A COMBINATORIAL APPROACH TO THE SEARCH FOR ANTICONVULSANT AGENTS FROM GOU TENG AND TIAN MA YANG HONG (B. Sc. (Pharm.)), Sichuan Univ. (M. Sc. (Pharm.)), Sichuan Univ. A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to give my sincere gratitude to my dear supervisor, Assoc. Prof. Chui Wai Keung. What I have learned besides his gentleness, kindness and generosity is the attitude towards scientific research, meticulous, skeptical and always openminded. I think I am lucky enough to be his student, who has been granted such a great freedom to experiment with wild ideas, guided not to lose the direction while exploring, and encouraged to confidently convey research findings in oral presentation. I hope I would be growing up as an independent researcher, since that will be the best gift a student could think of to give to her teacher. I would also like to thank Dr. Low Chian Ming, who helped me to plan my electrophysiological study and initiated so many insightful discussions regarding the results, Dr. Chew Eng Hui, who gave me valuable advice and shared with me her experiences in culturing cell lines, and Mr. Wee Xi Kai, who taught me how to use autodock program. I am grateful to the Pharmacy department lab technicians namely, Ms Ng Sek Eng, Ms Dyah Nanik Irawati and Ms Lye Pey Pey, for all your hard work in providing technical support. More importantly, I wish to express my deep gratitude to National University of Singapore for offering the research scholarship that supports my candidature. In addition, I would like to acknowledge the academic research grant (R148-000-049-112) for providing financial resources for the work carried out. My dear friends, Dr. Ma Xiang, Ms Pauline Ong, Ms Michelle Kok, Dr. Huang Meng, Ms Wang Chunxia, Mr. Zhang Yaochun, Ms Jin Jing, Ms Yang Shili, Mr. Wang Zhe and Ms Sim Hong May, I owe you all a big thank you. You all know how I can easily cry but you may not know I only cried in front of friends. The time spent with you was so precious and would be preserved in my memories. My dear parents and sister, I am indebted to all of you for being my first teachers and my best companions ever. My dearest husband, Mr. Lu Xiaohua, you have fulfilled my life outside the lab, and I am thanking you for always being there for me, appreciating me as I am in spite of my ill-temper and willfulness. This thesis is written for you. i CONTENTS ACKNOWLEDGEMENTS PAGE i CONTENTS ii SUMMARY v ABBREVIATIONS vii LIST OF SCHEMES xi LIST OF FIGURES xii LIST OF TABLES xv 1. INTRODUCTION 1.1. General introduction 1.2. Drug discovery from natural products 1.2.1. Crude, pre-fractionated and pure natural products library 1.2.2. The reductionist and holistic approach in drug discovery from natural products 10 1.2.2.1. Interplay between constituent in natural products 10 1.2.2.2. Bioassay-guided fractionation and systems biology based metabolomic approach 13 1.2.3. A proposition-purifying one single chemical class from natural products 16 1.2.4. Hyphenated instrumentation applied in the analysis of natural products 18 1.3. Two traditional Chinese Medicines – Gou Teng & Tian Ma 21 1.3.1. Phytochemical and pharmacological studies of Gou Teng 21 1.3.2. Phytochemical and pharmacological studies Tian Ma 26 1.4. Antiepilepsy, antiepileptogenesis and neuroprotection 28 1.4.1. Definition for antiepilepsy, antiepileptogenesis and neuroprotection29 1.4.2. In vitro & in vivo models for epilepsy, epileptogenesis and neuroprotection 30 1.5. The role of NMDA antagonism in antiepilepsy, antiepileptogenesis and neuroprotection 33 1.5.1. NMDA antagonism 33 1.5.2. Antagonists at the glycine site of NMDA receptor 34 1.6. Summary 39 1.7. Hypothesis and objectives 40 2. RESULTS & DISCUSSIONS 43 2.1. The standardized extracts derived from Gou Teng and Tian Ma 43 2.1.1. Methodology 43 2.1.2. The standardized alkaloidal extract from