Description
This detailed book explores protocols for a wide array of preclinical pharmacology and toxicology evaluations to be applied to chemical drugs and their development through in vitro, involving tissues and cell lines, and in vivo models, using animals as experimental systems, utilized to conduct pharmacological research. Written for the Springer Protocols Handbooks series, the methodologies included in this collection have been standardized by the authors through extensive use in the lab so that they are ready to be applied in the labs of readers around the world. Authoritative and practical, Bioassays in Experimental and Preclinical Pharmacology aims to assist undergraduate and postgraduate students, research scholars, scientists, and other academicians performing research in the vital field of drug discovery. 1. Overview 2. Cell Culture Assays 2.1 Cell Cycle Assay: Propidium Iodide Marking 2.2 Apoptosis Assay: Annexin V/Propidium Iodide Marking 2.3 Cytotoxicity in Caco-2 cells by the Alamar blue method 2.4 Viability in Caco-2 cells stimulated with lipopolysaccharide (LPS) 2.5 Determination of Nitric oxide (NO) by in vitro experiment 2.6 Analysis of specific cytotoxicity against RAW 264.7 for determination anti-inflammatory activity in vitro 2.7 Scratching of Fibroblast cells (scratch assay) 2.8 Evaluation of the in vitro inflammatory response in AGS cells 3. Phytochemical Analysis 3.1 Preliminary phytochemical assays 3.2 Quantitative analysis of secondary metabolites 3.3 Thin Layer Chromatography Analysis 3.4 Analysis by High Performance Liquid Chromatography (HPLC) 3.5 Mass spectrometry 3.6 Electrospray Ionization Mass Spectrometry (ESI-MS) analysis 3.7 Identification of phenolic compounds by HPLC-LC-ESI- MS/MS 3.8 Quantitative determination of total alkaloids content by HPLC 3.9 Quantitative determination of total alkaloids content by HPLC 4. Preclinical Drug Dose Calculation 4.1 Conversion based on normalization of dose-to body surface area 4.2 Determination of animal equivalent dose 5. Toxicology Studies (In Vitro and In Vivo) 5.1 Cytotoxicity evaluation using Alamar blue 5.2 Micronucleus test 5.3 Comet test 5.4 Effect on cell cycle 5.5 In vivo acute toxicity study 5.6 Subchronic toxicity (repeated doses) 6. Standard Procedure for Anesthesia in Preclinical Experiments 6.1 Planning and anesthetic care 6.2 Pre-Procedure 6.3 Experimental Procedure 7. General Considerations and Collection of Animal Blood 7.1 Vein blood collection 7.2 Tail vein blood collection 7.3 Cardiac puncture blood collection 7.4 Posterior vena cava 7.5 Orbital sinus 8. Animal Experiments on the Cardiovascular System 8.1 Obtaining electrocardiogram (ECG) and blood pressure (BP) signals 8.2 Myocardial ischemia induction 9. Animal Experiments on Ulcerative Colitis 9.1 Ulcerative colitis induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) 9.2 Chronic ulcerative colitis induced by Dextran Sodium Sulfate (SSD) 2% 9.3 Colitis with recurrence (Chronic) 10. Experiments on Anti-Bacterial Activities 10.1 Antibacterial activity by in vitro assays 10.2 Animal Experiments on antibacterial activity 11. Experiments on Anti-Fungal Activities 11.1 Agar Disc Diffusion Method 11.2 Minimum Inhibitory Concentration (MIC) 11.3 Sorbital test 11.4 Neurospora crassa assay 11.5 Effect of Ergosterol on MIC 11.6 Test of 1,3-D-Glucan Synthase (GS) 11.7 Virulence Factors Inhibition of Candida albicans 11.8 Germinative Tube Inhibition Test 11.9 Biofilm assay 11.10 Time-kill studies 