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© United Nations Industrial Development Organization and the International Centre for Science and High Technology, 2006 Earth, Environmental and Marine Sciences and Technologies International Centre for Science and High Technology ICS-UNIDO, AREA Science Park Padriciano 99, 34012 Trieste, Italy Tel.: +39-040-9228108 Fax: +39-040-9228136 E-mail: [email protected]

Biological Screening of Plant Constituents

Training Manual

Scientific Editors: Mahabir P. Gupta

Sukhdev Swami Handa Karan Vasisht

INTERNATIONAL CENTRE FOR SCIENCE AND HIGH TECNHOLOGY Trieste, 2006

Contributors Chapter 1 Bioassay Screening Methods in the Search for Pharmacologically Active Natural Substances Arnold J. Vlietinck Laboratory of Pharmacognosy, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium Chapter 2 Bioassays for Some Parasitic Protozoa: Screening Concepts and Standard In Vitro and In Vivo Laboratory Models Kristel Kuypers1, Paul Cos1, Eduardo Ortega-Barria2, Vanden Berghe Dirk1 and Louis Maes1

1 Laboratory for Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium 2 Institute for Advance Scientific Investigations & High Technology Services, P.O. Box 7250, Clayton Building 175, Panama, Republic of Panama Chapter 3 Bioassays for Antibacterial and Antifungal Activities Paul Cos1, Louis Maes1, Jean-Bosco Sindambiwe1, Arnold J. Vlietinck2 and Dirk Vanden Berghe1 1 Laboratory for Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium 2 Laboratory of Pharmacognosy, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium Chapter 4 Bioassays for Screening Antiviral Activity Paul Cos1, Louis Maes1, Dirk Vanden Berghe1, and Arnold J. Vlietinck2 1Laboratory for Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium 2Laboratory of Pharmacognosy, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium Chapter 5 Evaluation of Anti-inflammatory Activity of Natural Products Based on Enzyme Inhibitors Lars Bohlin1 and Ulrika Huss2

1Division of Pharmacognosy, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden 2International Foundation for Science, Karlavägen 108, SE-115 26 Stockholm, Sweden

Chapter 6 Pharmacological Screening Methods for Gastrointestinal System S. Oguz Kayaalp and K. Husnu Can Baser Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Yunus Emre Campus, 26470 Eskisehir, Turkey Chapter 7 Bioassay Models for Evaluation of Antihepatotoxic Activity Maninder Karan1, Jaswant Singh2 and Karan Vasisht1, 1 University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India 2 Regional Research Laboratory (CSIR), Canal Road, Jammu Tawi, India Chapter 8 A Practical Approach for Assessing Cardiovascular Activity of Natural Products Raimo Hiltunen, H. J. Damien Dorman and Yvonne Holm Faculty of Pharmacy, Division of Pharmacognosy, P.O. Box 56 (Viikinkaari 5E), FIN-00014 Helsinki, Finland Chapter 9 Bioassay Models for Antidiabetic Activity of Natural Products M. Iqbal Choudhary and Shamsun Nahar Khan International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

Preface

Natural products, derived from micro-organisms, insects, plants and animals are a source of food and medicine for mankind. Over the 4 billion years of evolution, competition of all life forms for survival has brought about a very rich biodiversity which has resulted in the formation of an impressive variety of chemicals by these organisms. This chemodiversity is a treasure house for phytochemists to find new chemicals and templates to develop new synthetic and semi-synthetic chemicals and for pharmacologists to discover biological activities. Microorganisms are the source of almost all potent antibiotics and antifungal agents. Higher plants are providing chemicals useful in treating a vast number of diseases ranging from simple disorders to complex diseases such as cancer. However, new leads are still needed to develop more effective medicines for a variety of known and incurable diseases. Therefore, research into in vivo and in vitro pharmacological techniques has intensified in the last three decades.

In the existing scenario of emerging global interest in medicinal and aromatic plants, it is worth exploring the traditional practices of medicine in developing countries for the invention of useful drugs or lead molecules. This is necessary to provide a scientific basis and differentiate safe and effective remedies from unsafe harmful practices.

Drug development requires a huge budget, modern facilities, multidisciplinary efforts and time consuming tasks. The patentability of the developed drug is of pivotal importance in financing drug development activities. The problems of patenting a natural product, protecting the intellectual property rights of the traditional knowledge of the country of origin, and paying the huge cost of development keep the involvement of the pharmaceutical companies in the development of natural products at a low, and frequently non-existent, level. For this reason, non-profit organizations such as WHO and UNIDO have so far had a leading role in encouraging and promoting the developmental activities of natural products.

Developing countries have a long tradition of using herbal medicines which provide the only medical care for most of the population. Developing a drug for global use in developing countries may be difficult but they can validate their own traditional remedies by simple techniques if provided with the means and the information. This will make folklore remedies safer and more reliable.

It is, therefore, pertinent to identify simple techniques of investigating traditional medicine and provide the necessary know-how and facilities. Bioassay guided pharmacological screening techniques have become an established protocol for the discovery of new bioactive compounds from plants and other sources. High throughput assays have automated and robotized the screening process allowing testing of millions of samples but these techniques are not readily available in developing countries. To satisfy the great capacity of high throughput screens based on molecular targets, large libraries of compounds are needed. Combinatorial chemistry was once thought to be the answer; however the expected output in terms of new chemical entities (NCE) for drug development has not yet materialized. Furthermore, a large number of drugs are known to be based on natural products and, therefore, exploration of nature for novel leads will always remain important to the development of drugs for various diseases.

The preparation of this manual is meant to consolidate the advances and needs of

bioscreening techniques that are relevant to diseases in developing countries. Experts agreed, at a two day workshop held in 2003 in Eskisehir, Turkey to discuss the issue, to put together a manual of easy to carry out techniques for modern screening methodologies of plant products for diseases in developing countries. The diseases requiring attention were prioritized and short-listed.

This manual focuses on these target diseases: infectious and parasitic diseases,

inflammatory, gastrointestinal, hepatic, cardiovascular disorders and diabetes. In order to encourage work on traditional medicines, the manual describes bioassay techniques applicable in most laboratories. The manual will contribute towards validating traditional claims and discovering bioactive molecules of natural origin. Gennaro Longo

Chief of Environment Area Special Adviser on Technology Development

Contents 1 Bioassay Screening Methods in the Search for Pharmacologically Active

Natural Substances

1

1.1 Introduction 1

1.2 Role of Bioassays 1

1.3 Classification of Bioassays 2

1.4 Selection of Target Diseases 3

References 4

2 Bioassays for Some Parasitic Protozoa: Screening Concepts and Standard In Vitro and In Vivo Laboratory Models

7

2.1 Introduction 7

2.2 The Drug Discovery Process 7

2.2.1 Subcellular Target Approach 8

2.2.2 Conventional Test Systems 8

2.3 ‘Hit’ Finding in Primary Screening 8

2.4 Description of Laboratory Models 10

2.4.1 Lieshmania 10

2.4.1.1 In Vitro Model 10

2.4.2.2 In Vivo Model 11

2.4.2 African Trypanosomes (Sleeping Sickness) 12



2.4.3 American Trypanosomes (Chagas Disease) 13



2.4.4 Plasmodium (Malaria) 14



2.4.5 Cytotoxicity 15

2.5 Efficacy Criteria for Active Compounds 16

2.6 Conclusions 16

References 17

3 Bioassays for Antibacterial and Antifungal Activities 19

3.1 Introduction 19

3.2 Overview of Antibacterial and Antifungal Assays 19

3.2.1 Diffusion Methods 20

3.2.2 Dilution Methods 21

3.2.3 Bio-autographic Methods 21

3.3 Compound Handling 21

3.4 Protocol of Broth Dilution Method 22

3.4.1 Preparation of Culture Medium 22

3.4.2 Preparation and Maintenance of Bacterial and Yeast Cultures 23

3.4.3 Preparation of Test Sample 24

3.4.4 Antimicrobial Evaluation Procedure 24

3.5 Protocol for Antifungal Test Method 25

3.5.1 Preparation of Culture Medium 25

3.5.2 Preparation and Maintenance of Fungal Cultures 25

3.5.3 Antifungal Evaluation Procedure 26

3.6 Conclusions 26

References 27

4. Bioassays for Screening Antiviral Activity 29

4.1 Introduction 29

4.2 Overview of Antiviral Assay 29

4.3 Virus Battery 30

4.4 Description of In Vitro Antiviral Screening Assay 31

4.4.1 Endpoint Titration Technique (EPTT) 31

4.4.1.1 Introduction 31

4.4.1.2 Propagation of VERO Cell Cultures 32

4.4.1.3 EPTT Method 32

4.4.1.4 Discussion 33

4.4.2 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay

33


4.4.2.2 Material and Equipment 34

4.4.2.3 Procedure 34

4.4.3 Description of an Animal Model for HSV Infection 34


4.4.3.2 Animal Model 34

4.4.3.3 Discussion 35

4.5 Conclusions 35

References 36

5. Evaluation of Anti-inflammatory Activity of Natural Products Based on Enzyme Inhibitors

37

5.1 Introduction 37

5.1.1 Inflammation 37

5.1.2 Neutrophils 38

5.2 The Enzyme Family of Cyclooxygenases 38

5.2.1 Cyclooxygenase-1 and -2: Similarities and Differences 39

5.2.2 Cyclooxygenase-2 Inhibitors 39

5.2.3 Brief Overview of Assay for Evaluating Cyclooxygenase-2 Inhibitory Effect of Natural Products

40

5.2.4 Determination of Cyclooxygenase-1 and -2 Enzymatic Inhibitory Activity

40

5.2.5 The Radiochemical In Vitro Assay 41

5.3 Protease Inhibitors 43

5.3.1 The Enzyme Family of Elastases 43

5.3.2 The Neutrophil Multitarget Assay 44

5.4 Safety Guidelines 45

5.5 Natural Products as Source of Anti-inflammatory Compounds 45

5.5.1 Phorbol Esters 46

5.5.2 Thapsiagargin 46

5.5.3 Ginkgolides 46

5.5.4 Cyclosporin A 47

5.5.5 Anti-inflammatory Activity of Certain Plants in Traditional Medicine 47

5.6 Conclusions 47

5.7 Acknowledgement 48

References 48

6 Pharmacological Screening Methods for Gastrointestinal System 51

6.1 Introduction 51

6.2 In Vitro Methods Using Gastrointestinal Tissues 51

6.2.1 Isolated Ileum Preparation (Magnus Method) 52

6.2.2 Electrically Stimulated Guinea Pig Ileum Preparation 53

6.2.3 Guinea Pig Mesenteric Plexus-longitudinal Muscle Preparation 54

6.2.4 Guinea Pig Ileum Preparation (Modified Method of Trendelenburg) 55

6.2.5 Rabbit Periarterial Sympathetic Nerve Jejunum Preparation (Finkelman Method)

55

6.2.6 Rat Gastric Fundus Preparation (Vane Method) 56

6.2.7 Isolated Guinea Pig Oddisphincter and Gall Bladder Preparation 56

6.2.8 Test for Histamine H2 Receptor Antagonist on Mucosal Adenylate Cyclase

57

6.2.9 Rat Uterus Preparation 58

6.2.10 Test for Proton Pump Inhibition 58

6.3 Methods to Evaluate Antiulcer Drugs 59

6.3.1 In Vivo Evaluation in Gastric Ulcers 59

6.3.2 Method Using Pylorus Ligation Ulcers 59

6.3.3 Method Using Immobilization Induced Stress Ulcers 60

6.3.4 Method Using Cold Exposure Induced Stress Ulcers 60

6.3.5 Method Using Indomethacin or Like Substances Induced Ulcers 61

6.3.6 Method Using Ethanol Induced Mucosal Lesions 61

6.3.7 In Vitro Evaluation of Antacids 61

6.3.8 In Vivo Evaluation in Duodenal Ulcers 62

6.4 Methods for Screening Antidiarrhoeal Activity 62

6.4.1 Castor Oil Induced Diarrhoeal Model 63

6.4.2 Enteropooling Assay in Rats 63

6.4.3 Diarrhoea Induced by Surgical Removal of Caecum in Rats 63

6.4.4 Diarrhoea Induced by Cold Restrain in Rats 64

6.5 Method of Evaluation of Effect on Propulsive Intestinal Motility 64

6.6 Anti-inflammatory Evaluation 65

6.6.1 Anti-inflammatory Evaluation in Ileitis 65

6.6.2 Anti-inflammatory Evaluation in Colitis 66

6.7 Methods for Evaluation of Emetic and Antiemetic Action 68

6.8 Conclusions 68

References 69

7 Bioassay Models for Evaluation of Antihepatotoxic Activity 73

7.1 Introduction 73

7.2 Bioassay Screening Techniques 74

7.3 In Vivo Bioassay Methods 74

7.3.1 Liver Toxins 75

7.3.1.1 Carbontetrachloride (CCl4) 75

7.3.1.2 Paracetamol (Pcml) 75

7.3.1.3 D-Galactosamine (GalN) 75

7.3.1.4 Ethanol 75

7.3.1.5 Thioacetamide 75

7.3.2 Biochemical Parameters for Evaluating Hepatic Functions 75

7.3.2.1 Transaminases (AST/ALT or GOT/GPT) 76

7.3.2.2 Alkaline Phosphatase (ALP) 76

7.3.2.3 Gamma-Glutamyl Transpeptidase (γ-GT) 76

7.3.2.4 Sorbitol Dehydrogenase (SDH) 76

7.3.2.5 Glutamate Dehydrogenase (GIDH) 76

7.3.2.6 Bilirubin (BRN) 76

7.3.2.7 Hepatic Triglycerides (HTG) 76

7.3.2.8 Hepatic Glycogen (HGN) 77

7.3.3 Test Protocols 77

7.3.3.1 Animals 77

7.3.3.2 Equipment 77

7.3.3.3 Chemicals, Solvents and Reagents 77

7.3.3.4 Procedure 78

7.3.3.4.1 Carbon Tetrachloride Model 78

7.3.3.4.2 D-Galactosamine Model 78

7.3.3.4.3 Paracetamol Model 78

7.3.3.5 Estimation of Biochemical Parameters 79

7.3.3.5.1 Sorbitol Dehydrogenase (SDH) 79

7.3.3.5.2 Glutamate Dehydrogenase (GIDH) 79

7.3.3.5.3 Glutamic Oxaloacetate Transaminase (GOT) 79

7.3.3.5.4 Glutamic Pyruvic Transaminase (GPT) 80

7.3.3.5.5 Alkaline Phosphotase (ALP) 80

7.3.3.5.6 Hepatic Triglycerides (HTG) 80

7.3.3.5.7 Hepatic Glycogen (HGN) 80

7.3.3.6 Histopathological Examination of the Liver 81

7.3.3.7 Barbiturate-induced Sleep Model 81

7.3.3.8 Estimation of Liver Protective Activity 81

7.3.3.9 Statistical Evaluation 82

7.4 In Vitro Bioassay Method Using Rat Hepatocyte Primary Monolayer Cell Culture

83

7.4.1 Biochemical Parameters for Evaluating Hepatic Functions in Cell Culture

83

7.4.1.1 Lactate Dehydrogenase (LDH) 83

7.4.1.2 Glutathione (GSH) 84

7.4.2 Requirements 84

7.4.2.1 Animals 84

7.4.2.2 Equipment and Apparatus 84

7.4.2.3 Chemicals, Solvents, Reagents and Test Solutions 84

7.4.3 Method 85

7.4.3.1 Preparation of Collagen and Collagen-coated Plates 85

7.4.3.2 Isolation of Hepatocytes 85

7.4.3.3 Primary Culture of Adult Rat Hepatocyte and Treatment with Toxicants

86

7.4.3.4 Estimation of Biochemical Parameters 86

7.4.3.4.1 Lactate Dehydrogenase (LDH) 86

7.4.3.4.2 Glutamic Pyruvic Transaminase (GPT) 86

7.4.3.4.3 Glutathione (GSH) 86

7.5 Conclusions 87

References 87

8 A Practical Approach for Assessing Cardiovascular Activity of Natural Products 91

8.1 Introduction 91

8.1.1 Cardiomyopathy and Heart Failure 91

8.1.2 Arrhythmias 91

8.1.3 Coronary Heart Disease 92

8.1.4 Atherosclerosis 92

8.1.5 Deep Vein Thrombosis and Chronic Venous Insufficiency 93

8.1.6 Hypertension 93

8.2 Toxicology and Safety 95

8.3 Bioassay and Evaluation 95

8.3.1 Synthetic Free Radical Scavenging Assessment 95

8.3.1.1 Preparation of the ABTS Free Radical 95

8.3.1.2 Preparation of Calibration Standards 96

8.3.1.3 Methodology 96

8.3.2 Nitric Oxide (NO) and Nitrite (NO2) Scavenging Assessment 96

8.3.2.1 Flow Injection Analysis 96

8.3.2.2 Treatment of the Blood Samples 97

8.3.2.3 Calculation of Nitrite Concentration 97

8.3.3 Experimental Procedure for the Investigation of the Potential Inhibitory Effect of a Plant Extract upon Experimental Cardiac Hypertrophy

98

8.3.3.1 Materials 98

8.3.3.2 Animals 98

8.3.3.3 Surgery 98

8.3.3.4 Administration of Extracts 98

8.3.3.5 Determination of Time Course of Blood Pressure Changes during Experimental Period

98

8.3.3.6 Assessment of Hemodynamic Parameters 99

8.3.3.7 Assessment of Cardiac Hypertrophy 99

8.3.3.8 Statistical Analysis 99

8.3.4 Determination of Isometric Contraction Force Using Guinea Pig Papillary Muscles, Aortic Strips, and Terminal Ilea and Chronotropic Activity Using Guinea Pig Right Atria

99

8.3.4.1 Solution 99

8.3.4.2 Tissue Isolation 100

8.3.4.3 Chronotropic Activity Tested on Guinea Pig Isolated Right Atria

100

8.3.4.4 Experimental Set Up 100

8.3.4.5 Methodology 100

8.3.4.6 Chronotropic Effect of Extract In Vivo after Intravenous Administration and In Vitro on Isolated Atria of Aortic Coarctated Rat

100

8.3.4.7 Induction of Hypertension 101

8.3.4.8 In Vivo Experiment: Extract Chronotropic Effect 101

8.3.4.9 Chronotropic Effect of an Extract in Isolated Atria 101

8.3.4.10 Data and Statistical Analysis 101

References 102

9. Bioassay Models for Antidiabetic Activity of Natural Products 105

9.1 Introduction 105

9.1.1 Pathophysiology and Syndrome of Type 2 Diabetes 105

9.1.2 Complications and Risk Factors in Diabetes 106

9.1.3 Treatment of Type 2 Diabetes 106

9.2 Bioassay Models 106

9.2.1 Antidiabetic Assay in Rats or Mice 107

9.2.2 Induction of Diabetes in Experimental Animals 107

9.2.2.1 Streptozotocin-induced Diabetes Model 107

9.2.2.2 Alloxan-induced Diabetes Model 108

9.2.3 Preparation and Administration of the Test Sample 108

9.2.4 Evaluation of Antidiabetic Activity 108

9.2.4.1 Glucose Estimation (GOPOD Method) 109

9.2.4.2 Estimation of Reducing Sugar in Urine 110

9.2.5 Statistical Analysis 110

9.3 Alpha Glucosidase Inhibitors (AGIs) 110

9.3.1 Method of Assessment of the α-Glucosidase Inhibitory Activity 111

References 112

BIOLOGICAL SCREENING OF PLANT CONSTITUENTS

1

1 Bioassay Screening Methods in the Search for

Pharmacologically Active Natural Substances

Arnold J. Vlietinck

1.1 Introduction

Nature with its infinite biodiversity still remains an unparalleled reservoir of new molecules with potential application in medicine, the food industry and agriculture. Plants, animals and micro-organisms synthesize a large number of 'secondary metabolites' with an enormous diversity of chemical structures. These compounds cannot be considered as waste products and are involved in the relationship of the organism with its environment e.g. to impart resistance against pests and diseases, as signal products or as attractants of pollinators. Consequently, they can play an economically important role as specialty chemicals. They afford, for example, prototype substances as leads or feedstock-products for the development of drugs or health products. Some of them can also be employed as flavours, fragrances, dyes, cosmetics, insecticides, antifeedants, pesticides and rodenticides.1-6

The starting materials, especially higher plants, have proved their value as

traditional medicines, dietary supplements, spices and foods. It is estimated that up to 80% of the population in developing countries rely on traditional medicine for primary health care. In many cases herbal medicinal products may be the only medicines available because of socioeconomic reasons. Moreover, in countries such as India and China, well developed systems of traditional medicine are in practice and are very popular.7 In Western countries a 'herbal remedy revolution' has taken place in recent years resulting in the registration in Europe of many herbal medicinal products as drugs for the treatment of mild to moderate symptoms of non life-threatening diseases.8, 9

Many of the most effective phytomedicines on the market are whole extracts of

plants and practitioners believe that synergistic interactions between the components of individual or mixtures of medicinal plants are vital for their therapeutic efficacy.10 Consequently, it is no longer necessary to isolate the active constituents of a medicinal plant, although it remains necessary to determine the identity of the principal active compounds so that chemical profiling and establishment of phytoequivalence can be more precise. Beyond this point phytotherapy separates from non-phytotherapy.11 1.2 Role of Bioassays

It is clear that bioassays play a crucial role in the isolation (or discovery) of new bioactive natural products. This is illustrated in a flow chart of the general sequence of steps in studying plants used in traditional medicine (Figure 1).