Gou Teng 44 2.1.2.1. Preparation of the alkaloidal extract 45 ii 2.1.2.2. Qualitative analysis of the alkaloidal extract by LC-ESI-MS 46 2.1.2.3. Isolation and characterization of single alkaloids from the alkaloidal extract 48 2.1.2.4. Quantitative analysis of the alkaloidal extract by LC-UV 56 2.1.2.5. Conclusions 60 2.1.3. The standardized phenolic extract derived from Tian Ma 62 2.1.3.1. The preliminary purification and analysis on GC-EI-MS 63 2.1.3.2. Isolation and characterization of single phenolic compounds in the EFME 70 2.1.3.3. Synthesis of some phenolic compounds present in Tian Ma 73 2.1.3.3.1. Retro-synthesis of target compounds 74 2.1.3.3.2. Synthesis of HPMP 75 2.1.3.3.3. Synthesis of BHE, HFC and CSD 79 2.1.3.3.4. Synthesis of HMP 81 2.1.3.4. Further purification on MAX cartridge and analysis on LC-UV 82 2.1.3.5. Conclusions 84 2.1.4. Summary 85 2.2. Antiepileptic activity investigation 85 2.2.1. MES and rotorod test 85 2.2.2. Implications for the results of the phytochemical and animal study 91 2.2.2.1. Justifications for choosing the strategy of the purification of the alkaloids and phenols over bioassay-guided fractionation 91 2.2.2.2. Explanations for the lack of antiepileptic activity possessed by the alkaloidal and phenolic extracts 93 2.3. In vitro neuroprotective activity assessed on neuro-cell line 95 2.3.1. Effects on cell viability in the absence of neurotoxins 96 2.3.2. Effects on cell viability in the presence of hydrogen peroxide 98 2.3.3. Effects on cell viability in the serum-deprived medium 99 2.4. Design and synthesis of potential antagonists at Gly/NMDA receptor and in vivo neuroprotective activity 103 2.4.1. Design of novel antagonists of Gly/NMDAR 103 2.4.2. Synthesis of 2-oxindole derivatives 107 2.4.2.1. Synthesis of 3-[(substituted furan-2-yl)methylidenyl] 2-oxindoles 107 2.4.2.2. Synthesis of substituted-3-benzoyl (furoyl)-2-oxindoles 110 2.4.3. Neuroprotective activity evaluation on SH-SY5Yneuroblastoma cell line 115 2.4.3.1. Effects of 2-oxindole derivatives on cell viability in the absence of neurotoxins 115 2.4.3.2. Effects of 2-oxindole derivatives on cell viability in the presence of hydrogen peroxide 116 2.4.3.3. DPPH free radical scavenging test 118 2.4.4. Electrophysiological characterization of 3112 on Xenopus oocytes expressing NR1/NR2B 120 2.4.5. Conclusions 125 3. CONCLUDING REMARKS 127 4. EXPERIMENTAL 134 4.1. General 134 4.2. Phytochemistry 135 iii 4.2.1. Prepare the standardized extracts 135 4.2.1.1. The alkaloidal extract from Gou Teng 135 4.2.1.2. The phenolic extract from Tian Ma 135 4.2.2. Instrumental analysis of the extracts 136 4.2.2.1. Qualitative analysis of the alkaloidal extract by LC-ESI-MS 136 4.2.2.2. Quantitative analysis of the alkaloidal extract by LC-DAD 137 4.2.2.3. Qualitative analysis of the EFME by GC-EI-MS 137 4.2.2.4. Quantitative analysis of the EFME by LC-DAD 138 4.2.3. Spectroscopic analysis of the isolated compounds 139 4.3. Computational method – Molecular docking 142 4.4. Synthetic chemistry 143 4.4.1. Synthetic procedures 143 4.4.2. Characterization data of synthesized compounds 149 4.4.3. X-ray crystallographic analysis of compound 18 162 4.5. Bioactivity investigations 163 4.5.1. DPPH free radical scavenging test 163 4.5.2. Determination of total free phenolic compounds by Folin-Ciocalteu test 164 4.5.3. MES and Rotorod test 164 4.5.4. Cell culture and cell viability test 165 4.5.5. Electrophysiological study of compound 20 (3112) on Xenopus oocytes expressing NR1/NR2B 166 4.5.6. Statistics 167 5. BIBILIOGRAPHY 168 iv SUMMARY Although current antiepileptic drugs can effectively relieve most of the epileptic patients from episodes of seizure, still 40% of the patients are refractory to the current therapeutics. Hence development of new antiepileptic agents is urgently needed. Gou Teng and Tian Ma are two well-known