation of GABA and glutamate Up ta ke an d bihding sys tems ??? Materials & Methods 11-α-hydroxy- erythravine erythravine Flowers from E. mulungu (5.0 kg Rifaina, São Paulo, Brazil) Erysothrine 2.1 mg % yield =4.2 x 10-5 Voucher specimen : UNAERP Herbarium under official number HPMU — 1330 Male Wistar rats (200–250 g) Surgery:implanted a stainless steel guide cannula 5-7 days recovery Control treated Positive control Erysothrine0.125- 3 μg/μL. i.c.v. group treated group, DZP : Saline: icvi.c.v. 2 mg/kg ; ipi.p. 1 μL/min 1 μL/min o.3 ml Surgery Stereotaxic co-ordination for the right lateral ventricle Antero- posterior (AP) – 0.9 cm Medio-Lateral – 1.6 cm Dorso-ventral -3.4 cm Male Wistar rats (200–250 g) Surgery:implanted a stainless steel guide cannula 5-7 days recovery Control treated group Erysothrine Positive control treated group, DZP Saline: i.c.v. 0.125- 3 μg/μL.:i.c.v. 2 mg/kg ; i.p. 1 μL/min 1 μL/min o.3 ml 10 min Chemical convulsants AitAnxiety tttests in doses adjusted to induce seizures in 97% of animals (CD97) Open field test Bicuculine NMDA PTZ kainic acid (0.9 μg/μL; i.c.v.) ( 17μg/μL; i.c.v.) (80 mg/kg ; i.p.) (0.8 μg/μL; i.c.v.) Neurochemical assays: 3H‐Glutamate and 3H‐GABA uptake and binding Anticonvulsant screening Anticonvulsant screening Male Wistar rats (200–250 g) Surgery:implanted a stainless steel guide cannula 5-7 days recovery Control treated group Erysothrine Positive control treated group, DZP Saline: i.c.v. 0.125- 3 μg/μL.:i.c.v. 2 mg/kg ; i.p. 1 μL/min 1 μL/min o.3 ml 10 min Chemical convulsants in doses adjusted to induce seizures in 97% of animals (CD97), Bicuculine NMDA PTZ kainic acid (0.9 μg/μL; i.c.v.) ( 17μg/μL; i.c.v.) (80 mg/kg ; i.p.) (0.8 μg/μL; i.c.v.) Observed the occurrence of seizures and the latencies to seizure onset or death 30 min The limbic seizures score proposedbd by Pi nel and dR Rovner 0 — No seizure; 1 —Orofacial movements; 2 — Head myoclonus; 3 — Hindlimb myoclonus; 4 — Elevation; 5 — Elevation and limbic fall; 6 — Head and ears myoclonus; 7 — Rotation, jumps and vocalization; 8 — Behaviors from class 7 and hypertonus Hypothesis Erysothrine + Prevent % of Protected animals Prolong Latency time to seizure Statistical analysis Chi-square test One way ANOVA, Newman-Keuls post test p<o.o5 Effect of Erysothrine to protected agianst seizures chemical convulsant induced Dose dependent 100% 100% 40% 20% 0%0% 0% 0% 100% 100% 60% 40% 0% 0% 0% 0% a*, a** p<0.05 ,p<0.01 compare with control vehicle treated gr. Effect of Erysothrine on latency to onset of seizures evoked chilhemical convul sant Å3x Proconvulsant effect – Æ threshold seizure a** a*** p<0.01 p<0.001 compare with control vehicle treated gr. b* b*** p<0.05 p<0.001compare with 1 μg/mL Erysothrine gr. Chemical Erysothrine Erythravine 11α-hydroxy- convulsant (Faggion SA et al erythravine 2011) (Faggion SA et al 2011) % of Kainic acid 100% at 0.5 μg/μL 100% at 2 μg/μL 100% at 2 μg/μL Protected animals bicuculine 100% at 3 μg/μL 75% at 3 μg/μL 100% at 1 μg/μL NMDA 100% at 3 μg/μL 25% at 3 μg/μL 100% at 1 μg/μL PTZ 100% at 3 μg/μL 100% at 2 μg/μ 60% at 3 μg/μL Latency time Kainic acid proconvulsant 1 μg/μL 0.5μg/μL to onset of seizure bicuculine 2 μg/μL 3 μg/μL 0.5μg/μL NMDA 2 μg/μL 2 μg/μL 0.5μg/μL PTZ 1 μg/μL 2 μg/μL - Anxiety test Male Wistar