In the first approach, a bioassay-guided isolation of a pharmacologically active plant

extract leads to the purification and structure elucidation of bioactive compound(s). Structure activity relationship (SAR) studies may result in more active and/or less toxic substances with improved bioavailability properties. These leads, after scale up, become normal competitors of synthetic leads for developing a new drug.

BIOASSAY SCREENING METHODS IN THE SEARCH FOR PHARMACOLOGICALLY ACTIVE NATURAL SUBSTANCES

2

In the second approach, active constituents are not isolated but are identified using several chromatographic and other techniques. Chemical profiling and quantitative determination of these active constituents afford a standardised extract, which can be screened for toxicity and safety before carrying out clinical tests to develop them into herbal drugs.

Figure 1: Flow chart showing sequence of steps for the study of plants used in traditional medicine

In both approaches not only are general requirements such as validity, lack of ambiguity, accuracy, reproducibility, simplicity and reasonable costs of the bioassays considered, but special considerations for screening plant extracts should also be taken into account.

The methodology should be adaptable to highly coloured, tarry, poorly water-soluble

and chemically complex materials. The bioassay should be selective to limit the number of leads for follow-up evaluation, specific to eliminate false-positives and sensitive to detect active compounds in low concentrations.12

Most of the aforementioned requirements are better met in in vitro testing.

Therefore, in most screening programmes only successful in vitro candidates are subjected to in vivo testing which is introduced during a secondary evaluation.

It should also be noted that screening programmes organized by a large

pharmaceutical company are substantially different from a consortium of several university research groups or an individual research group of a university or a small pharmaceutical firm. It is therefore necessary to consider the bioassays in the context of their usage before one can evaluate them or understand their benefits and weaknesses.13 1.3 Classification of Bioassays

The methods of detection of biological activity of natural products can be classified into two groups: general screening bioassays and specialized screening bioassays. Depending on the aim of a screening programme, either a general screening which can pick up many

MEDICINAL PLANT

APPROVAL AS DRUG(S)

GALENICAL PREPARATION(S)

INDUSTRIAL PRODUCTION OF STANDARDIZED EXTRACT(S)

CLINICAL TESTS

TOXICITY AND SAFETY STUDIES

SAR STUDIES

PURE ACTIVE COMPOUNDS

STANDARDIZED EXTRACT(S)

BIOASSAY(S)

EXTRACT(S)

INDUSTRIAL PRODUCTION OF ACTIVE COMPOUND(S)


3

different effects, or a specific assay which is directed at finding an activity against a specific disease has to be performed. A broad screening bioassay is more useful if random screening of organisms to select any kind of pharmacological activity is performed. The alternative to broad screening is setting up of a battery of specific test methods, which is cumbersome and expensive.

The drawback of using a general screening and monitoring is that it does not allow

us to know if the work is worth doing until the active compounds have been isolated. Most phytochemical laboratories engaged in the bioassay-guided isolation of active compounds from natural sources cannot use complex bioassays. Therefore, efforts have been made to introduce simple, inexpensive 'front-line' or 'bench-top' bioassays for the rapid screening of extracts and fractions. Care must be taken in interpreting and predicting the ability of these screening bioassays, but in general they provide interesting preliminary information on the pharmacological potential of plant extracts.14 Primary screening bioassays can be applied in-house by chemists, pharmacognosists, botanists and others, who lack the resources or expertise to carry out more elaborate bioassays. Many specialized screening bioassays have been or are being developed, however they are increasingly sophisticated and require the skills and expertise of biologic scientists.

Specialized screening bioassays can be subdivided according to the target organisms

used in the model.13 These can be lower organisms (micro-organisms, insects, molluscs, protozoa and helminths), isolated subcellular systems (enzymes, receptors and organelles), isolated human or animal cells, isolated organs of vertebrates, or whole animals.

In all cases, specialized bioassays have to be relevant which means that they should

predict the intended therapeutic indications. To be relevant or 'correlational' a bioassay has to fulfil some basic criteria. Firstly, the bioassay must be sensitive in a dose-dependent manner to standard compounds with the desired therapeutic effect. Secondly, the relative potency of known active agents in the bioassay should be comparable to their relative potency in clinical use. Thirdly, the bioassay should be selective i.e. the effects of known agents in this therapeutic indication should be distinguishable from the effects of drugs in other indications.15

It is clear that the degree of relevance increases from subcellular systems (molecular

assays) to cellular systems (cellular assays), to organs, to live animals and finally human volunteers. Although considerable discussion is going on about the necessity of animal experiments but there is little doubt of their necessity in the discovery and evaluation of drugs. However, they should be performed only when necessary and well conceived.16

Molecular bioassays which use isolated subcellular systems have some contrasting

characteristics with cellular bioassays which use intact cells. The primary feature of molecular bioassays is their high specificity. It is possible to find a small number of new compounds with a specific activity in a selective way in a relatively short period of time using either a high capacity receptor binding or enzyme inhibition assay. The hit rate is quite low, necessitating screening of large numbers of diverse samples. All compounds acting on a mechanism unrelated to the assay will be missed, whereas agents that do not enter the cells or are rapidly metabolised will be recorded as false positives. The cost of screening is low but the cost of the large number of samples required must be considered. This type of screening is ideal in the first step of testing a specific hypothesis for the potential of agonists or antagonists of a particular molecular target to demonstrate pharmacological activity.17

1.4 Selection of Target Diseases

Since an enormous number of specialized screening bioassays have been described in the literature, it was decided to limit our evaluation to those bioassays currently used to screen traditional medicines for target diseases which occur more frequently. Nevertheless, priority was

BIOASSAY SCREENING METHODS IN THE SEARCH FOR PHARMACOLOGICALLY ACTIVE NATURAL SUBSTANCES

4

given to target diseases which occur mainly in developing countries, because the traditional usage of natural drugs is still prominent in these countries. Moreover, integration of traditional remedies into existing health care programmes is one of the major aims of the World Health Organisation (WHO). Consequently, it is desirable to evaluate specialized screening bioassays used by different research groups to find new leads or standardised extracts for clinical investigations.

Many indices can be employed to select the target diseases to be dealt with but priority

should be given to health problems that cause a large disease burden and for which cost-effective interventions are not available. Health problems are often measured in terms of morbidity or mortality or a combination of both, but can also be considered by disease incidence and prevalence.

If mortality is an index of disease burden, infections remain the most common causes

of death. HIV, malaria and other infective agents account for more than 70% of the causes of death in many African countries although diabetes, stroke and cancer, previously considered to be diseases of the industrialized world, are now on the increase in developing countries.

Mortality as an indicator of health problems neglects ill health or disability and social

problems. A study undertaken jointly by the World Bank and the World Health Organization

introduced an indicator that measures the burden of disease by combining both mortality and disability.18 The losses from premature death and the loss of healthy life resulting from disability were expressed as the disability adjusted life years (DALY). The top ten causes of losses of DALY’s, however, were quite similar to those obtained in the aforementioned adult morbidity/mortality study, taking into account the life category from 15 to 44 years.

According to these basic criteria the following target diseases can be considered as

priority health problems in most developing countries. These are not in order of priority because besides basic criteria there are many biased criteria which differ from country to country: - HIV and other sexually transmitted diseases; - malaria and other parasitic infections; - tuberculosis and other microbial and fungal infections; - diabetes (type II); - cardiovascular diseases; - diarrhoeal and other gastro-intestinal diseases; - liver and kidney diseases; - healing of wounds.

Consequently, the specialized screening bioassays dealing with some of these target diseases will be discussed and critically evaluated in this manual. References 1 Clark, A. M., 1996, Natural products as a resource for new drugs, Pharmaceutical Research, 13, 1133-1144 2 Verpoorte, R., 2000, Pharmacognosy in the new millennium: lead finding and biotechnology, Journal of

Pharmacy and Pharmacology, 52, 253-262 3 Fabricant, D. S. and Farnsworth, N. R., 2001, The value of plants used in traditional medicine for drug

discovery, Environmental Health Perspective, 109 Suppl 1, 69-75 4 Kinghorn, A. D., 2001, Pharmacognosy in the 21st century, Journal of Pharmacy and Pharmacology, 53, 135-

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5 Blunden, G., 2001, Biologically active compounds from marine organisms, Phytotherapy Research, 15, 89-94 6 Rates, S. M., 2001, Plants as source of drugs, Toxicon, 39, 603-613 7 Yuan, R. and Lin, Y., 2000, Traditional Chinese medicine: an approach to scientific proof and clinical validation,

Pharmacology and Therapeutics, 86, 191-198 8 Bauer, R., 1998, Quality criteria and standardization of phytopharmaceuticals: can acceptable drug standards

be achieved?, Drug Information Journal, 32, 101-110 9 Benzi, G. and Ceci, A., 1997, Herbal medicines in European regulation, Pharmacological Research, 35, 355-

362 10 Williamson, E. M., 2001, Synergy and other interactions in phytomedicines, Phytomedicine, 8, 401-409 11 Tyler, V. E., 1999, Phytomedicines: back to the future, Journal of Natural Products, 62, 1589-1592 12 Vanden Berghe, D. A. and Vlietinck, A. J., 1991, Screening for antibacterial and antiviral agents, In:

Hostettmann, K. (Ed.), Methods in Plant Biochemistry, Vol. 6, Academic Press, London, pp. 47-69 13 Vlietinck, A. J. and Apers, S., 2000, Biological screening methods in the search for pharmacologically active

natural products, In: Tringali, C. (Ed.), Bioactive Compounds from Natural Sources: Isolation, Characterisation and Biological Properties, Taylor and Francis, London, pp. 1-29

14 Ghisalberti, E. C., 1993, Detection and isolation of bioactive natural products, In: Colegate, S. M. and

Molyneux, R. J. (Eds.), Bioactive Natural Products: Detection, Isolation and Structural Determination, CRC Press, Boca Raton, pp. 9-57

15 Vogel, H. G. and Vogel, W. H. (Eds.), 1997, Drug Discovery and Evaluation: Pharmacological Assays, Springer

Verlag, Berlin


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2 Bioassays for Some Parasitic Protozoa:

Screening Concepts and Standard In Vitro and In Vivo Laboratory Models

Kristel Kuypers, Paul Cos, Eduardo Ortega-Barria, Dirk Vanden Berghe and Louis Maes

2.1 Introduction

The control of many parasitic infections is still strongly dependent on the availability of safe and effective chemotherapeutics because vaccines are not yet available. Drug treatment is complex and prolonged administration is generally necessary to achieve clinical cure and prevent the development of resistance. Despite extensive research for new medications, the current armamentarium remains limited and is not devoid of adverse effects. Resistance to first-line medication for several diseases, is growing alarmingly in some areas of the world and the medication is frequently not available to patients for socio-economic reasons.1

Developing a new drug is a complex, lengthy and particularly expensive process. The

cost of drug development, as recent estimates suggest, averages over US$ one billion against the widely accepted figure of US$ 897 million in 2003, depending on the therapeutic area. This explains why the private sector (pharmaceutical industry) is withdrawing from the area of tropical diseases - the economic returns cannot be guaranteed. However, as tropical diseases gain importance as part of the ongoing globalization of society and as the discrepancy between developed and developing countries becomes more controversial, drug discovery research for these diseases must continue and become the joint responsibility of both the public and the private sector.2,3 Developing countries that are faced with particular disease/health problems should therefore join initiatives to explore local resources, such as natural products diversity and new drug discovery.4, 5

Some general approaches for drug screening are described here with reference to a

number of protozoal diseases that currently represent the greatest global medical need, i.e. malaria, African sleeping sickness, leishmaniasis and Chagas disease.6 The in vitro and in vivo laboratory procedures described can be implemented in laboratory settings with limited technical resources - a common situation in developing countries. 2.2 The Drug Discovery Process

Different screening approaches are available for identifying the primary pharmacological activity in plants, natural products or synthetic chemicals. The option chosen largely depends on the nature of the disease and on the availability of practical and biologically validated laboratory models. A rewarding screening strategy should at least opt for models that remain as close as possible to the final target, i.e. the patient. In that respect, some compromises will have to be made for throughput, labour-intensiveness, costs and compound requirements. Whenever possible, the activity discovered at one particular level should be confirmed using a model at the next, higher evaluation level. For example, results

BIOASSAYS FOR SOME PARASITIC PROTOZOA

8

obtained in a subcellular (enzymatic) screening should be confirmed in the whole organism. A good in vitro activity should be linked to a confirmation test in an animal model., This can easily be achieved for most bacterial, fungal and parasitic diseases as adequately validated in vitro and in vivo laboratory models are available.7

2.2.1 Subcellular Target Approach

A theoretical first step in drug discovery is to identify and validate specific molecular targets. With the expanding knowledge of parasite biochemistry and genetics, it has now become possible to identify important parasite enzymes, receptors and processes at the molecular level, which could be suitable targets for drug action. Compounds are identified that block the functioning of these targets and inhibit parasite growth. Genomic and proteomic research may enable a more rapid progression towards 3D-structural information and may create realistic possibilities for computer (in silico) drug design or screening. The initial ‘active’ compounds (hits) may result from high throughput screening (HTS) of large and diverse chemical libraries, or from a more rational approach if structural information on the target is available. Enzyme screens are frequently applied in HTS environments because of the ease of implementation and the minimal compound requirements, but their overall poor validation status limits the predictive value., Several targets have been identified for the listed protozoal diseases and new ones are still being discovered.8-11 However, relatively few enzyme screens have been validated for drug screening purposes as yet. Specific models for subcellular screens are therefore not discussed here. 2.2.2 Conventional Test Systems

Conventional screening systems using whole organisms have considerable advantages over the subcellular target-oriented options: they involve all targets and take bioavailability phenomena into account. Frequently, compounds that are potent inhibitors of enzymes in vitro ultimately fail to confirm against the whole organism because, among other reasons, they do not pass the cell membrane. Two additional arguments determine the selection of screening options and the adopted screening strategy: integration of screening to assess selectivity of pharmacological action; and the level of empirics in compound selection.

In Vitro Models

In vitro models using the whole organism are the standard and should be used whenever possible. The above mentioned protozoa can be cultivated easily either in cell-free medium or in cell cultures (Table 1). It is important to note that any drug action on the parasite should be discriminated from aspecific cell toxicity, necessitating the inclusion of a parallel evaluation in host cell lines (cytotoxicity evaluation) and/or by inclusion of other microbial screens such as bacteria and fungi or viruses.

In Vivo Models

Laboratory rodents as animal models are indispensable for confirmation of in vitro active drug candidates. The number of animal tests is generally kept to an absolute minimum because of animal welfare considerations, high costs and labour-intensiveness. A more profound pharmacological picture is obtained from these models, as pharmacokinetic, metabolic and toxicological phenomena are taken into account.

2.3 'Hit' Finding in Primary Screening

The overall purpose of screening is to selectively eliminate compounds that do not possess the targeted pharmacological characteristics (‘funnel’ approach). To achieve this several levels of ‘elimination’ testing can be envisaged. A general principle in primary screening is that the model(s) should be as sensitive as possible in order to enable it to pick-


9

up weakly active compounds. At the same time false positives should be avoided, rapidly recognized and eliminated. To achieve this, an ideal screening set-up should contain a standard in vitro component (for ‘hit’ finding) intrinsically linked to a simple in vivo component (for ‘hit’ confirmation).

Table 1: Standard laboratory models for in vitro and in vivo evaluation of antiprotozoal drug compounds

Target In vitro model In vivo model

Leishmania Intracellular L. infantum or L. donovani amastigotes in primary macrophages or receptive cell line

Visceral leishmaniasis model (L. infantum, L. donovani) in Balb/C mice or the golden hamster

African trypanosomes

Axenic culture of blood stage forms in HMI-medium

Survival model in Swiss mouse (T. gambiense, T. rhodesiense, T. brucei)

American trypanosomes

Intracellular T. cruzi amastigotes in a receptive cell line

Acute survival model in Swiss mice

Malaria P. falciparum culture in human erythrocytes

Acute mouse model with P. berghei (survival or 4-day test)

Host cells and general toxicity

Cytotoxicity evaluation against various cell lines: MRC-5, Vero, Hela, or others

Standard tests for acute, subacute and chronic toxicity evaluationa

Microbial test systems for profiling

Axenic models with bacteria, yeast and fungi and cellular models with viruses

A broad selection of animal models for viral, bacterial and fungal diseases is availableb

a study package outlined in detail in regulatory guidelines (FDA, EMEA) for new drug registration b for more detailed information on in vivo models, see reference 7

The following experimental design options can be considered to improve the overall

sensitivity of the selected model(s):

a) Use of highly drug-sensitive parasite species: the well-characterized strains should be used for drug screening. Clinical isolates should preferably be reserved for secondary profiling of established ‘hits’ at a later stage. Care in selection should be taken for diseases that can be caused by different parasite species, for example different Leishmania species are available but visceral species are to be preferred in view of their higher sensitivity to available reference drugs,12 the availability of validated laboratory models and because they represent a more relevant clinical target (greater medical need). It makes little difference if T. gambiense, T. rhodesiense or T. brucei are used for sleeping sickness, although the latter is preferred owing to its non-pathogenicity for man. P. falciparum is the only species for which an in vitro model is available for malaria, while rodent models with P. berghei or P. yoelli are used for in vivo evaluation. b) Use of the LILIT-principle (Low Infection – Long Incubation Test): will assure that the drug has maximal chances to exert its pharmacological action while being faced with a minimal infection pressure. This test principle has been adopted for in vitro screening against African trypanosomes13 and should be considered in every primary screening design. This involves initiation of treatment before establishment of the infection for in vivo testing. c) Use of high ‘in- test’ compound concentrations: about 100 µM (or µg/ml) should be regarded as a relevant upper limit for in vitro tests if too many false-positive results are to be avoided. Doses of about 40 to 50 mg/kg are retained for either single- or multiple-dose treatment regimens for in vivo testing. Parenteral dosing is preferred to minimize bioavailability problems after oral dosing. d) Use of sensitive endpoint reading techniques: evaluation of parasite multiplication and total parasite burdens can be performed using different methods and is specific for each parasite species, for example, simple microscopic reading of Giemsa-stained preparations for determination of the parasite burdens can be used for malaria, leishmaniasis and Chagas disease.


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Standardization across the different models will maximize efficiency, minimize cost and allow easy data acquisition in order that primary screens be part of an integrated operation. This could, for example, include the use of: - standard (20mM or 20 mg/ml) stock solutions in DMSO; - fixed concentrations in all screens (using 4-fold serial dilutions); - standard lay-out of 96-well microplates to facilitate plate production; - spectrophotometric or visual reading whenever possible; - standard templates (spreadsheet) for test result processing and reporting.

Within the option of integrated evaluation of test compounds, it is recommended to

include antibacterial and antifungal screens, and if possible also antiviral screens alongside the listed parasitological models. This will facilitate discrimination between specifically active compounds and aspecific cytotoxic compounds.

It is advisable for compounds with known molecular weight to express test

concentrations in µM. For natural products or compound mixtures where the exact molecular weight is not known, concentrations are expressed in µg/ml.

2.4 Description of Laboratory Models

The models described have been kept simple to allow implementation for primary drug screen in laboratory settings with limited technological logistics. In depth technical detail should be retrieved from the literature.

2.4.1 Leishmania

Leishmaniasis is a diverse group of clinical syndromes that are caused by parasitic protozoa of the genus Leishmania and are transmitted to humans (and animals) by the bite of female sandflies belonging to the genera Phlebotomus (Old World) and Lutzomyia (New World). The disease is endemic in tropical regions of America, Africa, the Indian subcontinent, the subtropics of southwest Asia and the Mediterranean. Some forms of the disease are anthroponotic (transmitted between humans), while others are zoonotic (involving an animal reservoir). There are about 20 known species of Leishmania that have been associated with various disease forms in humans. The severity of the disease ranges from the disfiguring cutaneous form in which nodules form at the site of infection, leaving scars upon healing, to the lethal visceral leishmaniasis. Based on the clinical disease patterns, leishmaniases are classified into three groups: cutaneous (CL), mucocutaneous (MCL) and visceral (VL) leishmaniasis. Reference drugs for treatment are antimonials, amphotericin B and pentamidine. Miltefosine was recently approved for oral treatment of VL.14

The most recent EC-biosafety directive15 categorizes most Leishmania species as

biohazard class-2 pathogens, except L. donovani and L. braziliensis. BSL-2 laboratory containment is adequate for in vitro laboratory work. In the case of in vivo work with L. donovani, BSL-3 laboratory containment is recommended. When handling Leishmania-infected materials, strict safety precautions should always be taken (work in a LAF safety cabinet, wear gloves, a safety mask and a laboratory safety gown). All materials and workspaces should be disinfected regularly with 70% ethanol. Special care should be taken with needles to prevent puncture accidents and all materials used should be autoclaved before disposal. 2.4.1.1 In Vitro Model

Parasite strains: as already mentioned, visceral species are best suited for drug screening and models with cutaneous or mucocutaneous species will therefore not be discussed. The use of promastigotes for screening should be discouraged, because of the


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lower level of validation. Reference strains of L. donovani or L. infantum should be used. Both can easily be maintained in the Syrian hamster (Mesocricetus auratus) by serial passage. To prepare the inoculum for infection, amastigotes are harvested from the spleen of an infected donor hamster. The amastigote burdens in the spleen can be assessed using the Stauber technique.16 Briefly, the spleen is aseptically removed, weighed and a small cut is used to make impression smears on a microscope glass slide. After fixation in methanol, staining is performed with 10% Giemsa in water for 45 minutes at room temperature. Slides are examined microscopically at 500 x magnification under oil immersion and the mean number of amastigotes per spleen cell is counted.