Traditional Chinese Medicines (TCMs), widely used in oriental medicine to treat epilepsy, hypertension and migraine, but their scientific evidences to support their therapeutic applications in the abovementioned areas are limited. NMDA (N-methyl-D-aspartic acid) antagonism is an important mechanism for many successful antiepileptic and neuroprotective agents, such as MK-801, CPP, ketamine etc. Nowadays NMDA receptor antagonism still remains as an effective strategy for discovery of new antiepileptic and neuroprotective agents. Our aim is to search for active small molecules or molecular combinations from Gou Teng and Tian Ma, and to design and synthesize new NMDA receptor antagonists for the benefit of controlling seizures and protecting against neuronal damages. Conventional extraction and purification techniques were employed to obtain bioactive fractions, which were subsequently subjected to careful qualitative and quantitative chemical analysis. The standardized fractions, derived from Gou Teng and Tian Ma respectively, were tested on the maximal electroshock-induced seizure (MES) model in mice and on cell cultures that were inflicted by either hydrogen peroxide or serum-deprivation. The indole-2-one (2-oxindole) template was utilized to prepare a series of 3-substituted derivatives which were later tested on cell cultures inflicted by hydrogen peroxide and on voltage-clamped Xenopus Oocytes expressed NR1/NR2B NMDA receptor. v The acquired fractions either alone or in combination did not prevent MES induced seizure activities on mice, but the phenolic fraction derived from Tian Ma was found to be rescuing cells from serum-withdrawal induced apoptosis. Two libraries based on 2-oxindole derivatives were synthesized with acceptable yields, and one of the libraries was found to be acting as free radical scavengers to protect against cell death inflicted by hydrogen peroxide. One synthesized compound, namely compound 20 (3112), was found to be a weak agonist acting at the glycine site of NMDA receptor based on the electrophysiological study on Xenopus Oocytes. Therefore, in this project it was found that Gou Teng and Tian Ma might be used more as neuroprotectants rather than as antiepileptic agents. In addition, some of the synthesized 2-oxindole derivatives that were initially designed as antagonists of Gly/NMDAR were acting as neuroprotectants against oxidative stress. In this project, the endeavour to search for potential antiepileptic and neuroprotective agents was made on both natural products and synthetic compounds. NMDA antagonism which was one of the mechanisms for Gou Teng to protect neuronal damage and also the rational basis on which the 2-oxindoles were synthesized served as the link. This was a trial that combined the passive process of isolating constituents from natural products and the active participation of design and synthesis of new compounds based on a well-defined target; this was an experiment that combined the diversified structures found in natural products and the synthetic library with limited structure diversity. It was believed that the most fruitful discovery of drugs should rely on both natural products and organic synthesis. Key words: Antiepileptic agents; Gou Teng (Ramulus Uncariae cum uncis); Tian Ma (Rhizoma Gastrodiae); NMDA receptor; 2-oxindole derivatives. vi ABBREVIATIONS 2CI 2-Carboxyindoles 4HA 4-Hydroxybenzyl Alcohol 4HD 4-Hydroxybenzaldehyde 5-HT 5-Hydroxtryptamine AEDs Antiepileptic Drugs AKU Akuammigine BBB Blood Brain Barrier BHE Bis-(4-hydroxybenzyl) Ether CE Collision Energy CI Combination Index CNS Central Nervous System CNMR Carbon Nuclear Magnetic Resonance COR Corynoxeine CSD Cirsiumaldehyde CT Corynantheine CTHQ 2-Carboxytetrahydroquinolines CUR Curtain Gas DAD Diode Array Detector DCKA 5,7-Dichlorokynurenic Acid DE Diethyl Ether DEPT Distortionless Enhanced