11.11 Determination of Antifungal Activity by in vivo 12. Animal Experiment on Wound Healing Activity 12.1 Excision wound healing model 12.2 Incision wound healing model 12.3 Evaluation of the effect of the extract on migration/proliferation in fibroblasts 12.4 Surgical wound infected in rats (in vivo) 13. Animal Experiments of Anti-inflammatory activities 13.1 Inflammatory Paw edema 13.2 Lipopolysaccharide (LPS)-induced peritonitis 13.3 Murine Model of Immediate Hypersensitivity Induced By OVA 13.4 Air pouch inflammation model 13.5 Cotton pellet inflammation model 14. Animal Experiment on Anti-Nociceptive Activities 14.1 Abdominal constriction by acetic acid 14.2 Formalin test 2.5% 14.3 Hot plate Test 15. Experiments on Anti-Oxidant Activities 15.1 DPPH free radical scavenging activity 15.2 Hydrogen peroxide radical scavenging activity 15.3 Free radical nitric oxide scavenging activity 15.4 Ferric Reducing Antioxidant Power assay 15.5 Determination of superoxide dismutase (SOD) 15.6 Glutathione peroxidase (GPx) activity 15.7 Determination of catalase (CAT) 15.8 Determination of reduced glutathione (GSH) 15.9 Determination of malonaldehyde (MDA) 15.10 Total protein dosage 15.11 Test of oxidative degradation of deoxyribose 16. Animal Experiments on Hepatoprotective Activities 16.1 Acute liver damage by paracetamol 16.2 Treatment of acute ethanol-induced injury 16.3 The induction of liver damage by carbon tetra chloride (CCl4) 17. Animal Experiments on Anti-Diarrheal Activities 17.1 Gastric emptying 17.2 Intestinal transit 17.3 Enter pooling induced by castor oil 17.4 Microvascular permeability 17.5 Activity on the H+/K+ pump – ATPase Animal Experiments on Central Nervous System (CNS) 18.1 Stress Induced experiment 18.2 Elevated Cross Maze (ECM) 18.3 Open Field Test (OFT) 18.4 Object Recognition Test (ORT) 18.5 Murinometric evaluation Experiments on Anti-Cancer Activities (In Vitro and In Vivo) 19.1 Cytotoxicity test 19.2 Cell cycle assay 19.3 Assessment of cell viability and proliferation using the MTT method 19.4 Evaluation of clonogenic survival of different cell lines 19.5 In vivo anti-tumor activity Animal Experiments on Anti-Diabetic Activities 20.1 Inhibition of a-amylase 20.2 Inhibition of a-glucosidase 20.3 In vivo Antidiabetic activity Animal Experiments on Gastric Ulcer 21.1 Gastric Injury Induced by Ethanol 21.2 Gastric Injury Induced by Acidified ethanol (Ethanol/HCl) 21.3 Indomethacin induced gastric injury 21.4 Gastric Injury Induced by Acetic Acid (chronic ulcer) in mice 21.5 NSAID ulcer experiment induced by Piroxicam 21.6 Pylorus ligation experiment in mice 21.7 Stress-induced gastric injury (water restraint) 21.8 Role of NO in a gastric ulcer model pretreated with L-NAME 21.9 Gastric Injury Induced by Ethanol/HCl for quantification of mucus 21.10 Ethanol/HCl-Induced Gastric Lesion with Indomethacin, L-NAME, Glibenclamide, Yohimbine 21.11 Ischemia-reperfusion-induced gastric ulcer 21.12 Evaluation of anti-H. pylori activity in vivo 21.13 Evaluation of anti-Helicobacter pylori activity in vitro Protein Extraction and Western Blot Analysis 22.1 Gel preparation 22.2 Samples application 22.3 Running 22.4 Preparing Membranes 22.5 Revelation Gel Electrophoresis and PCR Amplification 23.1 DNA Extraction 23.2 Dosage of RNA and DNA 23.3 Solutions for Electrophoresis and Western blotting Molecular Docking Methods for Drug Design 24.1 Biological activity calculation 24.2 Molecular docking between the inhibitors and target proteins