Cell culture: murine peritoneal macrophages are induced by intraperitoneal

administration of 2 ml 2% starch in water. The primary peritoneal cells are harvested about 24 to 48 hours later with cold RPMI-1640 supplemented with antibiotics. All cell cultures and screening assays are conducted at 37°C and 5% CO2/air.

Drug sensitivity assay: the stock solution of test compounds are 2-fold or 4-fold

serially pre-diluted in DMSO, followed by another dilution in water (the final test concentration of DMSO should not exceed 1%). Assays are performed in sterile 96-well tissue culture plates, each well containing 10 µl of the watery compound dilutions together with 190 µl of macrophage/parasite inoculum (1.5x105 macrophages/ml and 1.5x106 parasites/ml in RPMI-1640 medium, supplemented with 20 mM L-glutamine, 16.5 mM NaHC03, 5% foetal calf serum and 2% P/S-solution). After 5 days incubation parasite growth is microscopically assessed after Giemsa staining. The results are expressed as percent reduction of parasite burden compared to control wells and the IC50 and IC90 values are calculated. Sodium stibogluconate (Pentostam®) or amphotericin B (fungizone®) can be included as reference drugs. A pure substance is classified as inactive when the IC50 is higher than 32 µM (or µg/ml). When the IC50 is lower than 4 µM (or µg/ml), the compound is classified as active (= ‘hit’) and should be further evaluated in a secondary in vitro screening (full dose-titration using 2-fold serial dilutions) and primary in vivo evaluation (Balb/C or hamster model). The recent establishment of axenic amastigotes might allow the implementation of colorimetric or fluorometric methods without the interference of host cells.17

2.4.1.2 In Vivo Model

Parasite strains: it is preferable to use the same species/strains as for the in vitro experiments, i.e. L. donovani or L. infantum.

Animal model: BALB/c mice and golden hamsters can be used for laboratory

infections. Hamsters are generally used for strain maintenance while mice are used for drug screening (easier handling, lower body weight requiring less compound, less expensive). Mice are infected with 1-2 x 107 amastigotes in 0.2 ml inoculum via a lateral tail vein on day 0. Hamsters (under superficial ether anaesthesia) are infected intracardially with 2x107 amastigotes in 0.2 ml inoculum. Compounds for treatment are prepared in 10% DMSO/water and administered intraperitoneally at 40 mg/kg for 5 days. Sodium stibogluconate is the best reference and is used at a dose of 15 mg/kg subcutaneously for 5 days. Within one week after the last treatment, all animals are sacrificed for assessment of parasite burdens in liver and/or spleen to evaluate activity (impression smears made as described above). Efficacy of treatment is presented as a reduction in parasite burden compared to untreated controls. Compounds with good activity (>90% reduction of amastigote burden) are further evaluated over an appropriate range of doses (dose-titration) or by using different dosing regimens (prophylactic versus curative, oral versus parenteral).


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2.4.2 African Trypanosomes (Sleeping Sickness)

There are many trypanosome species that infect vertebrates. In sub-Saharan Africa, Trypanosoma brucei causes sleeping sickness or human African trypanosomiasis while other species cause veterinary diseases. T. brucei is divided into three subspecies of which two cause human diseases. In west and central Africa, T. gambiense causes a chronic form. In east and southern Africa, T. rhodesiense causes an acute form. Infection is generally fatal if left untreated. T. brucei is not infectious to man. At present four drugs are available for treatment and can be used as references in laboratory tests: pentamidine and suramin are used before CNS involvement; melarsoprol is used against late-stage disease. Eflornithine (DFMO) is only useful against T. gambiense, but the requirement for large doses limits its use. Diminazene (berenil) and isomethamidium are drugs for veterinary use.

African trypanosomes are in principle biohazard class-3 pathogens (T. gambiense, T.

rhodesiense). Given the non-pathogenicity for man, T. brucei is preferred for primary screening purposes becuase it only requires BSL-2 laboratory containment. The human-pathogenic species should be included for secondary evaluation. 2.4.2.1 In Vitro Model

Parasite: a drug sensitive T. brucei strain should be used. Strains of T. gambiense or

T. rhodesiense can only be used if appropriate laboratory containment can be guaranteed. The blood stream forms are axenically grown in Hirumi (HMI) medium18 supplemented with 10% foetal calf serum and 2% P/S-solution. All cultures and assays are conducted at 37°C under an atmosphere of 5% C02.

Drug sensitivity assays: assays are performed in 96-well tissue culture plates, each

well containing 10 µl of the watery compound dilutions together with 190 µl of the parasite suspension (5 x 104 parasites/ml). After 4 days incubation at 37°C/5% C02, parasite growth is assessed by adding 10µl of a 1/1Alamar Blue™ and fluorimetric reading (excitation 530 nm; emission 590 nm) after 4 hours at 37°C.19 The results are expressed as percentage reduction in parasite burden compared to control wells and IC50 and IC90 values are calculated for each compound. Suramin is included as the reference drug. A compound is classified inactive when the IC50 is higher than 8 µM (or µg/ml). 2.4.2.2 In Vivo Model

Parasite strains: preferably the same strains are used as for in vitro experiments. The blood stream forms may come from a stock of cryopreserved stabilates containing 10% glycerol or can be derived from an infected donor animal with high parasitaemia. The inoculum for infection is prepared in sterile PBS at 5 x 104 trypanosomes/ml.

Animal model: the laboratory infections with African trypanosomes can easily be

performed on Swiss mice (CD-1, NMRI). The mice are infected intraperitoneally with 0.2 ml inoculum (1 x 104 trypanosomes) on Day 0. Infected mice are treated with the test compound at 40 mg/kg for five consecutive days: treatment is given orally on day 0 (to avoid interference with the artificial infection) and intraperitoneally on the next four days. From Day 7 onwards, survival of the mice is checked daily. Suramin and diminazene are used as reference drugs. The evaluation endpoint is mean survival time (MST) and results are compared to the MST of non-treated controls. If mice are still alive on Day 30, the presence of parasitaemia is microscopically assessed in Giemsa-stained blood smears or a subpassage in an infection-naive mouse is performed. Activity criteria can be recommended as follows: inactive if MST <150%, highly active if MST >200% of the untreated infected controls. In secondary follow-up evaluations, different species, strains and treatment regimens (dose, frequency, route and time of administration) must be included.


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2.4.3 American Trypanosomes (Chagas Disease)

Chagas disease, also called American trypanosomiasis, occurs primarily in South and Central America and is caused by Trypanosoma cruzi. It is estimated that 16 to 18 million people are infected with this disease. It is spread by reduviid or kissing bugs that live in cracks and holes of substandard housing. Man can become infected by infective reduviid faeces contacting the eyes, mouth or open cuts, by infected mothers passing infection to their baby during pregnancy, at delivery or while breastfeeding, by blood transfusion or organ transplant or by eating uncooked food contaminated with infective faeces of kissing bugs. The early (acute) stage of infection is not usually severe, but sometimes it can cause death in infants. However, in about one-third of those infections, chronic symptoms develop after 10 to 20 years and the average life expectancy decreases by an average of 9 years (due to cardiac and digestive problems). Medication (benznidazole or nifurtimox) is usually effective when given during the acute stage of infection. Once the disease has progressed to later stages, medication may be less effective. In the chronic stage, treatment involves managing symptoms associated with the disease.

According to existing biosafety guidelines, T. cruzi is a biohazard class-3 pathogen

and all laboratory work should be carried out under BSL-3 containment. Strict safety precautions must be adopted (work in a LAF safety cabinet, wear gloves, a safety mask and a laboratory safety gown). All materials and workspaces should be disinfected regularly with 70% ethanol and extreme care has to be taken with needles to prevent puncture accidents.

2.4.3.1 In Vitro Model

Parasite and cell cultures: T. cruzi, (MHOM/CL/00/Tulahuen), transfected with β-

galactosidase (Lac Z) gene (C2C4-strain) is used.20 This nifurtimox-sensitive strain is maintained on MRC-5 (human lung fibroblast, diploid cell line) cells in MEM tissue culture medium, supplemented with 20 mM L-glutamine, 16.5 mM NaHC03, 5% foetal calf serum and 2% P/S-solution. All cultures and assays are conducted at 37°C under an atmosphere of 5% C02.

Drug sensitivity assays: Assays are performed in sterile 96-well tissue culture plates, each well containing 10 µl of the watery compound dilutions together with 190 µl of MRC-5 cell/parasite inoculum (2.5x104 MRC-5 cells/ml and 2.5x105 parasites/ml). After 7 days at 37°C, parasite growth is assessed after adding 50µl/well CPRG (chlorophenol red ß-D-galactopyranoside) as substrate [substrate solution = 15.19 mg CPRG + 250 µl Nonidet in 100 ml PBS]. Colour change is measured at 580 nm after 4 hours incubation at 37°C. The results are expressed as percentage reduction in parasite burden compared to control wells and IC50 and IC90 values are calculated. Nifurtimox is included as reference drug. A compound is classified as inactive when the IC50 is higher than 16 µM (or µg/ml).


Parasite strains: multiple T. cruzi strains can be used for in vivo infections, e. g. MHOM/CL/00/Tulahuen. The blood stream forms can be obtained from an infected donor animal with high parasitaemia. The inoculum for infection is prepared in sterile PBS and contains about 106 trypanosomes/ml.

Animal model: laboratory infections with T. cruzi can easily be performed in Swiss

mice (NMRI or CD1). Blood is collected from an infected donor mouse and parasitaemia is determined. The mice are infected intraperitoneally with 0.2 ml inoculum containing 2 x 105 trypomastigotes on Day 0. Infected mice (minimum 3 animals per group) are dosed at 40 mg/kg for 5 consecutive days (drug formulation in 100% DMSO). The treatment is given orally on Day 0 and intraperitoneally on the next four days. From day 14 onwards, survival of the mice is checked daily. Nifurtimox or benznidazole are used as reference drugs. The evaluation


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endpoint is mean survival time (MST) and results are expressed as percent MST compared to the MST of non-treated controls (MST = 16 to 18 days). If mice are still alive on Day 50, the presence of parasitaemia is microscopically assessed in Giemsa-stained blood smears. The criteria for activity can be recommended as follows: inactive if MST <125%, highly active if MST >200%. In secondary follow-up evaluations different strains and treatment regimens (dose, frequency, route and time of administration) must be considered.

2.4.4 Plasmodium (Malaria)

Malarial parasites (Plasmodium spp.) circulate between humans and female Anopheles mosquitoes. In humans, the parasites grow and multiply first in the hepatocytes and then in the erythrocytes. In the blood, successive generations of parasite develop inside the red cells and destroy them, releasing merozoites that continue the cycle by invading other red cells. The blood stages cause the symptoms of malaria, which may vary from asymptomatic, the classic symptoms (fever, chills, sweating, headaches, muscle pains) to severe complications (cerebral malaria, anaemia, kidney failure, death). The severity of the symptoms depends on several factors, such as the species and the human's acquired immunity and genetic background. The female Anopheles mosquito ingests gametocytes during a blood meal and infective sporozoites are present in the mosquito's salivary glands about 10 to 18 days later. When the Anopheles mosquito takes a blood meal on another human, the sporozoites are injected with the mosquito's saliva and start another human infection. Four malaria parasite species can infect humans: Plasmodium falciparum, P. vivax, P. ovale and P. malariae. The first two species cause the most infections worldwide. P. falciparum causes severe, potentially fatal malaria with an estimated 2 million deaths annually, mostly young children in Africa. P. vivax and P. ovale have dormant liver stage parasites (hypnozoites) which can reactivate (relapse) and cause malaria several months or years after the infecting mosquito bite. P. malariae produces long-lasting infections and if left untreated can persist asymptomatically for years, even a lifetime. Most drugs used in treatment are active against the blood forms and include chloroquine, sulfadoxine-pyrimethamine (Fansidar®), mefloquine (Lariam®), atovaquone-proguanil (Malarone®), quinine, doxycycline and artemisinin derivatives (such as artesunate). Primaquine is the only drug active against the hypnozoite liver forms and prevents relapses. The search for new antimalarials has become very intensive because drug resistance is spreading rapidly. Several drug discovery initiatives are currently running and standard drug finding and evaluation methodologies have been recently reviewed.21

2.4.4.1 In Vitro Model

Parasite: several P. falciparum strains can be used and it is advisable to include drug sensitive as well as drug-resistant strains in the screening panel. Drug-sensitive strains are, for example, GHA, FCR3, NF54 and D6 that are susceptible to chloroquine, quinine and pyrimethamine. Drug resistant strains include W-222 and K1, known to be resistant to chloroquine, quinine and pyrimethamine, but susceptible to mefloquine. The strains are maintained in continuous log phase growth in RPMI-1640 medium supplemented with 2% P/S-solution, 0.37 mM hypoxanthine, 25 mM Hepes, 25 mM NaHC03, and 10% A+ human serum together with 4% human O+ erythrocytes, as described by Trager.23 The human serum can also be replaced by lipid-rich bovine serum albumin (AlbuMAXII). All cultures and assays are conducted at 37°C under micro-aerophilic atmosphere (4% C02, 3% 02 and 93% N2). P. falciparum is a human pathogen and appropriate measures for personal safety in the laboratory must be adopted. It causes malignant tertian malaria which is the most severe clinical form of malaria. The biosafety guidelines classify P. falciparum as class-3 pathogen. However, the laboratory strains used for in vitro screening have been maintained for years by serial passage in vitro and have lost a lot of their natural pathogenicity. In addition, vector transmission is not used. All laboratory procedures involving P. falciparum can be carried out in BSL-2 laboratory containment for these reasons.


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Drug sensitivity assay: antimalarial activity can be assessed using an adaptation of the procedure described by Desjardings24 and Makler25 for the parasite lactate dehydrogenase assay. Assays are performed in 96-well tissue culture plates, each well containing 10 µl of the watery compound dilutions together with 190 µl of the parasite inoculum (1% parasitaemia, 2% haematocrit). After 72 hours of incubation at 37°C, plates are stored at –20°C until further processing. After thawing, 20 µl of haemolysed parasite suspension from each well is transferred into another plate together with 100 µl Malstat™ reagent and 10 µl of a 1/1 mixture of PES (phenazine ethosulphate, 2 mg/ml) and NBT (nitro blue tetrazolium grade III, 0.1 mg/ml). The plates are kept in the dark for 2 hours and change in colour is measured spectrophotometrically at 655 nm. Alternatives to the Malstat assay for quantification of parasite growth is [3H]-hypoxanthine incorporation assay (addition of 0.2 µCi and reading with liquid scintillation counter after 24 hours) or the use of the DNA fluorochrome Picogreen®.26 The results are expressed as percentage reduction in parasitaemia compared to control wells and the IC50 and IC90 values are determined. Artesunate and chloroquine are included as reference drugs. The compound is classified as inactive when the IC50 is higher than 8 µM (or µg/ml). Active hits have an IC50 below 0.5 to 1 µM or µg/ml.


Parasite: two rodent Plasmodium strains can be used: P. berghei (ANKA-strain) which is chloroquine-sensitive and P. yoelli (NS strain) which is chloroquine-resistant. The infection dose for mice is about 106 to 107 parasitized erythrocytes per animal.

Animal model: male Swiss albino mice (CD1 or NMRI) are infected on Day 0 with

about 5 x 106 infected erythrocytes, derived from an infected donor animal with high (30%) parasitaemia. The donor blood is appropriately diluted in physiological saline and 0.2 ml of this suspension is injected intravenously in the tail vein. In untreated control animals, parasitaemia rises to 30% within 3 to 4 days and deaths start to occur after about 6 to 7 days (>80% parasitaemia). Infected mice (minimum 3 animals per group) are dosed at 40 mg/kg intraperitoneally for five consecutive days (drug formulation in DMSO). Two evaluation endpoints are possible: (a) parasitaemia is microscopically assessed on a Giemsa stained blood smear on day 4 and results are expressed as percent reduction of parasitaemia compared to untreated control mice; (b) determination of mean survival time (MST) and results are expressed as the excess 5 MST compared to the MST of the untreated controls. If mice are still alive on day 25, the presence of parasitaemia is checked on a blood smear. Activity criteria are: inactive if <70% reduction in parasitaemia or if excess MST <50%; highly active if >90% reduction in parasitaemia or if excess MST >100%. In secondary follow-up evaluations different treatment regimens (dose, frequency, route and time of administration) must be considered. 2.4.5 Cytotoxicity

Cytotoxicity on host cells is a very important criterion for assessing the selectivity of the observed pharmacological activities and should always be included in parallel. Although many cell types theoretically can be used for cytotoxicity evaluation MRC-5 cells are used as an example.

Cell cultures: MRC-5 (human lung fibroblast) cells are cultured in MEM medium,

supplemented with 20 mM L-glutamine, 16.5 mM NaHC03, 5% foetal calf serum and 2% P/S-solution. All cultures and assays are conducted at 37°C and 5% C02.

Cytotoxicity assay: assays are performed in sterile 96-well tissue culture plates,

each well containing 10 µl of the watery compound dilutions together with 190 µl of MRC-5 cell inoculum (2.5 x 104 cells/ml). Cell growth is compared to untreated control wells (= 100% cell growth). After 7 days incubation, cell proliferation/viability is assessed after addition of Alamar Blue™ (5 µl of a 1/10 solution/well). After 4 hours at 37°C, fluorescence is measured


16

(550 nm excitation, 590 nm emission). Resazurin can be used as a much cheaper alternative.27 The results are expressed as percentage reduction in overall cell viability compared to control wells and a IC50 (50% inhibitory concentration) is determined. A compound is considered non-cytotoxic when the IC50 is >32 µM (or µg/ml). 2.5 Efficacy Criteria for Active Compounds

In antimicrobial in vitro models, the activity of compounds is generally expressed by numeric values (IC50, IC90, MIC, etc.) and correct interpretation of these efficacy variables is not easy because a profound knowledge of the model and protocol is required. This particular problem could be solved, for example, by using a set of semi-quantitative activity scores that not only reflect the level of activity but also contain the overall assessment of the pharmacologist who is familiar with the test. To enable a ‘normalized’ profiling of each compound, the following scores could be envisaged: Score-1: inactive, does not merit/require further interest; Score-2: marginally active, may require further interest if analogue structures show a similar trend for activity and Score-3: highly active, requires further follow-up and exploration of analogue structures.

As already outlined, a primary screen should be as sensitive as possible to enable

detection of weakly active compounds, but at the same time must not produce too many false positives. Consequently, dose-range selection becomes very important and will be largely dependent on the type of model. It is well known that different pathogens have specific sensitivities to the aspecific or toxic action of chemical molecules. Same cell type, for example, becomes much more sensitive to in vitro cytotoxic effects when cultivated in serum-free conditions. In random antiprotozoal screens, a consistent observation is that free-living organisms such as T. vaginalis are much more robust than the blood protozoa Plasmodium, Leishmania or Trypanosoma. Among the latter, Plasmodium tends to be the most sensitive.

Non-specific activity or toxicity, which shows up as false positives, must be kept

within reasonable limits. To check this, different in vitro screens are performed at appropriate dose levels, the more sensitive organisms being evaluated at relatively lower concentrations compared to the more resistant ones. However, in an integrated screen, one standard dose range is applied for all models for practical and logistic reasons (single compound dilution schemes, uniform test plate production). The primary screening example in the present publication included a standard 4-fold serial dilution range with 32 µM (or µg/ml) as the highest available test concentration. This dose range is, in principl, wide enough to calculate IC50 and IC90 values for most compounds in a random library. The parameters used to calculate the IC values are specific for the various test protocols and reflect the percentage reduction of growth in the treated cultures in comparison with untreated control cultures (100 % growth, i.e. 0 % inhibition). The proposed semi-quantitative activity scores should be based on the IC50 values.

While screening natural products, it is essential to identify the active constituent(s)

at an early stage to avoid isolation of molecules with known structure and known activity. The combination of literature search and exploratory analytical power is a prerequisite before any bioassay-guided fractionation should be initiated.

2.6 Conclusions

New drug discovery is a rather difficult exercise and the success of the operation is determined by several factors. Screening remains a cornerstone activity and the availability of validated laboratory models is pivotal. Along with the progress of genomics and proteomics, subcellular targets have moved into first-line screening in several disease areas (HIV, metabolic diseases). This has not, however, happened in parasitology because relatively few targets have been fully validated. Furthermore, enzyme screens generally require a high-tech


17

laboratory infrastructure and are consequently much less accessible when compared to whole organism or in vitro screens.