by Polarization Transfer DHPM 4,4’-Dihydroxyphenyl Methane DP Declustering Potential DPPH 2,2-Di(4-Tert-Octylphenyl)-1-Picrylhydrazyl vii EAA Excitatory Amino Acid EC50 Half-Effective Concentration) EDRF Endothelium-Derived Relaxing Factor EFME Ether Fraction of Methanolic Extract EGCG (-)-Epigallocatechin Gallate EI Electron Impact EIMS Electron Ionization MS ELSD Evaporative Light Scattering Detector EMS Enhance Q3 Single MS EP Entrance Potential ESI Electrospray Ionization FHPE 1-Furan-2-Yl-2-(4-Hydroxy-Phenyl)-Ethanone GABA γ-Aminobutyric Acid GC Gas Chromatography Gly/NMDAR Strychnine-Insensitive Glycine Site of NMDA Receptor GME Geissoschizine Methyl Ether GS1 Ion Source Gas HBAD 3-Hydroxy-1H-1-Benzazepine-2,5-Diones HFC 5-(4-Hydroxy-Benzyloxymethyl)-Furan-2-Carbaldehyde HME 4-Hydroxybenzyl Methyl Ether HMFC 5-Hydroxymethyl-Furan-2-Carbaldehyde HMP 2,4-Bis[(4-Hydroxyphenyl)Methyl]-Phenol HMQC Heteronuclear Multiple Quantum Coherence HNMR Proton Nuclear Magnetic Resonance HPLC High Pressure Liquid Chromatography viii HPMP 4-((4-hydroxymethyl)phenoxy)methyl) phenol HQ 4-Hydroxy-2-Quinolones HRMS High Resolution MS HS Hirsutine HT Hirsuteine HTS High-Throughput Screening IR Infrared Spectroscopy ISO-RHY Isorhynchophylline JNK C-Jun N-Terminal Kinases KA Kynurenic Acid KI Dissociation Constant LC Liquid Chromatography LEV Levetiracetam MCAO Middle Cerebral Arterial Occlusion MDRs Multidrug Resistance Pumps MES Maximal Electroshock-Induced Seizure MPP+ 1-methyl-4-phenylpyridinium cation MS Mass Spectrometry MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide MVDA Non-linear Multivariate Data Analysis MW Molecular Weights NCEs New Chemical Entities NGF Neuronal Growth Factor NMDA N-methyl-D-aspartic acid NMR Nuclear Magnetic Resonance ix Yellow crystals, yield 95%, mp: 141-143℃; 1H NMR (300MHz, CDCl3) δ (in ppm) 14.2 (s, 1H, OH), 8.32-8.37 (dd, 1H, H-4, J = 0.75 & 8.3 Hz), 8.23-8.26 (dd, 1H, H-7, J = 1.1 & 7.9 Hz), 7.82 (d, 1H, H-4’, J = 1.5 Hz), 7.38-7.39 (d, 1H, H-2’, J = 3.4 Hz), 7.18-7.30 (m, 2H, H-5 & 6), 6.69-6.71 (dd, 1H, H-3’, J = 1.5 & 3.4 Hz), 2.78 (s, 3H, COCH3). 13 C NMR (75MHz, CDCl3) δ (in ppm) 174.0, 170.5, 159.7, 148.3, 146.2, 135.3, 126.6, 124.7, 121.7, 121.3, 118.3, 116.0, 112.8, 99.4, 27.1. MS (APCI) m/z [M+1]+ 270.2. 4.4.3 X-ray crystallographic analysis of compound 18 Crystals of [Z]-3-[Hydroxy-(4-phenoxy-phenyl)-methylene]-6-chloro-1,3-dihydro- indol-2-one (compound 18) were grown in solvent mixture of methanol and acetone and mounted on glass fibers. X-ray data were collected with a Bruker AXS SMART APEX diffractometer, using Mo Kα radiation at 223K, with the SMART suite of Programs (SMART version 5.628 (2003), Bruker AXS Inc., Madison, WI). Data were processed and corrected for Lorentz and polarization effects with SAINT (SAINT + version 6.22a (2001) Bruker AXS Inc., Madison, WI), and for absorption effect with SADABS (SADABS, version 2.10 (2001), University of Göttingen). Structural solution and refinement were carried out with the SHELXTL, suite of programs (SHELXTL, version 6.14 (2000), Bruker AXS Inc., Madison, WI). The structure was solved by direct methods to locate the heavy atoms, followed by difference maps for the light, non-hydrogen atoms. All non-hydrogen atoms were generally given anisotropic displacement parameters in the final model whereas H-atoms were placed at calculated positions. 4.5 Bioactivity investigations 4.5.1 DPPH free radical scavenging test 164 The measurement of antioxidant activity was performed according to a procedure described previously with minor modification 124. Quercetine was used as the standard antioxidant sample. DPPH and EtOH were used as stable free radical agent and blank, respectively. The sample stock solutions were prepared in DMSO at concentration of 100 mmol, and subsequently diluted by EtOH to the test concentrations. 