rats (200–250 g) Surgery:implanted a stainless steel guide cannula 5-7 days recovery 30 min acclimatized to the experimental room Control treated group Erysothrine Positive control treated group, DZP Saline: i.c.v. 0.5- 3 μg/μL.:i.c.v. 2 mg/kg ; i.p. 1 μL/i/min 1 μL/mi n o.3 ml 10 min Elevated plus maze apparatus and a light/dark apparatus 5 min Elevated plus maze test Total time spent in the open arm Entries into the open and closed arms. Dark/light box Light Dark Dark/light transition Time spent in the light side Hypothesis ÅTotal time spent in the open arm ÅEntries into the open and closed arms. ÅDark/light transition+ ÅTime spent in the light side Åthe number of line crossing Åthe number of gggrooming Åthe number of rearing Effect of Erysothrine in EPM anxiety test a* a***p<0.05 p<0.00.1 compare with control vehicle treated gr. b** p<0.01 compare with 0.5 μg/μL Erysothrine gr Effect of Erysothrine in light/dark box NO significant Open field test Male Wistar rats Surgery:implanted a stainless steel guide cannula 5-7 days recovery 30 min acclimatized to the experimental room Control treated group Erysothrine Positive control treated group, DZP Saline: i.c.v. 0.5 ,1.5,3 μg/μL.:i.c.v. 2 mg/kg ; i.p. 1 μL/i/min 1 μL/mi n o.3 ml 10 min Open field test Locomotion activity : the number of line crossing Self care : the number of grooming Exploratory : the number of rearing Effect of Erysothrine on open-field test NO significant Neurochemical screening Male Wistar rats (200–250 g) OdOverdose o fthitlf thiopental (100mg/kg :. i.p.) Brains were removed Cerebral cortex *Synaptosome P2(glutamate/GABA uptake assay *Synaptic membrane( GABA/glutamate binding assay *protein assay by modified Lowry method(Hartree EF 1972 ) Hypothesis Erysothrine + Å Glutamate uptake ÆGABA uptake ÆGlutamate ÅGABA binding binding Effect of Erysothrine on GABA/glutamate uptake and blinding NO significant uptake binding Discussion: Anti-epileptic anxiety, insomnia and pain, anddtidfd as a sedative and for Hydroalcohoholic extract blood pressure control. Antinociceptive sedative activities Å Latency to death and convulsion Vasconcelos et al2007 Discussion: Anxiolytic effects Erythravine In the light–dark transition model at doses of 3 and 10 mg/kg 11-α-hydroxy-erythravine In the light–dark transition model at dose 10 mg/kg increase the number of transitions at dose 3 mggg/kg Flausino Jr OA et al 2007 GABA antagonists Glutamate agonists PTZ, Bicuculline NMDA, Kainic acid Anxiolytic(+)-erythravine drugs and (+)-11α-hydroxy-erythravine benzodiazepine Anticonvulsant Anticonvulsant Anxiolytic activities ÅOpen arm entries in EPM Anticonvulsant activity Bicuculline PTZ NMDA Kainic acid Erysothrine % of Protected 100% 100% 100% 100% animals at 3μg/μL at 3 μg/μL at 3 μg/μL at 0.5 μg/μL Latency time to 2 μg/μL 1 μg/μL 2 μg/μL proconvulsant onset of seizure Modulation of GABA and glutamate Uptake and bihding systems no modification Future study ¾Investigation of the mode of action of eryypsothrine as well as other plant alkaloids is needed. Possible mechanism of erysothrine Increased inward Na+, Ca++ currents Defective intracellular buffering of calcium DfDefec tive inward dCI CI-, outdK+ttward K+ currents ACKNOWLEDGEMENT Assist. Prof. Dr. Supaporn Muchimapura Thank you for your attention.