The protocols of the parasitic protozoa in vitro screens presented in this chapter

have been kept as simple as possible to create the possibility of successful implementation in laboratory settings which are less technologically sophisticated. The authors recognize that many deviations from the proposed standard protocols are possible and that newer and better techniques for endpoint (i.e. parasite growth) reading are available. Rather than focusing on the parasite model itself, integration of the models with other microbial screens (bacteria, fungi, viruses) is perceived as a more important need, given that selectivity of drug action is far more revealing in defining promising hits than mere pharmacological potency (IC50, IC90).

People from developing and developed countries are invited to supply compounds

and natural product extracts for integrated evaluation against the targeted diseases. Global networking between different drug discovery initiatives may enhance the chances of finding valid new drug leads, which may eventually lead to new medications for patients in need. Parasitic disease patients, who are currently being neglected by the private sector, would possibly benefit most from a more concerted action of this kind. References 1 Pecoul, B., Chirac, P., Trouiller, P. and Pinel, J., 1999, Access to essential drugs in poor countries: a lost battle,

Journal of American Medical Association, 281, 361-367 2 Trouiller, P., Torreele, E., Olliaro, P., White, N., Foster, S., Wirth, D. and Pecoul, B., 2001, Drugs for neglected

diseases: a failure of the market and a public health failure, Tropical Medicine & International Health, 6, 945-951

3 Webber, D. and Kremer, M., 2001, Perspectives on stimulating industrial research and development for

neglected infectious diseases, Bulletin of the World Health Organization, 79, 735-741 4 Gutierrez, M. O., 2002, Biodiversity and new drugs, Trends in Pharmacological Sciences, 23, 8 5 Quah, S. R., 2003, Traditional healing systems and the ethos of science, Social Science and Medicine, 57,

1997-2012 6 Remme, J. H., Blas, E., Chitsulo, L., Desjeux, P. M., Engers, H. D., Kanyok, T. P., Kayondo, J. F., Kioy, D. W.,

Kumaraswami, V., Lazdins, J. K., Nunn, P. P., Oduola, A., Ridley, R. G., Toure, Y. T., Zicker, F. and Morel, C. M., 2002, Strategic emphases for tropical diseases research: a TDR perspective, Trends in Microbiology, 10, 435-440

7 Zak, O. and Sande, M., 1999, Handbook of Animal Models of Infection: Experimental Models in Antimicrobial

Chemotherapy (1st ed.), Academic Press, London 8 Werbovetz, K. A., 2000, Target-based drug discovery for malaria, leishmaniasis, and trypanosomiasis, Current

Medicinal Chemistry, 7, 835-860 9 Brady, R. L. and Cameron, A., 2004, Structure-based approaches to the development of novel anti-malarials,

Current Drug Targets, 5, 137-149 10 Krauth-Siegel, R. L., Meiering, S. K. and Schmidt, H., 2003, The parasite-specific trypanothione metabolism of

trypanosoma and leishmania, Biological Chemistry, 384, 539-549 11 Opperdoes, F. R. and Michels, P. A., 2001, Enzymes of carbohydrate metabolism as potential drug targets,

International Journal of Parasitology, 31, 482-490 12 Neal, R. A. and Croft, S. L., 1984, An in-vitro system for determining the activity of compounds against the

intracellular amastigote form of Leishmania donovani, Journal of Antimicrobial Chemotherapy, 14, 463-475


18

13 Brun, R. and Lun, Z. R., 1994, Drug sensitivity of Chinese Trypanosoma evansi and Trypanosoma equiperdum isolates, Veterinary Parasitology, 52, 37-46

14 Sundar, S., Jha, T. K., Thakur, C. P., Engel, J., Sindermann, H., Fischer, C., Junge, K., Bryceson, A. and Berman,

J., 2002, Oral miltefosine for Indian visceral leishmaniasis, New England Journal of Medicine, 347, 1739-1746 15 Council directive 93/88/EEC of 12 October 1993 amending directive 90/679/EEC on the protection of

workers from risks related to exposure to biological agents at work, Official Journal of the European Communities, 29.10.93 - pp. L268 & 271.

16 Stauber, L. A., 1966, Characterization of strains of Leishmania donovani, Experimental Parasitology, 18, 1-11 17 Debrabant, A., Joshi, M. B., Pimenta, P. F. and Dwyer, D. M., 2004, Generation of Leishmania donovani axenic

amastigotes: their growth and biological characteristics, International Journal of Parasitology, 34, 205-217 18 Hirumi, H. and Hirumi, K., 1989, Continuous cultivation of Trypanosoma brucei blood stream forms in a

medium containing a low concentration of serum protein without feeder cell layers, Journal of Parasitology, 75, 985-989

19 Raz, B., Iten, M., Grether-Buhler, Y., Kaminsky, R. and Brun, R., 1997, The Alamar Blue assay to determine drug

sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro, Acta Tropica, 68, 139-147 20 Buckner, F. S., Verlinde, C. L., La Flamme, A. C. and Van Voorhis, W. C., 1996, Efficient technique for screening

drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase, Antimicrobial Agents and Chemotherapy, 40, 2592-2597

21 Fidock, D. A., Rosenthal, P. J., Croft, S. L., Brun, R. and Nwaka, S., 2004, Antimalarial drug discovery: efficacy

models for compound screening, Nature Reviews Drug Discovery, 3, 509-520 22 Oduola, A. M., Milhous, W. K., Weatherly, N. F., Bowdre, J. H. and Desjardins, R. E., 1988, Plasmodium

falciparum: induction of resistance to mefloquine in cloned strains by continuous drug exposure in vitro, Experimental Parasitology, 67, 354-360

23 Trager, W. and Jensen, J. B., 1976, Human malaria parasites in continuous culture, Science, 193, 673-675 24 Desjardins, R. E., Canfield, C. J., Haynes, J. D. and Chulay, J. D., 1979, Quantitative assessment of antimalarial

activity in vitro by a semiautomated microdilution technique, Antimicrobial Agents and Chemotherapy, 16, 710-718

25 Makler, M. T., Ries, J. M., Williams, J. A., Bancroft, J. E., Piper, R. C., Gibbins, B. L. and Hinrichs, D. J., 1993,

Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity, American Journal of Tropical Medicine and Hygiene, 48, 739-741

26 Corbett, Y., Herrera, L., Gonzalez, J., Cubilla, L., Capson, T. L., Coley, P. D., Kursar, T. A., Romero, L. I. and

Ortega-Barria, E., 2004, A novel DNA-based microfluorimetric method to evaluate antimalarial drug activity, American Journal of Tropical Medicine and Hygiene, 70, 119-124

27 McMillian, M. K., Li, L., Parker, J. B., Patel, L., Zhong, Z., Gunnett, J. W., Powers, W. J. and Johnson, M. D., 2002,

An improved resazurin-based cytotoxicity assay for hepatic cells, Cell Biology and Toxicology, 18, 157-173


19

3 Bioassays for Antibacterial and Antifungal Activities

Paul Cos, Louis Maes, Jean-Bosco Sindambiwe, Arnold J. Vlietinck and Dirk Vanden Berghe

3.1 Introduction

Infectious diseases are a major threat to human health, mainly due to the unavailability of vaccines or deficiencies in chemotherapy. Most of the current antibiotics have considerable limitations in terms of antimicrobial spectrum and side effects, and their widespread overuse has led to increasing clinical resistance of previously sensitive micro-organisms and to the emergence of previously uncommon infections. Moreover, due to progress made in the fields of intensive care, haemato-oncology and transplantation, the number of immuno-suppressed individuals is increasing. The rapidly growing number of HIV infections worldwide presents another group susceptible to so called opportunistic infections. These patients become susceptible to micro-organisms that would normally not affect a healthy individual. The emergence of more nosocomial infections among hospitalized patients constitutes another life threatening health problem. The need for new molecules with prominent activities against methicillin-resistant staphylococci (MRSA), vancomycin-resistant enterococci (VRE), gram negative bacilli including Pseudomonas sp., Serratia sp. and Acinetobacter sp. and acid fast bacilli such as Mycobacterium spp., and with no cross resistance with existing antibiotics is crucial to keep the threat from microbes under control.1-4

Opportunistic and nosocomial infections are not only caused by all major groups of

bacteria, but also by yeasts and fungi which are often difficult to treat and may be fatal as they become systemic. These opportunistic infections are not only becoming more frequent in immune-suppressed patients but unfortunately also in healthy individuals who receive long term treatment with broad spectrum antibiotics or hormones. Such treatments induce alterations in the normal microbial flora of the intestine, upper respiratory and genitourinary tracts, which may lead to secondary infections by Enterobacteriaceae, yeasts (Candida spp., Cryptococcus sp.) and other fungi (Aspergillus, Histoplasma, Coccidioides, Fusarium sp.). The latter are very difficult to control with the currently available antifungal drugs so the need for new antifungal drugs remains critical.5-7

This chapter will only focus on primary or general bioassay methods for antibacterial

and antifungal evaluation. Primary bioassays are designed for rapid screening of large numbers of natural product extracts or pure compounds. In general they are simple and easy to implement, produce results quickly and are inexpensive,8, 9 thereby allowing the user to set up high capacity screening. Compounds or extracts with a specific activity at a non-toxic dose, so called ‘hits’, can then be further evaluated in secondary or specialized in vitro bioassays and in animal models. 3.2 Overview of Antibacterial and Antifungal Assays

Antimicrobial activity of natural extracts and pure compounds can be detected by observing the growth response of various micro-organisms to samples that are placed in contact with them. Many methods of detecting activity are available, but because they are not equally sensitive or not based upon the same principle, the results obtained will be profoundly influenced

BIOASSAYS FOR ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES

20

by the method selected. The problems inherent to antimicrobial screening of plant extracts have already been discussed by a number of authors,1, 10-12 so focus will mainly be on the correct implementation of the different laboratory models.

Antibacterial and antifungal test methods are classified into three groups: diffusion,

dilution, and bioautographic methods.10 It should be emphasized that many research groups have modified these methods for specific samples, such as essential oils and non polar extracts and these small modifications make it almost impossible to directly compare results obtained by different groups. It is therefore a must to include at least one, preferably several reference controls in each assay. 3.2.1 Diffusion Methods

In the diffusion technique, a reservoir containing the test compound at a known

concentration is brought into contact with an inoculated medium and the diameter of the clear zone around the reservoir (inhibition diameter) is measured at the end of the incubation period. In order to lower the detection limit, the inoculated system is kept at lower temperature for several hours before incubation to favour compound diffusion over microbial growth, thereby increasing the inhibition diameter. Different types of reservoirs can be used, such as filter paper discs, stainless steel cylinders placed on the surface and holes punched in the medium (Figure 1). The whole plate method is the only suitable diffusion technique for testing aqueous suspensions of extracts, because the suspended particulate matter in the test sample is much less likely to interfere with the diffusion of the antimicrobial substance into the agar than in the other types of reservoirs. Nevertheless, great care should be taken to ensure that the sample does not leak from the hole under the layer of the agar. To prevent this, a small fixed amount of agar can be put on the bottom of the hole.12

Figure 1: Different types of reservoirs used in the agar diffusion method: (a) filter paper disc, (b) cylinder, and (c) hole plate

The small size of the sample and the possibility to test five or six extracts per plate

against a single micro-organism are the major advantages of the diffusion method.10 On the other hand, this method is not the best choice for testing non polar samples or samples that do not easily diffuse into the medium. For example, aqueous dispersions containing high molecular weight solubilizers (mol. Wt > 100.000) cannot diffuse into 1% agar. The relative antimicrobial activity of different samples cannot always be compared using the agar diffusion method due to potential differences in physical properties of the antimicrobial agents, such as solubility, volatility, and diffusion characteristics in agar. In other words, it is not possible to determine the actual concentration of the test sample that affects the micro-organism. Furthermore, agar-diffusion methods may be rather difficult to run in high capacity screening environments.


21

3.2.2 Dilution Methods In the dilution methods, test compounds are mixed with a suitable medium that has

previously been inoculated with the test organism. It can be carried out in liquid as well as in solid media. After incubation, growth of the micro-organism can be measured in a number of ways. In the agar dilution method, the Minimal Inhibitory Concentration (MIC) is defined as the lowest concentration able to inhibit any visible microbial growth. In liquid or broth dilution methods, turbidity and redox indicators are most frequently used. Turbidity can be estimated visually or obtained more accurately by measuring the optical density with a spectrophotometer (405 nm). However, the accuracy of turbidity measurements can be limited by interference from the test samples. This is a particular problem with extracts that are sometimes incompletely soluble in the aqueous broth. It also emphasizes the need for a negative control or sterility control, i.e. the extract dissolved in the medium without micro-organisms.

At present, redox indicators, such as 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-

tetrazolium bromide (MTT) and resazurin are more frequently used to quantify bacterial growth.13,

14 Easy and reproducible measurements can be obtained with a microtitre plate reader, but visual reading may also be used in cases where spectrophotometric equipment is not available. Recently, microbial growth in a 96-well plate broth dilution assay was measured using fluorescein diacetate.15 The assay is based on the principle that only living cells convert fluorescein diacetate to fluorescein, a yellowish green compound which fluoresces under UV light. It can be questioned if this technique will be rapidly implemented in developing countries, since it requires a more significant investment in equipment. Furthermore, the validation of the fluorescein diacetate assay is not easy.16 For example, Sabouraud liquid medium can quench up to 95% of fluorescence, while sodium phosphate buffers hydrolyse fluorescein diacetate to fluorescein. Fluorescent constituents present in crude plant extracts may also interfere.

In general, dilution methods are appropriate for assaying polar and non polar extracts

and pure isolates/compounds for determination of MIC values. The use of micro dilution methods in microtitre plates allow the setting up of a primary high capacity screening battery for antibacterial and antifungal activities at a relatively low cost. 3.2.3 Bio-autographic Methods

Bioautography is a method to localize antimicrobial activity on a chromatogram. It can be divided into three groups: (a) direct bioautography, where the micro-organism grows directly on the thin layer chromatographic (TLC) plate; (b) contact bioautography, where the antimicrobial compounds are transferred from the TLC plate to an inoculated agar plate through direct contact; and (c) agar overlay bioautography, where a seeded agar medium is applied to the TLC plate.17 In direct bioautography, inhibition zones can be visualized after spraying with a tetrazolium salt.18 Despite its high sensitivity, its applicability is limited to micro-organisms that can grow directly on TLC plates. Another problem is the complete removal of residual solvents of low volatility, such as n-butanol, trifluoroacetic acid and ammonia. As with contact bioautography, the agar overlay technique requires the transfer of the active compounds from the stationary phase into the agar layer by diffusion. Bioautography is a valuable method in the search for new antimicrobial agents from plants through bioassay-guided isolation because it allows the localization of antimicrobial activities of an extract on a chromatogram.

3.3 Compound Handling

Plant extracts are generally prepared by maceration or percolation of fresh green plants or dried powdered plant material with water or organic solvents. Sometimes, a fractionation of the total extract is carried out prior to testing in order to separate polar from non polar compounds and acid and neutral from basic substances.1 It is advisable to extract the plants and to dry the extracts at low temperature to avoid destruction of thermolabile


22

(antimicrobial) constituents. Non polar extracts can be dissolved in organic solvents, such as dimethylsulfoxide (DMSO) and in methanol or ethanol. Given that most plant extracts and purified compounds dissolve well in DMSO, stock solutions of test samples (concentration: 20 micromolar or microgram/ml for pure compounds or extracts, respectively) are prepared in 100% DMSO and stored frozen until use. An added advantage of storage in 100% DMSO is that it eliminates microbial contamination of the test samples, so that sterilization of the samples by autoclaving or other strenuous methods is no longer necessary. In view of automation and integrated high capacity screening, choosing a single solvent for the stock solutions becomes an advantage. To avoid later interference in the biological test systems, the in-test concentration of DMSO should not exceed 1%. Therefore, an intermediate dilution step in water is generally introduced.

Antimicrobial testing of essential oils is difficult19 because of their volatility, water

insolubility and complexity. At present there is still no standard validated method. In our method an emulsifier is incorporated into the test system to ensure contact between the test micro-organism and the agent for the duration of the experiment. The agents most commonly used are Tween-80 and Tween-20.

The use of a single standard test plate layout template for all the test models is time

saving and minimizes human errors during the bioassay screen. Furthermore, it facilitates implementation of a high capacity system with regard to plate reading and data processing. The proposed plate layout, as shown in Section 4, is not restrictive and can be modified depending on the specific objectives of the test. 3.4 Protocol of Broth Dilution Method

In this chapter, a detailed protocol for a broth dilution method is presented to evaluate plant extracts and pure compounds for antibacterial and anti-yeast activity. 3.4.1 Preparation of Culture Medium Materials and Equipment

Mueller-Hinton broth, dry powder (commercial source), growth medium for bacteria and Sabouraud dextrose broth, dry powder, (commercial source) for yeast. Other materials include distilled or demineralised water, glass flasks and sterile tubes of 10 ml capacity with sterile caps.

Equipment

Autoclave and incubator of 37°C. Methods

Weigh the appropriate amount of Mueller-Hinton broth powder or Sabouraud dextrose broth powder, as recommended by the manufacturer, and dissolve in 1000 ml distilled or demineralised water. Stir for 10 minutes at room temperature, until complete dissolution. Distribute the medium in glass flasks, e.g. of 100 ml (volumes are chosen according to the number of tests to be performed) and sterilize at 121°C for 15 minutes and allow the medium to cool.

If no biological indicator is used to check the sterilization procedure, a flask containing

the broth medium is incubated at 37°C for 24 hours. After incubation, the medium should be completely clear (cloudy aspect indicates non-sterility).


23

3.4.2 Preparation and Maintenance of Bacterial and Yeast Cultures Background

The choice of test organisms depends on the purpose of the investigation. In a general primary screening of plant extracts, plant derived molecules or pure chemical substances, drug sensitive reference strains should be used and represent common pathogenic species of different classes (Table 1). Although ATCC strains are well defined and very popular, defined clinical isolates may also be used as test organisms.

Table 1: Prototype of a bacterial and yeast battery for primary screening purposes

Bacteria Species

Gram positive bacteria:

Cocci Staphylococcus aureus Streptococcus pyogenes

Spore-forming rods Bacillus cereus Gram negative rods:

Enterobacteriaceae – non-encapsulated Escherichia coli Salmonella enteritidis

Enterobacteriaceae – encapsulated Klebsiella pneumoniae Non-enterobacteriaceae Pseudomonas aeruginosa

Acid fast bacteria Mycobacterium fortuitum

Yeast Species

Yeast Candida albicans

Materials

Cryo tubes 1.5 ml (Nunc), glycerol, Mueller-Hinton broth for bacterial medium, Sabouraud dextrose broth for yeast medium, sterile tubes of 10 ml with sterile caps, and inoculation loops (öse) in platinum or sterile plastic (disposable) loops.

Equipment

Biofreezer (-80°C) for storage, incubator of 37°C, microbiological safety cabinet class II and Bunsen burner. Methods

Starting from an existing biofreeze stock

Pipette 20 µl of the biofreeze stock into 10 ml Mueller-Hinton or Saubouraud dextrose broth in a sterile tube. Vortex for 5 seconds and incubate at 37°C for 18 to 24 hours.

Starting from an agar plate

Heat sterilize a platinum loop in a flame and cool in sterile Mueller-Hinton or Sabouraud dextrose broth for a few seconds or use a disposable loop. Touch gently the surface of a bacterial colony with the wet loop. Dip the loop into Mueller-Hinton or Sabouraud dextrose broth (10 ml) in a tube, vortex for 5 seconds and incubate at 37°C for 18 to 24 hours.

Storage of basic cultures in biofreezer

Add 1 ml sterile glycerol to 9 ml of a fully grown bacterial or yeast culture. Vortex for at least 10 seconds. Divide into labelled cryo tubes, 1 ml or 500 µl per tube and store at -80°C.


24

3.4.3 Preparation of Test Sample

Please refer to chapter on bioassays for screening antiviral activities. 3.4.4 Antimicrobial Evaluation Procedure Materials

Fully grown bacterial/yeast cultures, Mueller-Hinton broth and Sabouraud dextrose broth as prepared above. DMSO/water intermediate dilutions of the test samples and the reference compounds, sterile tubes of 10 ml with sterile caps, sterile 96-well plates and multichannel pipettes of 10 to 100 µl and 20 to 200 µl. Equipment

Incubator of 37°C, microplate OD reader with 405 nm filter and microbiological safety cabinet class II. Methods

Preparation of the different inocula for screening

Take a bacterial/yeast culture and vortex for 5 seconds. Pipette 20 µl of this culture per 10 ml Mueller-Hinton broth (for bacteria) or Sabouraud dextrose broth (for Candida albicans). For Mycobacterium fortuitum pipette 200 µl culture per 10 ml Mueller-Hinton broth.

Test plate layout (Figure 2)

In the proposed plate layout, 16 samples are tested at final concentrations 128, 32, 8, 2, and 0.5 µM (pure compounds) or µg/ml (extracts). Column-11 is reserved for a positive control and column-12 for bacterial or yeast control.

A different plate should be used for each micro-organism tested. This allows the use of different incubation procedures, without changing the test plate layout.