20 µL of each compound at five concentrations (10-100 µM) was transferred to the vials in the 96-well plates which contained DPPH solution (0.2 mg/mL, 180 µL/vial). The reaction was kept in the dark for hrs. The antioxidant activity was measured as the decrease of the absorbance of DPPH and expressed as the percentage of the absorbance of a control DPPH solution with the addition of EtOH 20 µL. 4.5.2 Determination of total free phenolic compounds by Folin-Ciocalteu test The amount of total phenolic compounds in the samples was determined using FolinCiocalteu method125. Briefly, a calibration curve was prepared from gallic acid solution with concentration ranged from 0.03-0.09 mg/mL. In this method, mL distilled water was added into a 10 mL volumetric flask. Samples each mL with average concentrations of 1.25 mg/mL was transferred into the volumetric flask to obtain absorbance in the range of the prepared calibration curve. 0.2 mL of FolinCiocalteu reagent was added and mixed well for oxidation. After min, the reaction was neutralized with 0.4 mL satd. sodium carbonate (Na2CO3) solution. The solution is mixed well and made up to volume with distilled water. After a hr reaction in the dark, the absorbance was measured at 725 nm using Shimadzu UV-1601 UV-Visible Spectrophotometer. The results were expressed as gram of gallic acid equivalent (GAE) per 100 g of dry weight (DW) of the extracts 126. 4.5.3 MES and Rotorod test 165 Animals and drug administration Swiss albino mice (male, 20-30 g) were obtained from University Laboratory Animal Holding Unit at least two days prior to experimentation. They were kept under a natural light-dark cycle with food and water available. All animal experiments under the project No. R-148-000-049-112 were formally approved by the NUS institutional animal care and use committee (IACUC). Test samples were dissolved in dimethylsulfoxide (DMSO) and administered by intraperitoneal injection at mL/kg body weight. Mice were then subjected to Rotorod and MES tests for pharmacological activities 15-30 after injection. Prior to drug administration, the mice were put on an accelerating rotating treadmill which accelerated from 2-20 rotations per in min, and those that could pass this test by staying on the rotating rod for more than of the testing period were considered qualified for the following tests. Rotorod test 20 after drug administration, mice were put on the rotating rod, and the time duration when the mice could stand on the accelerating treadmill was recorded and used as a measure of impact on motor coordination. MES test Mice were shocked electrically with auricle electrodes at 40 mA for 0.2 sec. At his magnitude, all control (vehicle-injected) mice exhibited characteristic clonic-tonic convulsion which can be briefly described by four stages: clonus, tonic flexor, full tonic extensor (hindlimbs), and a pre-recovery period with loss of righting reflex. Under the influence of a drug with anticonvulsant activity, mice may show seizure 166 activities up to different stages depending on the dose and the efficacy of the drug used. Therefore, mice that showed the full range of seizure activities were arbitrarily assigned an “anticonvulsant score” of 1, while mice with seizure up to the flexor phase, 2; clonus, 3; and no seizure, 4. 