Figure 2: Microtitre plate presenting 16 test samples

0.528321280.52832128µg/ml or µM

168H

157G

146F

135E

124D

113C

102B

91A

121110987654321

0.528321280.52832128µg/ml or µM

168H

157G

146F

135E

124D

113C

102B

91A

121110987654321


25

Assay procedure

Use a multichannel pipette to transfer 10 µl of the intermediate test sample dilution per well of the 96-well plate. Column-11 is filled with 10 µl of a positive control. Column-12 does not contain test sample (bacterial/yeast control). Add to each well of columns 1 to 12, 190 µl of the bacterial or yeast inoculum suspension using a multichannel pipette. In one plate, the negative control or sterility control plate, the bacterial or yeast inoculum suspension is replaced by bacterial or yeast growth medium without micro-organisms. Incubate at 37°C for 24 to 48 hours, depending on the micro-organism. It is advisable to place the plates in a closed box with some water or in a humidified incubator to prevent desiccation.

Endpoint reading

Before reading, allow the plates to acclimatize at room temperature to avoid condensation phenomena which may interfere with the reading. Read the plates with the OD reader at 405 nm. In the absence of an OD reader, visual observation of growth is also possible. Visual examination can be facilitated by the use of a viewing mirror. Growth must be absent in the sterility control and present in the bacterial and yeast control. The MIC value is the lowest concentration at which no (visual) bacterial or yeast growth is observed.

3.5 Protocol for Antifungal Test Method

A detailed protocol for an agar dilution method is presented in order to evaluate plant extracts and pure compounds for their antifungal activity.20 3.5.1 Preparation of Culture Medium

Sabouraud dextrose broth is used as a culture medium. For materials, equipment and methods refer to the section on protocol for broth dilution method. 3.5.2 Preparation and Maintenance of Fungal Cultures Background

The choice of test organisms depends on the purpose of the investigation. For a general primary screening it is preferable to use a small set of reference micro-organisms, as proposed in Table 2. Trichophyton mentagrophytes and Epidermophyton floccosum are selected as representatives of the dermatophytes, which is a distinct group of fungi infecting the skin, hair, and nails in animals and man. As opportunistic filamentous fungi, Aspergillus niger and Fusarium solani are included in the proposed battery.

Table 2: Prototype of a fungal battery

Group of fungi Micro-organism

Dermatophytes Trichophyton mentagrophytes Epidermophyton floccosum Opportunistic filamentous fungi Aspergillus niger Fusarium solani

Materials

Sabouraud dextrose broth, Sabouraud dextrose agar, sterile distilled or demineralized water, sterile tubes of 10 ml with sterile caps and inoculation loops (öse) in platinum or sterile plastic (disposable) loops. Equipment

Incubator of 25°C, titanium probe sonicator, Bunsen burner and microbiological safety cabinet class II.


26

Methods

Starting from an agar slant

Filamentous fungi are grown on ten-fold diluted Sabouraud agar slants at room temperature for 15 days. Spores and mycelial fragments are collected in sterile distilled water (10 ml) and then homogenized by ultrasonication using a titanium probe sonicator at low power and 10 µm amplitude. The titres of fungal suspensions are determined by an endpoint dilution method. Ten-fold dilutions are prepared in Sabouraud broth and are incubated for 4 to 7 days at room temperature. Sometimes it is necessary to prepare the suspension just before use, e.g. Fusarium poae; otherwise they are better kept at room temperature. 3.5.3 Antifungal Evaluation Procedure

Two different procedures can be used but both are based on the same test principle. In high capacity screens the use of broth-based systems (Sabouraud broth) is advantageous because of easy pipetting of small volumes. If preference is given to solid test systems, Sabouraud agar is used. Materials

Fungal suspension, Sabouraud dextrose agar or Sabouraud broth, DMSO/water intermediate dilutions of the test samples and the reference compounds, sterile tubes of 10 ml with sterile caps, sterile 96-well plates and multichannel pipettes of 10 to 100 µl and 20 to 200 µl. Equipment

Incubator of 25°C and microbiological safety cabinet class II. Methods

Test plate layout (Figure 2)

Use an individual test plate for each micro-organism tested. This permits specific incubation conditions (temperature, duration, etc.) for each test organism within the same test plate layout. The test plate layout shown in Figure 2 is used.

Assay procedure

Aliquots of test compound dilutions (10 µl) are thoroughly mixed with 190 µl of either Sabouraud broth (in liquid system) or melted Sabouraud agar (in solid system). The plates are allowed to cool. Columns 1 to 10 are used for the test samples, while column 11 is reserved for a positive control. Column 12 can be used as growth medium control. The wells are inoculated with 100 µl of 103 or 104 CFU/ml fungal suspension. One plate, serving negative control or sterility control, is not inoculated with the fungal suspension.

The plates are incubated at 25°C (or room temperature) for 15 days or depending upon the specific strains or species.

Endpoint reading

Growth must be absent in the sterility control and present in the fungal control. The MIC is defined as the lowest concentration at which no visible fungus development is observed. Reading is performed microscopically or visually. In the broth-based test turbidimetric reading (405 nm) can also be considered. 3.6 Conclusions

Antibacterial and antifungal dilution methods are very suitable to screen plant extracts, plant derived molecules and pure chemical substances. They are easy to perform, do not require


27

expensive equipment, reagents or high-skilled expertise. This method can therefore be recommended for antibacterial and antifungal drug discovery initiatives in laboratory settings with limited research capabilities, a situation which occurs most frequently in developing or low income countries. On the other hand, these countries generally have a wide natural biodiversity which unfortunately remains largely unexplored due to lack of appropriate resources. The proposed drug screening methods offer a low cost solution and support local initiatives to access their natural resources for antimicrobial drug discovery.

A key factor in the integrated screening approach is the use of a single solvent (100%

DMSO) for stock solutions of test compounds and the use of a single template for test plate production for all microbial screens, including the viral models and the parasitological models discussed elsewhere in this book. The integrated approach will also limit the consumption of test substance (which may be available only in minute quantities), reduce costs, eliminate errors and facilitate data processing and reporting.

Although the individual models may seem simple to perform, interpretation of

screening results and identification of promising ‘hits’ may prove to be more problematic. One of the more important criteria for activity is selectivity of action and this aspect can only be investigated if the antimicrobial screens are run in an integrated manner, i.e. against a broad panel of related (other bacteria and fungi such as those presented in Tables 1 and 2) and unrelated organisms (e.g. viruses and parasites). It is important to stress that evaluation of in vitro cytotoxicity on cell lines is an essential component but cell culture technology may not be available to all laboratories. Furthermore, potency criteria such as IC50- and MIC-values may have a different meaning, depending on the micro-organism involved. For example, it has been well established that Gram positive bacteria are much more sensitive to drug action than Gram negative bacteria and this can be reflected by a higher number of ‘hits’ in the screen. However, prominent active plant extracts against Preferably, Gram positive cocci are selected for testing against MRSA and VRE. To correct for too many false positives, more stringent endpoint criteria are needed. As a rule of thumb, extracts or compounds with selective activity and IC50- or MIC-values below 1 to 10 µM (pure compounds) or µg/ml (extracts) can be considered as active ‘hits’ for most organisms. For the Gram negative bacteria, mycobacteria and fungi, 10 to 100 µM (pure compounds) or µg/ml (extracts) may be more appropriate as endpoint criterion. References 1 Vanden Berghe, D. and Vlietinck, A. J., 1991, Screening methods for antibacterial and antiviral agents from

higher plants, In: Hostettmann, K. (Ed.), Methods in Plant Biochemistry, Vol. 6, Academic Press, London, pp. 47-69

2 Cizman, M., 2003, The use and resistance to antibiotics in the community, International Journal of

Antimicrobial Agents, 21, 297-307 3 Chopra, P., Meena, L. S. and Singh, Y., 2003, New drug targets for Mycobacterium tuberculosis, Indian Journal

of Medical Research, 117, 1-9 4 Kauffman, C. A., 2003, Therapeutic and preventative options for the management of vancomycin-resistant

enterococcal infections, Journal of Antimicrobial Chemotherapy, 51 Suppl 3, 23-30 5 Eckermann, S. J. and Graham, K. J., 2000, Using chemical ecology to locate new antifungal natural products,

In: Atta-ur-Rahman, A. (Ed.), Studies in natural products chemistry, Vol. 22, Elsevier Science B. V., Amsterdam, pp. 55-92

6 Afeltra, J. and Verweij, P. E., 2003, Antifungal activity of nonantifungal drugs, European Journal of Clinical

Microbiology and Infectious Diseases, 22, 397-407


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7 Torres, H. A., Rivero, G. A., Lewis, R. E., Hachem, R., Raad, II and Kontoyiannis, D. P., 2003, Aspergillosis caused by non-fumigatus Aspergillus species: risk factors and in vitro susceptibility compared with Aspergillus fumigatus, Diagnostic Microbiology and Infectious Disease, 46, 25-28

8 Vlietinck, A. J. and Apers, S., 2000, Biological screening methods in the search for pharmacologically active

natural products: isolation, characterisation and biological properties, In: Tringali, C. (Ed.), Bioactive Compounds from Natural Sources, Taylor and Francis, London, pp. 1-29

9 Atta-ur-Rahman, A., Choudhary, M. I. and Thomsen, W. J., 2001, Bioassay Techniques for Drug Development,

Harwood Academic Publishers, Amsterdam 10 Hadacek, F. and Greger, H., 2000, Testing of antifungal natural products: methodologies, comparability of

results and assay choice, Phytochemical Analysis, 11, 137-147 11 Rios, J. L., Recio, M. C. and Villar, A., 1988, Screening methods for natural products with antimicrobial activity:

a review of the literature, Journal of Ethnopharmacology, 23, 127-149 12 Cole, M. D., 1994, Key antifungal, antibacterial and anti-insect assays - a critical review, Biochemical

Systematics and Ecology, 22, 837-856 13 Gabrielson, J., Hart, M., Jarelov, A., Kuhn, I., McKenzie, D. and Mollby, R., 2002, Evaluation of redox indicators

and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates, Journal of Microbiological Methods, 50, 63-73

14 Eloff, J. N., 1998, A sensitive and quick microplate method to determine the minimal inhibitory concentration

of plant extracts for bacteria, Planta Medica, 64, 711-713 15 Chand, S., Lusunzi, I., Veal, D. A., Williams, L. R. and Karuso, P., 1994, Rapid screening of the antimicrobial

activity of extracts and natural products, Journal of Antibiotics, 47, 1295-1304 16 Clarke, J. M., Gillings, M. R., Altavilla, N. and Beattie, A. J., 2001, Potential problems with fluorescein diacetate

assays of cell viability when testing natural products for antimicrobial activity, Journal of Microbiological Methods, 46, 261-267

17 Rahalison, L., Hamburger, M., Hostettmann, K., Monod, M. and Frenk, E., 1991, A bioautographic agar overlay

method for the detection of antifungal compounds from higher plants, Phytochemical Analysis, 2, 199-203 18 Hamburger, M. O. and Cordell, G. A., 1987, A direct bioautographic tlc assay for compounds possessing

antibacterial activity, Journal of Natural Products, 50, 19-22 19 Janssen, A. M., Scheffer, J. J. and Baerheim Svendsen, A., 1987, Antimicrobial activity of essential oils: a 1976-

1986 literature review. Aspects of the test methods, Planta Medica, 53, 395-398 20 Sindambiwe, J. B., Calomme, M., Geerts, S., Pieters, L., Vlietinck, A. J. and Vanden Berghe, D. A., 1998,

Evaluation of biological activities of triterpenoid saponins from Maesa lanceolata, Journal of Natural Products, 61, 585-590


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4 Bioassays for Screening Antiviral Activity

Paul Cos, Louis Maes, Dirk Vanden Berghe and Arnold J. Vlietinck

4.1 Introduction

The coronavirus and HIV retrovirus are the respective causing agents of severe acute respiratory syndrome (SARS) and acquired immune-deficiency syndrome (AIDS). The steadily increasing incidence of infections is caused by the human herpes virus family (HSV), the growing pathogenic role of a number of viruses such as human papilloma virus (HPV) in primary hepatocellular carcinoma and the socioeconomic impact of viral infections of the respiratory (influenza, adeno, corona and rhinoviruses) and gastrointestinal tracts (rotaviruses). All have a pivotal role in boosting the search for new antiviral agents and new modalities of antiviral chemotherapy.1-4

However, the concept of antiviral drugs has been accepted slowly due to the

peculiar nature of viruses, which totally depend upon the host cell for multiplication and survival; so many antiviral compounds are likely to injure the host cell.5-8 Moreover, in contrast to a bacteriostatic compound, an effective antiviral drug should not only display considerable specificity but should also irreversibly block viral synthesis to stop host cell death due to viral infection and restore its normal functions. In addition, the antiviral agents should have a broad spectrum of activity and favourable pharmacodynamic properties without being immunosuppressive. In an ideal situation, the antiviral drug should initiate clearance of infection while the immune system prepares to destroy the last virus particles. This point is critical for immunocompromised patients of AIDS, cancer or drug therapy in transplants and cancer. Viral infections are a frequent cause of death in these patients and adjuvant antiviral chemotherapy may be vital. There is also a growing need for new substances with extracellular virucidal activity, because the number of disinfectants and antiseptics is very limited and many of them also interfere with the growth of normal skin flora.

4.2 Overview of Antiviral Assay

Evaluation of in vitro antiviral activity and cytotoxicity is the initial step in antiviral drug discovery. Different viruses grow in different cell systems and as such it is almost impossible to design a single antiviral test that can be applied to all viruses. Moreover, methodologies of assessment of antiviral agents may vary greatly from one laboratory to another, thereby often making direct comparisons of test results difficult.

Various cell culture-based assays are currently available and can be successfully

applied for the antiviral (A) or virucidal (V) activity determination of single substance (S) or mixture of compounds (M) e.g. plant extracts (Table 1). Antiviral agents interfere with one or more dynamic processes of virus biosynthesis and are consequently candidates for clinically useful antiviral drugs. Virucidal substances extracellularly inactivate virus infectivity and are potential candidates for development of biocides, exhibiting a broad spectrum of germicidal activity.

BIOASSAYS FOR SCREENING ANTIVIRAL ACTIVITY

30

The methods commonly used for evaluation of in vitro antiviral activity are based on the different abilities of viruses to replicate in cultured cells. Some cause cytopathic effects (CPE) or form plaques while others are capable of producing specialized functions or cell transformation. Virus replication in cells may also be monitored by the detection of viral products, i.e. viral DNA, RNA or polypeptides. A survey of the various in vitro antiviral tests and their possible suitability for antiviral and/or virucidal screening of plant extracts and plant products is presented in Table 2. An intrinsic component of antiviral screening is the determination of a selectivity index (SI) towards the host cell. The SI refers to the ratio of the maximum drug concentration at which x% (50%, 90% or 99%) of the growth of normal cells is inhibited (CCx) and the minimum drug concentration at which x% of the virus is inhibited (ICx). It should be emphasized that the calculation of the SI of a mixture of compounds, e.g. crude extracts, may seem irrelevant since cytotoxicity and antiviral activity of the mixture are not necessarily due to the same components in the mixture. On the contrary, without cytotoxicity data, report of antiviral activity of a single compound even at very low concentration is of limited value.

Table 1: In vitro antiviral screening assaysa

Test Comment Application

Plaque inhibition assay Only for viruses which form plaque in suitable cell systems.

Titration of a limited number of viruses using non-toxic dose of the test substance.

A-S

Plaque reduction assay Only for viruses which form plaque in suitable cell systems.

Titration of residual virus infectivity after extracellular treatment with test substance(s). Cytotoxicity should be eliminated e.g. by dilution, filtration before the titration.

V-S; V-M

Inhibition of virus-induced cytopathic effect (CPE)

For viruses that induce CPE but do not readily form plaque in cell cultures.

Determination of virus-induced CPE in monolayers infected with a limited dose of virus and treated with a non-toxic dose of the test substance.

A-S; A-M

Virus yield reduction assay Determination of the virus yield in tissue cultures infected with a given amount of virus and treated with a non-toxic dose of the test substance.

Virus titration is carried out after virus multiplication by the plaque test (PT) or the 50% tissue culture dose end point test (TCD50).

A-S; A-M

Endpoint titration technique (EPTT)

Determination of virus titre reduction in the presence of two-fold dilutions of test compound.

A-S; A-M

Assay based on measurement of specialized functions and viral products

For viruses that do not induce CPE or form plaque in cell cultures.

Determination of virus-specific parameters, inhibition of cell transformation, immunological tests detecting antiviral antigens in cell cultures.

Reduction or inhibition of the synthesis of virus-specific polypeptides in infected cell cultures, determination of the uptake of radioactive isotope labelled precursors or viral genome copy numbers.

A-S; A-M; V-S; V-M

a Determination of the viral infectivity in cultured cells during virus multiplication in the presence of a single compound (A-S) or a mixture of compounds e.g. plant extracts (A-M) or after extracellular incubation with a single compound (V-S) or a mixture of compounds (V-M)

4.3 Virus Battery

There is a great variety of viruses that can be used for antiviral screening. In

general, human immunodeficiency virus (HIV) and herpes simplex virus (HSV) are the most commonly used in antiviral screening assays. Since anti-HIV screening should be performed in


31

a laboratory conforming to biosafety level-3, the number of research groups working with HIV is limited. Therefore, in this chapter viruses requiring biosafety level-2 are considered.

Many combinations of test viruses are possible but a battery of five viruses seems

to be quite acceptable (Table 2). Virus types and strains may vary in sensitivity but they have to be selected as a function of their ability to multiply in the same cell culture. In this way an objective comparison of antiviral activities is possible whereas toxicity tests are minimized. Moreover, virus multiplication must cause a visible CPE within a reasonable period of time, preferably within a week after infection.

Table 2: An example of a virus panel for screening

Virus DNA/RNA Capsid Envelop Serotypes

Coxsackievirus Single-stranded RNA Icosahedral No A: 25 B: 6

Vesicular stomatitis virus (VSV)

Single-stranded RNA Helical Yes 1

Semliki forest virus (SFV)

Single-stranded RNA Polyhedral Yes 2

Herpes simplex virus (HSV)

Double-stranded DNA Icosahedral Yes 2

Adenovirus Double-stranded DNA Icosahedral No 34

The virus battery shown in Table 2 consists of HSV and adenovirus as

representatives of DNA viruses, while coxsackievirus, Semliki forest virus (SFV), and vesicular stomatitis virus (VSV) are good prototypes of the RNA virus group. More specifically, coxsackievirus represents the Picornavirus family which includes rhinoviruses, the causative agents of common cold and enteroviruses. SFV and VSV represent the family Togaviridae and Rhabdoviridae respectively. The latter is transmitted by anthropods and is able to multiply both in insect and animal cells. All these viruses cause a visible CPE on monolayers of VERO cells within three days of infection. High titres, 105 TCID50 ml-1 or higher can easily be obtained which markedly increases the sensitivity of the testing system. The TCID50 (50% tissue culture infective dose) is defined as that dilution of virus required to infect 50% of inoculated cells. Suitable animal models for most of these viruses are available, enabling in vivo antiviral testing of pure compounds isolated from the selected plant extracts. 4.4 Description of In Vitro Antiviral Screening Assay

Various methods are available for evaluation of antiviral activities in vitro. In this chapter the endpoint titration technique (EPTT) and a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method will be discussed. Compared to the MTT method the EPTT is more suitable for testing complex samples such as plant extracts but cannot be implemented in a high throughput screening (HTS) for screening of a large number of samples. 4.4.1 Endpoint Titration Technique (EPTT) 4.4.1.1 Introduction The EPTT is performed on confluent monolayers of VERO cells in microtitre plates which are infected with serial tenfold dilutions of a virus suspension (Figure 1).9 Starting with monolayers containing 104 cells per well and a virus suspension (e.g. of 107 TCID50 ml-1), the first monolayers of cells are infected with a multiplicity of infection (MOI) of 10. Further serial tenfold dilutions of the virus suspension decrease the MOI from 10 to 10-4. The virus is allowed to absorb for 60 minutes at 37°C, after which serial twofold dilutions of plant extract or test compound in maintenance medium supplemented with 2% serum and antibiotics are


32

added. The plates are incubated at 37°C and the viral CPE is recorded daily by light microscopy for at least one week. Cytotoxicity controls (uninfected but treated cells) and cell controls (uninfected untreated cells) are run at each treatment concentration, and virus controls (infected but untreated cells) at each viral dilution. Toxic dose of the extract (T) is considered to be the dilution that causes destruction and degeneration of the monolayer, so that no virus titre can be determined. The antiviral activity is expressed as the virus titre reduction at the maximal non toxic dose (MNTD) of the test substance, i.e. the highest concentration that does not affect the monolayer under the conditions of the antiviral test procedure.

Viral titre reduction factor (RF, the ratio of the viral titre reduction in the absence, virus control and presence of the MNTD of the test sample) of 103 or higher indicates a pronounced antiviral activity and is one of the many criteria for further investigation.