4.5.4 Cell culture and cell viability test SH-SY5Y cell culture and experimental treatment of hydrogen peroxide Human neuroblastoma SH-SY5Y cells were obtained from American Type Culture Collection (Rockville, MD, USA). Cells were routinely cultured in MEM-Eagle/F-12 (HAM) (1:1), containing 10% FBS and a mixture of 2.5% sodium bicarbonate. Cell cultures were incubated at 37℃ in a humid 5% CO2-95% air environment. For experiments with H2O2, SH-SY5Y cells (6x103/well) were seeded in the cultured medium and allowed to attach for 24 hrs before drug treatment. Test samples were prepared at 100 µM in DMSO as stock. The sequential dilution using culture medium was to attain the required concentration. Test samples were added to the culture medium hrs before H2O2 treatment and cells were further incubated for 20 hrs. Vehicle-treated cultures were used as controls. Experimental treatment of serum deprivation SH-SY5Y cells were seeded (5x103/well) in 96-well plates and allowed to attach for 24 hrs before drug treatment. Serum-free medium was applied after removing medium in each well, and drugs dissolved in serum-free medium were given simultaneously. Cells were further incubated for 48 hrs. Vehicle-treated cultures were used as controls. Cell viability assay (MTT assay) 167 The MTT assay is based on the conversion of MTT to purple formazan crystals by viable cells. Briefly, after treatment the medium was removed and replaced with a fresh medium (100 µL/well). 10 µL of MTT (5 mg/mL) was added to each well. After incubation at 37 ℃ for hrs, the medium was removed and replaced with 100 µL DMSO. Absorbance was measured at 570 nm using a Tecan® microplate reader (Switzerland) after automatic subtraction of background readings. Cell viability was expressed as a percentage of control, untreated cells. 4.5.5 Electrophysiological study of compound 20 (3112) on Xenopus oocytes expressing NR1/NR2B cRNA for rat NR1-1a (hereafter NR1) and NR2B were synthesized in vitro and injected (5-10 ng) into stage V-VI Xenopus laevis oocytes, isolated as previously described127. Two electrode voltage-clamp current recordings were made 2-7 days post injection. The recording solution contained (in mmol) 90 NaCl, KCl, 10 HEPES, 0.5 BaCl2, 0.01 EDTA, 0.05 glycine (23 ℃); pH was adjusted to 7.3 with NaOH. EDTA (10 µM) was added to chelate contaminant extracellular divalent ions, including trace amounts of Zn2+. Solution exchange was computer controlled through an 8-modular valve positioned (Digital MVP Valve, Hamilton Company, Reno, NV). Voltage and current electrodes were filled with 0.3 and 3.0 M KCl, respectively, and current responses recorded at a holding potential of -50 mV. Data acquisition and voltage control were accomplished with a two-electrode voltage-clamp amplifier (OC-725, Warner Instruments, Hamden, CT). Current response was expressed as the percentage of maximal of induced current in the same oocyte and pooled among oocytes. 4.5.6 Statistics 168 ED50 values were calculated using Chou’s median-effect equation84. The seizure score obtained in the MES test were statistically evaluated using Kruskal-Wallis nonparametric ANOVA. In other cases, parametric ANOVA followed by Dunnett’s test was performed to compare the effects of drug treatment with the control. Difference between control and treatment would be considered statistically significant if P value was less than 0.05. All statistical tests were performed using commercially available SPSS 11.0 for Windows. 169 5. 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The Journal of Neuroscience 1998, 18 (16), 6163-6175. 179 [...]... project, the endeavour to search for potential antiepileptic and neuroprotective agents will be made on natural products, exemplified by both Gou Teng and Tian Ma, and synthetic compounds tentatively targeting at NMDA receptor In the following sections, the current trend in drug discovery from natural products, previous phytochemical and pharmacological studies on Gou Teng and Tian Ma, NMDA antagonism as a. .. used for the treatment of epilepsy constitute both Gou Teng and Tian Ma Moreover, a recent investigation has clearly indicated that co-application of extracts derived from Gou Teng and Tian Ma would significantly delay the onset time of wet dog shakes (WDS) in kainic-acid treated rats3 Besides, polypharmacy is a widely employed strategy in the pharmacotherapy of epilepsy due to the complexity in classification... 