Figure 1: The endpoint titration technique (EPTT) as antiviral evaluation method using a 96-well microtitre plate 4.4.1.2 Propagation of VERO Cell Cultures Materials and Equipment

The propagation of VERO cell cultures requires trypsin/EDTA, growth medium (medium E199 + 6% foetal calf serum), sterile calcium and magnesium free phosphate-buffered saline solution (PBS), cell culture flasks, 96-well tissue culture plates, sterile plastic pipettes, cell counting chamber (e.g. Bürker), CO2 incubator, inverted light microscope and containment of BSL-2. Procedure

Warm the PBS, trypsin/EDTA and growth medium in a water bath to 37°C. Observe the cell monolayer under an inverted light microscope. If the cells are healthy and appear to be free of contamination proceed with trypsinization. Aspirate the spent medium and wash the cell layer with PBS. Decant the wash solution and add trypsin/EDTA to the flask. If the cells do not detach easily incubate the flask at 37°C for a few minutes. When the cells detach add a small amount of growth medium to the flask. Suspend the cells by repeatedly pipetting up and down. Perform a cell count and seed new flasks for propagation of cell cultures and/or 96-well microtitre plates for antiviral assays. Incubate the newly seeded vessels in the CO2 incubator at 37°C. 4.4.1.3 EPTT Method Materials and Equipment

The technique requires maintenance medium (Plaisner medium + 2% foetal calf serum + 50 IU/ml penicillin and 50 µg/ml streptomycin), virus stock solution (titre of minimal 105 TCID50 ml-1), positive control (e.g. acyclovir for anti-HSV assays), 96-well tissue culture plates with confluent VERO cell monolayer, sterile plastic tubes for test sample and virus


33

dilution, sterile plastic pipettes, 1- and 8-channel pipettes and sterile tips, incubator, inverted light microscope and BSL-2 laboratory containment. Procedure

Warm the maintenance medium in a water bath to 37°C. Observe the cell monolayer under an inverted light microscope. If the cells are healthy and appear to be free of contamination proceed with antiviral assay. Dissolve the test sample in maintenance medium and make a two-fold serial dilution in the same medium. DMSO can be used to dissolve the test samples but the final concentration of DMSO is limited to 1%. Make 10-fold dilutions of virus stock solution in maintenance medium. Aspirate the medium from the 96-well microtitre plate with confluent VERO cell monolayer. Add 100 µl of the serial 10-fold dilution of the appropriate virus suspension to rows 1 to 6. Add 100 µl of maintenance medium to rows 7 and 8 and incubate plates for 60 minutes at 37°C. Add 100 µl of the appropriate two-fold dilution of the test sample to columns 2 to 11. Add maintenance medium to columns 1 and 12 (virus controls) and row 8 (cell control). Incubate all plates at 37°C. Examine the plates daily for CPE using an inverted light microscope for at least one week.

Antiviral activity is determined as reduction factor (RF) of the viral titre (ratio of the

virus titre in absence and presence of the test sample). 4.4.1.4 Discussion

The EPTT method is suitable to evaluate complex samples such as plant extracts (9). It uses serially diluted extracts and the cells are subsequently infected with different MOI. This system allows the correlation of all possible MOI values in the same microtitre plate with decreasing amounts of plant extract so that the non-toxic concentration of plant extract can be determined. A correlation between extract toxicity and antiviral activity according to the corresponding MOI can be determined in the same microtitre plate. It can be stated, as a general rule, that the detected antiviral activity should be stable in at least two subsequent dilutions of non-toxic concentration of the extract. Otherwise, the activity is directly correlated with the toxicity of the extract or is only virucidal. Moreover, a true antiviral product has to protect the cells infected with low virus dilutions. Finally, it should be stressed that all possible steps of virus replication are included in the EPTT because cells are inoculated with virus before the plant extracts are added. This means that an antiviral product that protects the cell monolayer at the highest concentrations of virus, under non-toxic conditions, must act on virus replication steps after uncoating. When the cells are completely protected only in the lower MOI, replication processes before uncoating may be involved in the activity.

Disadvantages of this method are the need for high virus titres (at least 104 TCID50

ml-1) and the possibility of overlooking activity in situations where the antiviral activity is not specific for all individual virus particles, due to a high degree of resistance of the virus to the corresponding extract. However, it is quite possible to obtain sufficiently high titres with all selected test viruses. Furthermore, we are mainly interested in specific antiviral substances active against all individual virus particles. 4.4.2 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay 4.4.2.1 Introduction

In the EPTT method, CPE and cytotoxicity are evaluated under an inverted light microscope. Therefore, the method is not suitable for HTS and for integrated screening. When a large number of samples are to be screened the viability of cells can be assessed spectrophotometrically by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The MTT assay is based on the reduction of the yellow coloured MTT by mitochondrial dehydrogenases of metabolically active cells to a blue formazan.10


34

4.4.2.2 Material and Equipment

The method requires maintenance medium (Plaisner medium + 2% foetal calf serum + 50 IU/ml penicillin and 50 µg/ml streptomycin), virus stock solution, MTT, isopropanol to solubilize MTT, positive control (e.g. acyclovir for anti-HSV assays), 96-well tissue culture plates with confluent VERO cell monolayer, sterile plastic tubes for test samples and virus dilution, sterile plastic pipettes, 1- and 8-channel pipettes and sterile tips, CO2 incubator, microtitre plate reader, inverted light microscope and BSL-2 laboratory containment. 4.4.2.3 Procedure

Warm the maintenance medium in a water bath to 37°C. Observe the cell monolayer under an inverted light microscope. If the cells are healthy and appear to be free of contamination proceed with antiviral assay. Add maintenance medium with or without test sample at the appropriate concentration and according to the standard test plate layout template (see chapter on bioassays for antibacterial and antifungal assays). Incubate plates for 60 minutes at 37°C and add 100 TCID50 of virus to all wells, except wells of cytotoxicity control and cell control. Incubate all plates at 37°C in a 5% CO2 incubator. After about three days, depending on the virus type, add 10 µl of MTT in PBS to all wells and incubate for 4 hours at 37°C in a 5% CO2 incubator. Add isopropanol to solubilize the formazan crystals. Mix and read the plates in a microtitre plate reader at a wavelength of 540 nm.

4.4.3 Description of an Animal Model for HSV Infection 4.4.3.1 Introduction

A general concept in drug screening and evaluation is the confirmation of primary in vitro results in a corresponding in vivo animal model, preferably with the same pathogen species. The latter allows the establishment of a stronger ‘proof-of-concept’ of promising pharmacological activity, because the gap between the in vitro and in vivo is extraordinarily large. While in vivo evaluation is easily achievable with most bacteria, yeasts and parasites it is certainly much more difficult for viruses in view of their generally higher host-specificity and the lack of validated animal models. The in vitro screening tests remain undoubtedly the first choice to screen crude plant extracts and to guide the isolation of antiviral constituents but in vivo assays in a suitable animal model will remain indispensable before drug testing can be initiated in man. An exception to this rule may be HIV where the enzyme targets (protease, reverse-transcriptase) and the in vitro screening technique on MT-4 cells have proven to be quite reliable for predicting pharmacological potency and efficacy in man. Fortunately, for a number of viruses animal models are available. Animal testing is helpful in confirming not only if the candidate compound is an effective viral inhibitor but also if it: (1) reaches the target organ(s); (2) is metabolically stable and/or is cleared within a reasonable time; (3) does not interfere with the normal cell processes of the host; (4) is systemically available after oral administration and; (5) does not adversely affect the immune system. The last two aspects are particularly relevant for antiretrovirals.

In the next section an animal model for herpes encephalitis is briefly described.

Detailed information on this model and on some other animal models of viral infection can be found in the literature.11

4.4.3.2 Animal Model Animals, Materials and Equipment

The model uses 21 day old female Balb/c mice. It requires normal biology lab equipment and wares such as CO2 incubator, microtitre plate reader, inverted light


35

microscope, sterile syringes and needles, pipettes and tips besides specific requirements of test organisms (Herpes simplex virus with known titre), a standard drug (acyclovir), vehicle for drug and test compounds (sterile PBS) and animal facilities and containment of biosafety level-2.

Procedure

After approval of the animal experiments by the local ethical committee, female Balb/c mice are purchased from an official laboratory or animal supplier. Mice are housed in animal facilities conforming to animal biosafety level-2. They are maintained under artificial diurnal lighting conditions (12 hours each of light and dark cycle) and with free access to food and water. They are handled according to the animal care guidelines. After arrival, mice are kept in quarantine for at least seven days before starting the experiment. Table 3 lists the minimal number of experimental groups for in vivo evaluation of anti-HSV activity. Depending on the test objectives the number of groups may increase.

Table 3: Experimental groups for in vivo evaluation of anti-HSV activity

Experimental group Infected Treated

Vehicle or placebo group Yes PBS Positive control Yes Acyclovir in PBS Test group Yes Test compound in PBS Negative control No PBS

Infections are initiated by intranasal or intraperitoneal inoculation of mice. For

intranasal inoculation virus is administered in a 0.04 ml volume containing 104 to 105 pfu/animal with a 20 µl Eppendorf pipette tip. The mouse is allowed to inhale 20 µl in each nostril. Intraperitoneal injection uses 0.1 ml of 102 to 103 pfu of HSV using sterile syringes and needles. All inoculated animals are examined daily for signs of clinical disease such as weight loss. Death and survival time are also recorded. After death, virus titres are determined in target organs, such as brain, spleen, liver and lung. 4.4.3.3 Discussion

It is advisable to use more than one experimental animal model whenever possible

because each model may show a different pattern of antiviral efficacy. Moreover, familiarity with the pathogenesis of a virus infection in a given animal model system is essential prior to its application for antiviral evaluation.

A major advantage of an HSV model is that HSV isolated from humans will readily

infect mice, rats and most other small animals. However, the herpes encephalitis model should not be oversimplified because there are many possible tissue sites for interactions between the antiviral compound and virus-infected cells. Therefore, mortality is not a very sensitive endpoint and should be accompanied by determination of virus titres in the critical target organs.

4.5 Conclusions

This chapter is primarily meant to describe two practical methods for the discovery of new antiviral agents. The EPTT method is developed for testing complex samples such as plant extracts while the MTT method is suitable for testing larger number of samples. Besides potency a pivotal criterion to select a compound for further evaluation is the selectivity index. Very often, the originally proposed antiviral activity of plant substances as reported in the literature is too low, non-specific or only detectable in toxic concentrations for the host. Moreover, many so called antivirals have an antiviral activity on extracellular viruses which means that they irreversibly denaturate virus proteins or glycoproteins so that the viruses are


36

no longer able to invade living cells. These compounds do not act on one of the replication steps of the viruses and high concentrations are needed for their activity. Hence, they cannot be considered as putative antiviral chemotherapeutics. References 1 Holmes, K. V., 2003, SARS coronavirus: a new challenge for prevention and therapy, Journal of Clinical

Investigation, 111, 1605-1609 2 De Clercq, E., 2002, Strategies in the design of antiviral drugs, Nature Reviews Drug Discovery, 1, 13-25 3 Lee, S. S., 2003, Review article: indicators and predictors of response to anti-viral therapy in chronic hepatitis

C, Alimentary Pharmacology and Therapeutics, 17, 611-621 4 Uhnoo, I., Linde, A., Pauksens, K., Lindberg, A., Eriksson, M. and Norrby, R., 2003, Treatment and prevention of

influenza: Swedish recommendations, Scandinavian Journal of Infectious Diseases, 35, 3-11 5 Vlietinck, A. J. and Vanden Berghe, D. A., 1991, Can ethnopharmacology contribute to the development of

antiviral drugs?, Journal of Ethnopharmacology, 32, 141-153 6 Vlietinck, A. J., de Bruyne, T. and Vanden Berghe, D. A., 1997, Plant substances as antiviral agents, Current

Organic Chemistry, 1, 307-344 7 Vlietinck, A. J., De Bruyne, T., Apers, S. and Pieters, L. A., 1998, Plant-derived leading compounds for

chemotherapy of human immunodeficiency virus (HIV) infection, Planta Medica, 64, 97-109 8 Cos, P., Vanden Berghe, D. A., de Bruyne, T. and Vlietinck, A. J., 2003, Plant substances as antiviral agents: un

update (1997-2001), Current Organic Chemistry, 7, 1163-1180 9 Vanden Berghe, D. A., Vlietinck, A. J. and Van Hoof, L., 1986, Plant products as potential antiviral agents,

Bulletin of Pasteur Institute, 84, 101-147 10 Mosmann, T., 1983, Rapid colorimetric assay for cellular growth and survival: application to proliferation and

cytotoxicity assays, Journal of Immunological Methods, 65, 55-63 11 Kern, E. R., 1999, Animal models for central nervous system and disseminated infections with herpes simplex

virus, In: Zak, O. and Sande, M. A. (Eds.), Handbook of Animal Models of Infection, Vol. 107, Academic Press, New York, pp. 899-906


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5 Evaluation of Anti-inflammatory Activity of

Natural Products Based on Enzyme Inhibitors

Lars Bohlin and Ulrika Huss

5.1 Introduction

Cures for a number of severe diseases are not yet available despite rapid progress

in medical research over the past decades. Many chronic diseases such as rheumatoid arthritis, psoriasis, and ulcerative colitis, all with an inflammatory and/or immunological background, are treated symptomatically - no drug is available to effectively cure them.

A number of drugs with different therapeutic effects are marketed for the treatment

of inflammatory diseases such as corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), and the recently marketed selective cyclooxygenase-2 (COX-2) inhibitors. Corticosteroids have an important role in the treatment of inflammatory diseases but due to their severe side-effects/toxicity, they can only be used for short periods of time and in serious cases. NSAIDs, such as acetylsalicylic acid derivatives, pyrazolidine derivatives, and propionic acid derivatives have been cornerstones in anti-inflammatory drug therapy. However, these drugs are often not sufficiently effective in cases of chronic inflammation, and are prone to cause severe gastrointestinal side effects such as ulcers and gastrointestinal bleeding.

Selective COX-2 inhibitors have been marketed as drugs with comparable anti-

inflammatory benefits as traditional NSAIDs but with a reduced risk of gastrointestinal complications. For the treatment of inflammatory diseases there are drugs which modify the course of the disease and are called disease modifying anti-rheumatic drugs (DMARDS). Immunosuppressive compounds are used in some cases/stages of the disease but treatment with these drugs involves a greater risk of serious side effects.

Advances in molecular biology have opened up the field for new approaches in the

search for anti-inflammatory drugs. One novel approach has been to evaluate the contribution of peptide mediators in inflammation and the possible role of antibodies and peptide antagonists as inhibitors, for instance of leukocyte surface receptors. 5.1.1 Inflammation

The inflammatory response in humans can be activated by several stimuli, including

mechanical or chemical injuries or as a defence against the invasion of micro-organisms. The stimuli initiate the inflammatory process by activating various enzymatic pathways and the release of different inflammatory mediators, for instance prostaglandins synthesised via the cyclooxygenase-2 enzyme. These chemical mediators participate in different ways in the inflammatory process, and thus contribute to the classical symptoms of inflammation, e.g. redness, heat and swelling.

After activation of the inflammatory process, the blood supply to the injured area

increases due to dilatation of small blood vessels. Further, increased permeability of the blood vessels generate a leakage of fluid to the neighbouring tissues. Phagocytic cells such as

EVALUATION OF ANTI-INFALAMMATORY ACTIVITY OF NATURAL PRODUCTS BASED ON ENZYME INHIBITORS

38

neutrophils and macrophages start migrating from the blood into the injured tissues. At the site of injury the phagocytic cells start engulfing the pathogens and cell debris and in consequence the inflammatory process contributes to tissue healing. Nevertheless, under some circumstances, the inflammatory response is the underlying cause of severe diseases, e.g. rheumatoid arthritis where the inflammatory response turns against healthy tissues.

5.1.2 Neutrophils

In humans the neutrophils are one of the essential phagocytic cells which play a

central role in the inflammatory process. Neutrophil functions are activated by various stimuli/chemotactic factors including: the bacterial product, N-formyl methionyl-leucyl-phenylalanine (fMLP); the platelet activating factor (PAF), a product of phospholipid metabolism; and leukotriene B4 and cytokines such as interleukine 8. Phagocytic cells such as neutrophils and macrophages, guided by chemotactic factors, migrate to the sites of tissue damage and infection. Neutrophils at the site of inflammation release toxic products such as proteolytic enzyme, reactive oxygen species and cationic proteins (e.g., defensins and cathelicidins) with the purpose to kill invading pathogens.

The general targets of the neutrophil include: bacteria, fungi, viruses, virally infected

cells and tumour cells. However, healthy tissue can also be injured in cases when neutrophils accumulate in too high concentration or they receive inappropriate stimuli and/or when the activity of their products is not adequately controlled. Activated human neutrophils and its secreted products such as the proteolytic enzyme, elastase are involved in the various chronic inflammatory disorders including chronic bronchitis, chronic obstructive pulmonary diseases and reumathoid arthritis.

5.2 The Enzyme Family of Cyclooxygenases

Among other regulatory factors, a wide variety of enzymes are involved in the

inflammatory process. For instance, enzymes such as the cyclooxygenases, proteases such as neutrophil elastase, lipoxygenases, phospholipases and nitric oxide synthases, contribute in various ways to the complex process of regulating the course of events. When tissue damage occurs, phospholipases contribute to the release of arachidonic acid from the cellular membranes. This fatty acid is rapidly converted by cyclooxygenases and lipoxygenases to various bioactive products such as prostaglandins, thromboxanes or leukotrienes.

The cyclooxygenase family is comprised of a number of enzymes which play a key

role in the regulation of inflammation by catalysing the prostaglandins biosynthesis. These enzymes catalyse the first two steps in the conversion of arachidonic acid (AA) to prostaglandins (PGs) which in turn mediate inflammation, fever and pain.

There exist at least three different isoforms of cyclooxygenases, namely,

cyclooxygenase-1, -2, and -3 (COX-1, COX-2 and COX-3) which have the capability to catalyse the prostaglandin production. The two first discovered enzymes, COX-1 and COX-2 are today very well characterised. It remains unclear where the recently detected COX-3 is produced in vivo and how it contributes to physiological and pathophysiological conditions. COX-1, the first identified member of the COX family, is normally constitutively expressed, and is present in almost all tissues at constant levels. The metabolites of arachidonic acid, derived via COX-1, are responsible for maintaining basic physiological conditions in the body as in the case of cytoprotection of the gastric mucosa. The second isoenzyme, COX-2, in contrast to COX-1, is mainly an inducible isoform which is triggered by various stimuli including inflammatory agents, growth factors and tumour promoters. A few tissues such as the brain and kidney have constitutive expression of COX-2. The induced COX-2 enzyme is involved in the production of prostaglandins that promote the inflammatory process. In recent studies, several splice


39

variants of COX-1 mRNA were detected and a second protein (translated) was found which possesses catalytic activity.1, 2 This enzyme was named COX-3.

5.2.1 Cyclooxygenase-1 and -2: Similarities and Differences

COX-1 and -2 exhibit two distinct enzymatic functionalities, the cyclooxygenase and

the peroxidase activities. Because of their two different enzymatic activities, these enzymes should actually be referred to as prostaglandin H synthases (PGHS). Nevertheless, they are commonly called cyclooxygenases (COX) which in general include both enzymatic activities. Both enzymes catalyse the conversion of arachidonic acid via a two-step process. In the first step two molecules of O2 are added to AA by cyclooxygenase activity generating prostaglandin G2 (PGG2). In the second step PGG2 is rapidly reduced to prostaglandin H2 (PGH2) by the peroxidase activity of the enzyme. The PGH2 is quickly converted to various PGs (e.g. prostaglandin D2, E2, PGF2α), thromboxane A2 (TXA2), or prostacyclin (PGI2) by cell and tissue specific synthases and isomerases.

The overall structures of the enzymes are very similar. The most notable difference

between COX-1 and COX-2 is at their cyclooxygenase active site, in the amino acid position 523. In COX-1, the amino acid 523 consists of isoleucine which is replaced with the smaller amino acid valine in COX-2. Thus a side-pocket opens up at the active site of COX-2.3 Noticeably, this minor difference of an amino acid plays a major role in the shape of the active site. This structural feature has been exploited in the search for selective COX-2 inhibitors (larger molecules have the possibility to enter the active site of COX-2 due to the extra space in the side-pocket).

5.2.2 Cyclooxygenase-2 Inhibitors

At present, a variety of natural and synthetic COX-2 inhibitors have been reported. A

number of compounds (called coxibs) have been launched commercially as selective COX-2 inhibitory drugs prescribed for arthritis and rheumatoid arthritis. These selective COX-2 inhibitors are marketed as drugs with the same anti-inflammtory benefits as traditional NSAIDs but with reduced risk of gastrointestinal complications. Although recently one of the COX-2 selective drugs, namely Vioxx® (rofecoxib), was hastily withdrawn from the market due to its severe side-effects (cardiac infarction).

The search for natural compounds with COX-2 inhibitory activity is rapidly

progressing. In 2001 we presented a review of literature for the period 1991-99 where reports of natural compounds and plant extracts with effect on COX-2 were summarised.4 The compounds are reported to affect COX-2 at several levels including inhibition of enzymatic activity or altering the levels of COX-2 mRNA or protein. These compounds represent a variety of substances including common compounds such as fatty acids, flavonoids, terpenes and some unusual compounds such as: ajoene, a thioester in garlic (Allium sativum L.);5 C-phycocyanin, a biliprotein from the blue green algae Spirulina platensis;6,7 and; tryptanthrine, an alkaloid found in Isatis tinctoria L.8

In recent years, interest in anti-inflammatory activity of plant extracts and pure

constituents has increased dramatically due to the discovery of inducible enzyme COX-2. This enzyme, besides being an important target in the search for new anti-inflammatory drugs is also considered to be a potential target in the field of cancer research. COX-2 is known to contribute to a number of pathophysiological processes9, including involvement in different types of cancer.10-12


40

5.2.3 Brief Overview of Assay for Evaluating Cyclooxygenase-2 Inhibitory Effect of Natural Products

A wide variety of bioassays have been developed to assess the effect of synthetic or

natural compounds on COX-1 and COX-2. There is a special interest to find compounds that preferentially inhibit COX-2. Consequently, variation in experimental conditions is high between different bioassays which nevertheless can be divided into two main categories: (1) bioassays for finding compounds that inhibit enzymatic activity of COX-2 and (2) bioassays for finding compounds that can suppress de novo synthesis of COX-2.

The assays used for search of inhibitors of COX-2 enzymatic activity differ

substantially in several respects including reaction time, concentration of substrate, source and amount of enzyme, use of co-factors and incubation time with drugs.13 Also, the detection systems used for measuring the inhibition of a drug on enzymatic activity vary greatly. For example, the inhibition of enzymatic activity could be measured continuously in real-time using a polarographic oxygen electrode to register the amount of oxygen that is used during conversion of AA to PGG2.14 However, often a stop-time assay is used instead with a separate step for detection of produced prostaglandins. Methods frequently used to measure the amount of produced prostaglandins include HPLC, radioimmunoassay (RIA), enzyme immunoassay (EIA) or scintillation counting (if a radiolabelled substrate is used).15-17 The inhibitory effect of a compound on enzymatic activity can also be measured in an intact cell system.18

In the assays used in the search for inhibitors of de novo synthesis of COX-2 there

are large variations, as previously mentioned, including cell types and the stimuli used to induce COX-2.19 A variety of human and animal cells are used along with a variety of stimuli which commonly are lipopolysaccharide (LPS) or cytokines, such as interleukin-1.19 Inhibitors of protein synthesis may interact at several levels by inhibiting transcription by affecting post-transcriptional processes (including mRNA transport, control of stability, and translation) and by regulating the rate of protein degradation. Commonly used methods to detect the effects of compounds in cells include the COX-2 luciferase assay to evaluate COX-2 promoter activity,20 northern blot to identify and quantify the amount of produced mRNA in cells, and western blot to evaluate COX-2 protein levels.21, 22

5.2.4 Determination of Cyclooxygenase-1 and -2 Enzymatic Inhibitory Activity

As already mentioned, a large number of assays are available to detect compounds

that affect COX-2 enzymatic activity. The techniques differ but all those assays aim to monitor the effect of plant extracts or pure compounds on the enzymatic activity.

One commonly used method for measuring inhibition of cyclooxygenase catalysed

prostaglandin biosynthesis is the radiochemical in vitro assay originally described by White and Glassman.23 The method described in this chapter has been further developed and systematically optimized for various parameters such as incubation time, concentration of co-factors and possible effects of solvents.16

The radiochemical assay in brief relies on COX-1 or COX-2 catalysed conversion of

[1-14C]-AA to radiolabelled PGs. The PGs produced are separated from the unmetabolized AA by column chromatography and the total amount of PGs is quantified as a group using scintillation counter.

To estimate the inhibitory effect of a natural product the enzyme is incubated with

the plant extract or pure compound before addition of [1-14C]-AA. An inhibitory effect of the natural product is observed if the amount of produced PGs is reduced compared with the amount of PGs produced by untreated enzyme. The inhibition of COX-2 catalyzed prostaglandin


41

biosynthesis is calculated as the relative decrease in radioactivity (disintegrations per minute) of the samples containing the test substance as compared to the solvent, i.e. untreated enzyme or enzyme treated with solvent vehicle.

The test compounds can affect the enzyme in various ways causing a decreased

capacity of converting arachidonic acid into prostaglandins. For example, a compound may compete with arachidonic acid for the binding site or may modify the structure of the enzyme and a conformation change of the binding site for arachidonic acid may occur which disables AA from entering into the catalytic site. Any of these actions may cause decreased production of prostaglandins.

The following section describes a bioassay suitable for monitoring the inhibition of

COX enzymatic activity, a method which is suitable for bioassay-guided fractionation of plant extracts. Literature references for additional information on COX-1 and COX-2 and in vitro method of radiochemical assay are available.4, 24-26

5.2.5 The Radiochemical In Vitro Assay Stock Solutions

A number of stock-solutions have to be prepared in advance, such as a 0.1 M Tris buffer (pH 8.0), stock-solution of AA (see below), 0.1 M K2HPO4 with 3 mM MgCl2, 0.02 M Na2CO3, 2 M HCl (stop-reagent) and the eluents: heptane-dioxane-glacial acetic acid (70:30:1) and EtOAc-MeOH (85:15).

Preparation of Enzymes

The purified COX-2 (from sheep placental cotyledons) obtained from the Cayman Chemical Company (Ann Arbor, MI, USA), immediately after arrival, is dispensed into aliquots suitable for one experiment and kept at -80°C until use. The COX-1 enzyme originates from microsomes isolated from bovine seminal vesicles according to Takeguchi et al.27 Fresh bovine seminal vesicles from bulls are immediately placed on ice and trimmed free from fat and connective tissue. Two volumes of Tris buffer are added and the seminal vesicles are gently homogenised by cutting. These steps should be performed on ice. Centrifuge the homogenate (12000 x g for 10 minutes, 4°C) and filter the supernatant through cheesecloth followed by centrifugation (48000 x g for 60 minutes, 4°C). The supernatant should be carefully siphoned off and the pellet is re-suspended in a minimal amount of Tris buffer and dispensed into aliquots of 20 µl and stored at -80°C until use. The protein concentration is determined by the Bradford protein assay.28 Alternatively, purified COX-1 could be purchased from Cayman Chemical Company.

Preparation of Stock Solution of Arachidonic Acid

The arachidonic acid [1-14C] (50 µCi) from Amersham (Uppsala, Sweden) is dissolved in toluene on arrival. To prepare a stock solution of AA, evaporate the toluene under a soft stream of N2. Thereafter, add 65.1 µl of unlabelled AA (10 mg/ml, dissolved in 99.5% EtOH) to the pure [1-14C] radiolabelled AA followed by 934.9 µl of EtOH (99.5%). Fill the bottle with N2 (i.e. degas with a soft stream of N2 for 20 s) and store at -20°C.

Prepare a working solution of AA (30 µM) from the stock solution a few minutes before start of the enzymatic reaction to avoid denaturation of AA. Working solution for one experiment is prepared in an eppendorf tube by mixing 14 µl of 0.02 M Na2CO3 with 1372 µl of the buffer containing 0.1 M K2HPO4 and 3 mM MgCl2. Add 14 µl of the stock solution of AA and fill the tube with N2 (i.e. degas with a soft stream of N2 for 20 s). Keep the solution on ice until use.


42

Plant Extracts and/or Pure Compounds

Dissolve the plant extract to a concentration of 0.5 mg/ml (which yields a final concentration of 0.1 mg/ml in the wells) in a suitable solvent. The selection of solvent depends on the polarity of the plant extract. Dimethylsulfoxide (DMSO) can be used for non-polar extracts such as hexane or dichloromethane, whereas ethanol, water or TRIS-buffer could be suitable for polar extracts. In our experience a final concentration of 4 % DMSO can be used without affecting the enzymes. However, the effect of solvents on the enzymes should be carefully evaluated and solvent controls should always be included in each experiment. Moreover, a compound with a known COX-1 or COX-2 inhibitory effect (e.g. indomethacin) should be included as a control (reference compound) in each experiment.

Bioassay Scheme

The experiments are performed in 96-well microtiter plates (Figure 1), with each well containing a final volume of 100 µl (20 µl of sample or solvent, 60 µl of enzyme-cofactor solution, and 20 µl of arachidonic acid working solution). Keep the microtiter plate on ice while dispensing solvent controls, reference compounds and test compounds/plant extracts. A total of 48 wells are used for each experiment. Add 20 µl of the solvent to the first six wells (solvent control). Add 20 µl of the test substance or extract solution to next wells in triplicates for each sample. Save the last nine wells, three for addition of a reference compound (e.g. indomethacin in a concentration yielding 50 % inhibition) and the remaining six wells for addition of solvent controls (20 µl each).

Figure 1: Schematic view of the radiochemical in vitro assay experiment

The Reaction

Take out Tris buffer from the refrigerator and degas for at least 15 minutes to decrease the amount of oxygen. Prepare the stock solutions of cofactors; l-epinephrine (6.5 mg/ml), reduced glutathione (1.5 mg/ml), and hematin (1.27 mg/ml) in Tris buffer. To facilitate dissolution of hematin, add 1 drop of NaOH. Transfer 600 µl each of l-epinephrine and glutathione stock solution and 3 µl of hematin stock solution into a glass vial. Add Tris buffer to make the final volume 3 ml (requires 1797 µl). The concentrations of l-epinephrine, reduced glutathione, and hematin in the final cofactor solution are 1.3 mg, 0.3 mg, and 1.3 µg per ml, respectively.

Take out the enzyme from the freezer and dilute it in Tris buffer with a volume that gives an enzyme concentration of 0.3 units/µl. Gently mix the solution by pipetting to obtain a homogenous enzyme-TRIS solution (vortexing has to be avoided). Transfer 600 µl of enzyme-TRIS solution to the cofactor solution and incubate for 5 minutes on ice. Dispense 60 µl of


43

COX-2 (3.0 units/well) or COX-1 (7 to 8 µg protein/well) to each well which already contain 20 µl of test compound/plant extract or solvent control. Pre-incubate the enzyme and samples for 10 minutes on ice. Meanwhile, add 10 µl HCl (2 M) to the first and last three wells to inactivate the enzyme. These wells will represent an estimate of the background radioactivity. During this 10 minutes incubation time prepare the working solution of [1-14C] AA (30 µM) and keep on ice until one minute of the incubation time remains. Then load a multidispenser with the AA-solution and start the reaction by rapid addition of 20 µl to each well. Gently shake the plate before placing it in a shaking water bath. The reaction is carried out for 3 minutes at 37°C. Terminate the reaction by putting the microtiter plate on ice and rapidly adding 10 µl of HCl (2 M) to each well (except to the wells which already contain HCl). Thereafter, gently shake the plate and leave it on ice until starting the separation of prostaglandins. Separation of the Unmetabolised Arachidonic Acid from the Prostaglandins

Precondition the silica columns (silica gel 60, particle size 0.063 to 2 mm, Merck, Darmstadt, Germany, packed in Pasteur pipettes) with 1 ml x 2 of heptane-dioxane-glacial acetic acid (70:30:1). Transfer 90 µl from the reaction wells to the silica gel columns to separate the unmetabolised arachidonic acid from the prostaglandins. Elute the unmetabolised arachidonic acid with 1 ml x 5 heptane-dioxane-glacial acetic acid (70:30:1). Place a rack with scintillation vials under the columns and elute the prostaglandins with 1 ml x 3 ethylacetate-methanol (85:15). Add scintillation liquid to the vials and count the samples in a Packard scintillation spectrometer. Each experiment should be repeated at least three times. Calculate the percent inhibition of prostaglandins with the following formula:

Percent inhibition = 100)(

)()( 321

backgroundMax

backgroundtestbackgroundMax

PGPGPGPGPGPG

−−−−

1Maximum production of prostaglandins in presence of solvent, measured in disintegrations per minute (DPM) 2Background measured in DPM (in presence of solvent and HCl) 3Amount of prostaglandins produced with test compound present, measured in DPM

5.3 Protease Inhibitors

The control of synthesis of protein turnover and function enable proteases to

regulate different physiological processes and is crucial for the propagation of a number of diseases. The importance of proteases as targets for drugs has increased over recent years and is evident from the ongoing intensive search for potent and selective protease inhibitors with therapeutic uses in diseases such as cancer, inflammatory disorders, viral infections, malaria and hepatitis.29 Several protease inhibitors of natural origin have been developed. The polypeptide hirudin isolated from the leech is an inhibitor of thrombin, a trypsin like serine protease.30 Another example is the discovery of a non-peptide, teprotide, isolated from the Brazilian snake Bothrops jararaca. This natural product showed a significant inhibition of the metallo protease angiotensin converting enzyme. Matrix metallo-proteinases are a family of structurally related zinc containing enzymes which are involved in the breakdown of connective tissue. These enzymes are important targets for therapeutic inhibitors in several inflammatory, malignant and degenerative diseases. Inhibitors of natural origin also include tetracyclines such as aranciamycin and catechin- and futoenone-derivatives.31 A review by Mattheé et al.32 describes a diverse number of HIV reverse transcriptase inhibitors of natural origin exemplified by calanolide A (IC50= 0.07 µM) isolated from the plant Calophyllum lanigerum.

5.3.1 The Enzyme Family of Elastases

The elastase family consists of a group of proteases which have the ability to cleave

the protein elastin of connective-tissue. The elastase enzyme in human neutrophils is a potent serine protease which possesses the ability to cleave a variety of substrates including elastin


44

and all other major connective-tissue proteins. The elastases under normal physiological conditions are carefully regulated by endogenous inhibitors to avoid destruction of connective tissue proteins. In chronic inflammatory diseases such as lung emphysema, arthritis and reumathoid arthritis, activated human neutrophils secrete elastase which is a major contributor of tissue destruction. Consequently, to monitor the amount of released elastase is an important tool in the search for natural compounds which affect the progress of chronic inflammatory diseases.

The following section describes a neutrophil multitarget assay suitable for detection

of plant extracts or isolated compounds which interact with the inflammatory process of activated neutrophils. The method monitors mainly the release of elastase as a function of formation of p-nitroanilide. More information on the subject is available.33 5.3.2 The Neutrophil Multitarget Assay

Stock Solutions

The stock-solutions should be prepared in advance. These include: elastase substrate cytochalasin B (Sigma) (dissolve 0.965 mg in 200 µl DMSO and add 800 µl water); 300 mM N-succinyl-L-alanyl-L-alanyl-L-vaniline-L-p-nitroanilide (SAAVNA) (Sigma) (108 mg in 750 µl DMSO); 10-2 M N-formyl methionyl-leucyl-phenylalanine (fMLP) (Sigma) (4.38 mg in 1 ml EtOH); platelet activating factor (PAF) (Sigma) 10-2 M (5.42 mg in 1 ml EtOH); phosphate buffered saline (PBS) containing 0.9 mM CaCl2 and 0.49 mM MgCl2; 0.9 % NaCl; and citric acid 2 %.

Preparations before Obtaining the Blood Samples

All plant extracts or test compounds are dissolved before obtaining the blood samples from the hospital. Plant extracts and compounds can be dissolved in DMSO, EtOH or MeOH to yield a final concentration of 0.1 %.

The dextran solution (10 % dextran-T-500 in 0.9% NaCl) used for erythrocyte

sedimentation should be dissolved in advance as it dissolves with difficulty. Also prepare in advance 0.25 % bovine serum albumin (BSA) in PBS containing 0.9 mM CaCl2 and 0.49 mM MgCl2 by adding 300 mg on the surface of 120 ml PBS. Leave it to settle, do not mix.

Neutrophil Isolation

The blood sampling step is performed in the hospital by trained personnel. Blood samples from healthy volunteers are obtained in heparinized test tubes (containing 10 000 U of heparin/100 ml blood) and are immediately transported to the laboratory. Do not mix blood from different donors until isolation of the PMS.

The blood samples (21 ml from each donor) are transferred from the test tubes into syringes and are further mixed with 2.1 ml of the dextran solution (10 %) for sedimentation of the erythrocytes. After approximately 30 minutes, carefully transfer the cells from the upper leukocyte layer into large centrifuge tubes. Add PBS (without Ca and Mg) up to 40 ml and 10 ml Ficoll-Paque in the bottom of the tubes. Centrifuge the cell suspension through the Ficoll-Paque (500 x g for 30 minutes, 20°C). Remove the supernatant and treat the obtained cell-pellet with one volume of ice cold sterile water for 21 sec, before the addition of 9 volumes of PBS, (without Ca and Mg) to lyse the remaining red blood cells. Centrifuge the samples (500 x g for 10 minutes, 4°C), remove the supernatant and re-suspend the obtained cell-pellet in PBS containing 0.9 mM CaCl2; 0.49 mM MgCl2 to obtain a suspension of 10 to 30 x 106 cells/ml. The pellets from two different donors can now be mixed and suspended in 6 ml PBS.


45

Determination of Released Elastase

Use concentrations of 106 neutrophils in each test tube (100 µl of cell suspension). The PMN suspension in each test tube is pre-incubated for 5 minutes at 37°C with 5 µl cytochalasin B stock solution (final concentration 5 µg/ml), 750 µl of a mixture of 154 µl SAAVNA stock sloution in 45 ml 0.25 % BSA (final concentration 0.8 mM), and the test solutions or the solvent used for dissolving the samples.

Dilute the inducers PAF and fMLP by mixing 25 µl of the PAF or fMLP stock solutions with 2475 µl of 0.25 % BSA. 50 µl of this mixture is further diluted to 5000 µl with 0.25 % BSA.

Start the challenge with one of the following agents: 100 µl PAF or 100 µl fMLP (of final dilutions) making the final concentration 0.1 µM. To allow a direct comparison samples are tested in parallel for both PAF- and fMLP-induced exocytosis using PMN from the same preparation. Test tubes without the addition of PAF/fMLP are also run in parallel.

After incubation at 37°C for 10 minutes, the reaction is stopped by the addition of

2 % citric acid and the tubes are centrifuged (500 x g for 10 minutes, 20 ºC).

The reaction of elastase with its substrate SAAVNA leads to formation of a coloured product, p-nitroanilide (pNA) that can be quantified spectrophotometrically (Figure 2).

Figure 2: Schematic view of the neutrophil assay

Measure the absorbance of each sample at 405 nm using a spectrophotometer. All samples are run in duplicate. The absorbance in the corresponding background tube (without inducer) is subtracted from that of the sample. The percent inhibition of fMLP- or PAF-induced pNA formation is calculated from the relative decrease in absorbance using the following formula.

% Inhibition= {1-[(ABS(S+I)-ABS(S+B))/(ABS(I)-ABS(B))]} × 100

ABS = absorbance; S = sample; I = inducer; B = buffer.

The plant extracts and pure compounds should also be evaluated for their hemolytic

activity in order to determine possible cell damaging/cytotoxic effects.33

5.4 Safety Guidelines

Safety regulations should be strictly followed when handling radiolabeled isotopes. Human blood collection should strictly follow ethical guidelines besides general safety regulations. Take all necessary precautions for proper disposal of radioactive and blood wastes. 5.5 Natural Products as Source of Anti-inflammatory Compounds

Plants, animals and micro-organisms constitute a self-rejuvenating and unique

source of new molecules with enormous variation in chemical structure and biological activity.

SAAVNA pNA

PAF/fMLP Neutrophil

Elastase


46

These bioactive substances exist in nature for various purposes including maintenance of physiological functions and defence against enemies such as bacteria, fungi, attacking insects, and plant eating animals. Many of these natural products are so elegantly constructed that not only the molecular backbone but also the placement of the functional groups has been done to produce the optimal effect. It is difficult for man to exceed nature's ability to design new biological molecules. The challenge in this area of research now lies in understanding the underlying mechanism of action that forms the basis for their activity in human beings. Several strategies have been developed to improve the likelihood of finding potential bioactive compounds. The rationale in the search for natural products with a biological activity is based on three major fundamentals i.e. selection, isolation and bioassays.

Two commonly used selection approaches are based on ethnopharmacological and

ecological observations. In addition, chemotaxonomic relationships have been used as a tool to identify possible naturally occurring analogues of active substances. Today, progress in botany has opened up the possibility of using phylogeny in the selection of plants.

A wide variety of bioassays have been developed in order to discover new anti-

inflammatory compounds. Traditionally anti-inflammatory studies relied on animal in vivo models such as carragenan-induced rat paw oedema etc. The rationale today is to use a bioactivity guided fractionation (i.e. the activity of the plant extract is successively evaluated after each separation step until the pure active compound is isolated) of plant extracts using an in vitro assay as a model to monitor the anti-inflammatory effect.

In medical research natural products are important tools, for instance when

studying the physiological/pathological functioning of receptors and enzymes. The next section presents some important compounds of natural origin which have been reported to affect the inflammatory process.