3 Pre-fractionated natural products library prepared from plants 9 4 Methodology adopted to obtain the standardized extracts from Gou Teng and Tian Ma 43 5 Preparation of the alkaloidal extract 45 6 Preparation of the EFME from Tian Ma 63 7 Retro-synthesis of BHE, HFC and CSD 75 8 Retro-synthesis of HPMP 75 9 Synthesis of HPMP 76 10 Conversion of 4,4’-diaminophenyl methane to DHPM by diazotization/hydrolysis... chromatography on silica column Followed by polyamide cartridge Flash fractions 2-5 Flash fractions 6 Parallel four-channel prep-HPLC 40 sub-fractions for each flash fraction Parallel eight-channel HPLC-ELSD-MS analysis HTS Active sub-fractions Purification and characterization Novel pharmaceutical lead Scheme 3 Pre-fractionated natural products library prepared from plants 11 The organic and aqueous... physicochemical properties which are beneficial to group and analyze them when extraction and separation are carried out However, the drawbacks of this approach are apparent as well First, purification of one chemical class from natural products may not be suitable for the HTS Secondly, it may also ignore the possible interaction present in these structurally related structures Last but not the least, the assumption... FIGURE PAGE 1 Qualitative pharmacophore for antagonists at Gly/NMDAR based on the structure of CTHQ 36 2 A developed pharmacophore model 37 3 Substituted 2-oxindoles proposed as potential antagonistic effect at Gly/NMDAR 38 4 EMS scans for peak 1 in TIC of the alkaloidal extract 46 5 Total ion chromatogram (TIC) of the alkaloidal extract (A) ; UV spectrum of the alkaloidal extract (B) 47 6 General structure... site as a partial agonist 123 xiv LIST OF TABLES TABLE PAGE 1 in vitro & in vivo model for epileptogenesis 32 2 Estimated MW and proposed structures for each component in alkaloidal extract 47 3 13 54 4 Quantities of five major alkaloids in the crude extract and in the alkaloidal extract of Gou Teng 59 5 Proposed structures and their characteristic ion fragments 64 6 ED50 of the standardized extracts for. .. of alkaloids1 The constituents of Tian Ma which are mainly small phenolic compounds were studied since the 1980s 2 On the basis of these phytochemical investigations, other researchers have either employed crude extracts 1 or single major constituents to study the pharmacological actions of these two herbs The reported activity can vary greatly, from antiepileptic to antiproliferative; from vasodilating... agents, two most famous traditional Chinese medicines (TCMs) which are monographed in Chinese Pharmacopoeia with antiepileptic effects, Gou Teng (Latin name: Ramulus Uncariae Cum Uncis) and Tian Ma (Latin name: Rhizoma Gastrodiae) have drawn our attention Since 1970s, J.D Phillipson and his co-workers have started the phytochemical study on the genus of Uncaria, and their focus was mainly on defining interesting... chemistry that a great variety of structures with “druglikeness” can be generated with less effort than natural products has diverted the attention of large pharmaceutical companies; third, the decision-makers in the large pharmaceutical companies has placed less focus on research in the area of infectious diseases, which traditionally has been a strong area of discovering new antibiotics from natural products7 . Methodology adopted to obtain the standardized extracts from Gou Teng and Tian Ma. 43 5 Preparation of the alkaloidal extract 45 6 Preparation of the EFME from Tian Ma. 63 7 Retro-synthesis. new antiepileptic and neuroprotective agents. Our aim is to search for active small molecules or molecular combinations from Gou Teng and Tian Ma, and to design and synthesize new NMDA receptor. A COMBINATORIAL APPROACH TO THE SEARCH FOR ANTICONVULSANT AGENTS FROM GOU TENG AND TIAN MA YANG HONG (B. Sc. (Pharm.)), Sichuan Univ. (M. Sc. (Pharm.)), Sichuan Univ.