5.5.1 Phorbol Esters

The group represents a class of polyhydroxylated diterpenes which occur as esters

in plants belonging to the family Euphorbiaceae. These substances are highly toxic inducing inflammation in the skin and are active in nM concentrations. Furthermore, phorbolesters are active as tumour promoters the most active of which, tetradecanylphorbol acetate (TPA), is used as a biochemical tool. The phorbol esters act as a substitute for the cells' own mediator, 1,2-diacylglycerol in the activation of calcium- and phospholipid-dependent protein kinase C (PKC). The phorbol esters are important in investigations of these enzymes due to their central role in understanding the inflammatory process.34

5.5.2 Thapsiagargin

Is a sesquiterpene originally isolated from a Mediterranean plant, Thapsia garganica

L.,which has been identified as an active principle responsible for the skin-irritating effects of this plant. Thapsiagargin activates cells in the immune system, in addition to acting as a tumour promoter. Extensive studies have shown that thapsiagargin releases intracellularly stored calcium ions by a specific inhibition of Ca2+-ATPase.35 5.5.3 Ginkgolides

These are a group of unique terpenes consisting of 20 carbon atoms arranged in a

complex ring structure with six 5-membered rings. They have been isolated from the fossil tree Ginkgo biloba, which is used in traditional Chinese medicine, for instance in the treatment of asthma. The ginkgolides have been shown to be specific and potent antagonists of PAF, an inflammatory mediator acting on different types of cells. The ginkgolides inhibit the binding of PAF to its receptor on the blood platelets. PAF can affect different immunoprocesses where


47

the ginkgolides can inhibit the suppressive effect of PAF on the proliferation of T-lymphocytes and their cytokine production. Furthermore, the ginkgolides have shown an ability to increase the survival of heart transplant in rats, acting synergistically together with cyclosporin A. These PAF-antagonists have also revealed that PAF has an important role in pathology of diseases such as asthma, shock, ischemia, anaphylaxis, rejection of transplants and inflammation in the kidneys.36, 37

5.5.4 Cyclosporin A

Is a neutral hydrophobic peptide consisting of 11 amino acid residues (Mw 1202)

isolated from the microscopic fungus Tolypocladium inflatum. It is the first of a new generation of immunosuppressive substances that act specifically and reversibly on lymphocytic T-cells. This property has had revolutionary consequences for organ transplantation.38 Cyclosporin A-based immunosuppression has been adopted world wide for protection of transplanted organs. However, its severe side effects e.g. nephrotoxicity are well documented and it is therefore necessary to continue the search for less toxic and more potent immunosuppressants.

5.5.5 Anti-inflammatory Activity of Certain Plants in Traditional Medicine

A number of plants used in traditional medicine to treat inflammatory disorders

were evaluated by the authors with a primary objective of isolating and characterizing the active substances through bioassay-guided fractionation to find new active substances for further structure-activity studies. Table 1 summarizes some of findings of the authors.

Table 1: Evaluation results of some traditionally used anti-inflammatory plants

Plant Target disease

Model Results Reference

Skin transplantation Increase in rejection time

39

PAF-model Inhibition of PAF

Polypodium decumanum Willd. Fam. Polypodiaceae

Psoriasis

Exocytosis model Inhibition of exocytosis In vivo rat paw & rat ear oedema

Inhibitory effect on oedema

40-42 Ipomoea pes-capra (L.)R. Br. Fam. Convolvulaceae

Dermatitis

Guinea pig ileum Antispasmodic activity 43

Choerospondias axillaris Roxb. Fam. Anacardiaceae

Burns In vitro anti-inflammatory

Inhibition of prosta-glandin formation

-

In vivo rat paw & rat ear oedema

Dose dependent inhibition of oedema

Menyanthes trifoliata L. Fam. Menyanthaceae

Glomerulo nephritis

In vitro anti-inflammatory

Inhibition of prostaglandins, leukotriene B4, PAF and exocytosis

44

In vivo rat paw oedema

Reduction of oedema

In vitro anti-inflammatory

Inhibition of prostaglandins, leukotriene B4, PAF and exocytosis

Alphitonia zizyphoides A. Gray Fam. Rhamnaceae

General inflammation

In vitro and in vivo antispasmodic test

Muscle relaxant activity

45, 46

5.6 Conclusions

The interpretation of the use of medicinal plants and understanding the clinical

observations are difficult tasks. It is of great importance to painstakingly identify the indications that form the basis for choice of the plants, the processing methods and the


48

bioassays to be utilized. We believe that plants with a well documented traditional use and with a verified effect in clinical studies on man have great potential to yield new lead structures for drug development.

In this chapter we describe two assays, one based on a purified enzyme model (the

radiochemical in vitro assay for studying effects on COX-1 and COX-2 catalysed prostaglandin biosynthesis) and a cell model (the neutrophil multitarget assay). These bioassays have been adapted for bioactivity guided fractionation of plant extracts. Consequently, these methods are valuable tools in the search for novel anti-inflammatory compounds of natural origin.

5.7 Acknowledgement

The authors gratefully acknowledge collegaues at the Division of Pharmacognosy,

Uppsala University for their contribution including the development and optimization of the bioassays described in this chapter. References

1 Chandrasekharan, N. V., Dai, H., Roos, K. L., Evanson, N. K., Tomsik, J., Elton, T. S. and Simmons, D. L., 2002,

COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression, Proceedings of the National Academy of Sciences, 99, 13926-13931

2 Schwab, J. M., Schluesener, H. J. and Laufer, S., 2003, COX-3: just another COX or the solitary elusive target of

paracetamol, Lancet, 361, 981-982 3 Browner, M., 1996, X-ray crystal structure of human cyclooxygenase-2, In: Bazan, N., Botting, J. and Vane, J.

(Eds.), New Targets in Inflammation-inhibitors of COX-2 or Adhesion Molecules, Klywer Academic Publishers and William Harvey Press, Dordrecht, pp. 71-74

4 Perera, P., Ringbom, T., Huss, U., Vasänge, M. and Bohlin, L., 2001, Search for natural products with effect on

cyclooxygenase-2., In: Tringali, C. (Ed.), Bioactive Compounds from Natural Sources, Taylor & Francis, London, pp. 433-472

5 Dirsch, V. M. and Vollmar, A. M., 2001, Ajoene, a natural product with non-steroidal anti-inflammatory drug

(NSAID)-like properties, Biochemical Pharmacology, 61, 587-593 6 Reddy, C. M., Bhat, V. B., Kiranmai, G., Reddy, M. N., Reddanna, P. and Madyastha, K. M., 2000, Selective

inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina platensis, Biochemical and Biophysical Research Communications, 277, 599-603

7 Reddy, M. C., Subhashini, J., Mahipal, S. V., Bhat, V. B., Srinivas Reddy, P., Kiranmai, G., Madyastha, K. M. and

Reddanna, P., 2003, C-Phycocyanin, a selective cyclooxygenase-2 inhibitor, induces apoptosis in lipopolysaccharide-stimulated RAW 264.7 macrophages, Biochemical and Biophysical Research Communications, 304, 385-392

8 Danz, H., Stoyanova, S., Wippich, P., Brattstrom, A. and Hamburger, M., 2001, Identification and isolation of

the cyclooxygenase-2 inhibitory principle in Isatis tinctoria, Planta Medica, 67, 411-416 9 O'Banion, M. K., 1999, Cyclooxygenase-2: molecular biology, pharmacology, and neurobiology, Critical Reviews

in Neurobiology, 13, 45-82 10 Ermert, L., Dierkes, C. and Ermert, M., 2003, Immunohistochemical expression of cyclooxygenase isoenzymes

and downstream enzymes in human lung tumors, Clinical Cancer Research, 9, 1604-1610 11 Koki, A. T. and Masferrer, J. L., 2002, Celecoxib: a specific COX-2 inhibitor with anticancer properties, Cancer

Control, 9, 28-35 12 Soslow, R. A., Dannenberg, A. J., Rush, D., Woerner, B. M., Khan, K. N., Masferrer, J. and Koki, A. T., 2000, COX-

2 is expressed in human pulmonary, colonic, and mammary tumors, Cancer, 89, 2637-2645


49

13 Pairet, M. and van Ryn, J., 1998, Experimental models used to investigate the differential inhibition of

cyclooxygenase-1 and cyclooxygenase-2 by non-steroidal anti-inflammatory drugs, Inflammation Research, 47 Suppl 2, S93-101

14 Wang, H., Nair, M. G., Strasburg, G. M., Chang, Y. C., Booren, A. M., Gray, J. I. and DeWitt, D. L., 1999,

Antioxidant and antiinflammatory activities of anthocyanins and their aglycon, cyanidin, from tart cherries, Journal of Natural Products, 62, 294-296

15 Kase, Y., Saitoh, K., Ishige, A. and Komatsu, Y., 1998, Mechanisms by which Hange-shashin-to reduces

prostaglandin E2 levels, Biological and Pharmaceutical Bulletin, 21, 1277-1281 16 Noreen, Y., Ringbom, T., Perera, P., Danielson, H. and Bohlin, L., 1998, Development of a radiochemical

cyclooxygenase-1 and -2 in vitro assay for identification of natural products as inhibitors of prostaglandin biosynthesis, Journal of Natural Products, 61, 2-7

17 Subbaramaiah, K., Chung, W. J., Michaluart, P., Telang, N., Tanabe, T., Inoue, H., Jang, M., Pezzuto, J. M. and

Dannenberg, A. J., 1998, Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells, Journal of Biological Chemistry, 273, 21875-21882

18 Kim, Y. P., Yamada, M., Lim, S. S., Lee, S. H., Ryu, N., Shin, K. H. and Ohuchi, K., 1999, Inhibition by

tectorigenin and tectoridin of prostaglandin E2 production and cyclooxygenase-2 induction in rat peritoneal macrophages, Biochimica et Biophysica Acta, 1438, 399-407

19 Pairet, M. and van Ryn, J., 1999, Measurement of differential inhibition of COX-1 and COX-2 and the

pharmacology of selective inhibitors, Drugs Today, 35, 251-265 20 Subbaramaiah, K., Bulic, P., Lin, Y., Dannenberg, A. J. and Pasco, D. S., 2001, Development and use of a gene

promoter-based screen to identify novel inhibitors of cyclooxygenase-2 transcription, Journal of Biomolecular Screening, 6, 101-110

21 Guan, Z. and Morrison, A. R., 1999, Assessment of cyclooxygenase RNA expression by northern hybridization,

In: Lianos, E. A. (Ed.), Methods in Molecular Biology, Humana Press Inc., Totowa 22 Guan, Z. and Morrison, A. R., 1999, Assessment of cyclooxygenase protein expression by western blotting, In:

Lianos, E. A. (Ed.), Methods in Molecular Biology, Humana Press Inc., Totowa 23 White, H. L. and Glassman, A. T., 1974, A simple radiochemical assay for prostaglandin synthetase,

Prostaglandins, 7, 123-129 24 Noreen, Y., 1997, Separation and bioassay in identification of cyclooxygenase inhibitors from plants, with

emphasis on flavonoids, Doctoral thesis, Uppsala University, Uppsala 25 Huss, U., 2003, Studies on the effects of plant and food constituents on cyclooxygenase-2, -Aspects in

inflammation and cancer, Doctoral thesis, Uppsala University, Uppsala 26 Ringbom, T., 2002, Bioassay development for identification of cyclooxygenase-2 inhibitors of natural origin,

Doctoral thesis, Uppsala University, Uppsala 27 Takeguchi, C., Kono, E. and Sih, C. J., 1971, Mechanism of prostaglandin biosynthesis. I. Characterization and

assay of bovine prostaglandin synthetase, Biochemistry, 10, 2372-2376 28 Bradford, M. M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein

utilizing the principle of protein-dye binding, Analytical Biochemistry, 72, 248-254 29 Leung, D., Abbenante, G. and Fairlie, D. P., 2000, Protease inhibitors: current status and future prospects,

Journal of Medicinal Chemistry, 43, 305-341 30 Verstraete, M., 1996, Modulating platelet function with selective thrombin inhibitors, Haemostasis, 26 Suppl 4,

70-77 31 Whittaker, M., Floyd, C. D., Brown, P. and Gearing, A. J., 1999, Design and therapeutic application of matrix

metalloproteinase inhibitors, Chemical Reviews, 99, 2735-2776 32 Matthee, G., Wright, A. D. and Konig, G. M., 1999, HIV reverse transcriptase inhibitors of natural origin, Planta

Medica, 65, 493-506 33 Johansson, S., 2001, Studies on cytotoxic and neutrophil challenging polypeptides and cardiac glycosides of

plant origin, Doctoral thesis, Uppsala University, Uppsala


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34 Evans, F. J., 1991, Natural products as probes for new drug target identification, Journal of

Ethnopharmacology, 32, 91-101 35 Brogger Christensen, S., Andersen, A., Lauridsen, A., Moldt, P., Smitt, U. W. and Thastrup, O., 1992,

Thapsiagargin: A lead to design of drugs with the calcium pump as target, In: Krogsgaard-Larsen, P., Brogger Christensen, S. and Kofod, H. (Eds.), New Leads and Targets in Drug Research: Alfred Benzon Symposium 33, Munksgaard, Copenhagen, pp. 243-252

36 Braquet, P., Esanu, A., Buisine, E., Hosford, D., Broquet, C. and Koltai, M., 1991, Recent progress in ginkgolide

research, Medicinal Research Reviews, 11, 295-355 37 Braquet, P. and Hosford, D., 1991, Ethnopharmacology and the development of natural PAF antagonists as

therapeutic agents, Journal of Ethnopharmacology, 32, 135-139 38 Kahan, B. H. (Ed.). 1984, Cyclosporin-Biological activity and clinical applications, Grune and Stratton, London 39 Vasänge, M., 1996, Characterisation of compounds with Platelet activating factor related activity from

Polypodium decumanum, a fern with clinical use in psoriasis, Doctoral thesis, Uppsala University, Uppsala 40 Pongprayoon, U., Bohlin, L., Baeckström, P., Jacobsson, U. and Lindström, M., 1992, Inhibition of ethyl

phenylpropiolate-induced rat ear oedema by compounds isolated from Ipomoea pes-caprae (L.) R. Br., Phytotherapy Research, 6, 104-107

41 Pongprayoon, U., Bohlin, L., Soonthornsaratune, P. and Wasuwat, S., 1991, Antiinflammatory activity of

Ipomoea pes-caprae (L.) R. Br., Phytotherapy Research, 5, 63-66 42 Pongprayoon, U., Baeckstrom, P., Jacobsson, U., Lindstrom, M. and Bohlin, L., 1991, Compounds inhibiting

prostaglandin synthesis isolated from Ipomoea pes-caprae, Planta Medica, 57, 515-518 43 Pongprayoon, U., Baeckstrom, P., Jacobsson, U., Lindstrom, M. and Bohlin, L., 1992, Antispasmodic activity of

beta-damascenone and E-phytol isolated from Ipomoea pes-caprae, Planta Medica, 58, 19-21 44 Tunón, H., 1995, Evaluation of Swedish medicinal plants as a source of anti-Inflammatory drugs and mosquito

repellents, Doctoral thesis, Uppsala University, Uppsala 45 Perera, P., Andersson, R., Bohlin, L., Andersson, C., Li, D., Owen, N. L., Dunkel, R., Mayne, C. L., Grant, D. M.,

Pugmire, R. J. and Cox, P. A., 1993, Structure determination of a new saponin from the plant Alphitonia zizyphoides by NMR spectroscopy, Magnetic Resonance in Chemistry, 31, 472-480

46 Andersson-Dunstan, C., 1995, Isolation and structure elucidation of pharmacologically active compounds from

Alphitonia zizyphoides (Spreng.) A. Gray, with emphasis on inflammation, Doctoral thesis, Uppsala University, Uppsala


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6 Pharmacological Screening Methods for Gastrointestinal

System

S. Oguz Kayaalp and K. Husnu Can Baser 6.1 Introduction

The main parts of the gastrointestinal system are the stomach, duodenum, jejunum,

ileum and colon. These organs, together with the endocrine and exocrine secretory glands including the liver, are involved in the digestion of food, absorption of nutritional ingredients and regulation of these functions in an integrated manner for energy intake, storage and consumption.

The intestinal wall is composed of three main layers: mucous membrane around the

lumen, smooth muscle layer of inner circular and outer longitudinal smooth muscle tissue, and serous membrane covering the inner layers. A nerve plexus is located between the circular and longitudinal smooth muscle layers. It regulates the motility and secretion of the intestine together with release of gut hormones from the endocrine cell clusters located within the intestinal wall. The peristaltic movements are induced by the pacemakers within the local excitable tissues and are modified by the intrinsic and extrinsic neurons and paracrine and endocrine factors. The somas (cell bodies) of the extrinsic motor and sensory nerve endings are located outside the gastrointestinal system such as the parasympathetic neurons of the brain stem. The excision of extrinsic nerves does not paralyse the intestinal motility. The organization of the neural and smooth muscle tissues within the oesophagus, stomach and colon is essentially similar to that described for the intestine.

Screening methods related to the gastrointestinal system are divided into two types

depending on the objectives: bioassays based on gastrointestinal tissues as a matrix and in vivo methods to evaluate effects in the gastrointestinal disorders.

6.2 In Vitro Methods Using Gastrointestinal Tissues

Gastrointestinal smooth muscle - its innervation and secretory tissues - are

employed as study objects in various in vitro bioassays to test drugs for the following actions: - spasmodic and antispasmodic actions; - actions on cholinergic parasympathetic innervation; - actions on postganglionic sympathetic innervation; - actions on muscarinic and nicotinic cholinergic, adrenergic, histaminergic, serotonergic

and peptidergic receptors; - action on gastric acid secretion; - action on histamine release upon antigen challenge in sensitized tissue.

These actions, except that on gastric acid secretion, may also be tested in

preparations employing the tissues of the other systems, such as vascular and bronchial. However, the gastrointestinal tissues are commonly used to study drug actions on the parasympathetic cholinergic system. Furthermore, the relatively larger mass of the

PHARMACOLOGICAL SCREENING METHODS FOR GASTROINTESTINAL SYSTEM

52

gastrointestinal tissues provides the opportunity to set up a large number of experiments from one animal. The bioassay preparations of gastrointestinal origin are addressed below. 6.2.1 Isolated Ileum Preparation (Magnus Method)

It is most widely used in vitro smooth muscle and smooth muscle-nerve preparation

used for isolated organ studies. The original method was described by Magnus.1 Guinea pigs, preferably male, weighing 250 to 500 g are used after fasting for 24

hours. The method requires scissors, forceps, petri dishes, 20 ml syringe or pipette, micropipette, a 15 to 20 ml isolated organ bath and a polygraph. Tyrode's solution oxygenated with a mixture of 95 % oxygen and 5 % carbon dioxide at 37 to 38°C is used as a physiological medium. The tone and movement of the isolated ileum are recorded on a polygraph via an isometric force displacement transducer sensitive to a tension of a few grams.

The fasted guinea pigs are sacrificed by a blow on the head and cutting the throat.

The abdomen is opened by a long midline incision from the tip of the xyphoid to the symphysis. The ileum is found by lifting the caecum on its back to locate the ileo-caecal junction. The ileum is cut close to the caecum. A portion of the ileum, up to 40 cm long, depending on the number of preparations, is removed by gently cutting the mesentery along the line of contact with the ileum. The portion of the ileum close to the ileocaecal junction is the most sensitive to the contractile action of drugs. The ileum should be cleared of mesenteric tissue as far as possible using forceps and scissors. The ileum is placed in the petri dish containing Tyrode’s solution and then cut into small pieces of 2 to 3 cm long. The lumen of the ileum is cleared by gently pushing through a glass rod taking care not to stretch its walls. The lumen of the ileum is usually clean in fasted animals. The piece of ileum is transferred to another petri dish containing Tyrode’s solution. The lumen is washed gently with Tyrode’s solution using a syringe or pipette until the effluent is clear. Care should be taken not to damage the ileum tissue with forceps. It is preferable to handle the ileum gently with the fingers rather than forceps. The small part of the cut end of the ileum which is unavoidably squeezed by the forceps may be removed by cutting it out with scissors.

The piece of ileum is suspended in Tyrode’s solution in the isolated organ bath. One

end is tied to the stationary hook at the lower tip of the holder to be immersed into the bath and the other end is tied to the tip of the force displacement transducer. A tension of 0.5 to 1 g is applied to the ileum and the experiment starts after an equilibrium period of 30 minutes.

During the experiment the calculated amount of test drug is added to the bath in a

volume of 10 to 100 µl with a micropipette to give a preset drug concentration in the Tyrode’s solution. The volume of the dose should be less than 1/100 of the physiological solution in the bath. The drug solution is commonly prepared in distilled water at an appropriate concentration to produce the intended concentration after dilution with Tyrode’s solution in the bath. The bath solution is continuously bubbled with a mixture of oxygen and carbon dioxide. Bubbling helps the quick dispersion of the drug solution in the bath fluid.

When the contractile response is attained to a maximal level, the bath fluid is

discharged and washed 2 or 3 times to remove the residual drug from the tissue. An equilibration period of about 5 to 30 minutes is allowed before adding the next dose. A cumulative concentration-response curve for certain drugs may also be obtained by adding them successively to the bath, without washing the organ between one addition and the next. The contractile response of agonistic drugs is recorded to plot a concentration-response curve. EC50 value (concentration producing half maximal response to agonist) may be calculated from this curve.

Antagonistic drugs are tested against the corresponding agonist by adding them a

few minutes before the agonist. Another approach is to produce a submaximal contractile


53

response to agonist (precontraction) and to challenge it with increasing cumulative concentration of the antagonist.2 Competitive antagonists (atropine-like substances) and physiological antagonists (α- and β-adrenergic blockers) can be tested on the ileum precontracted by cholinergic agonists. Precontraction may also be produced by barium chloride solution or by depolarization with high concentration of potassium chloride (above 20 mM) or by prostaglandin E2 (1-10 µM). This general approach allows calculation of IC50 value (concentration producing a 50 % reduction in the maximal response to agonist) of the antagonist. Furthermore, after calculating the IC50 value of an antagonist against a constant concentration of agonist (C), the dissociation constant of the antagonist (Ki) may be calculated from the following equation:

IC50 Ki = --------------------

1 + (C/EC50)

Indicators of the gravimetric potency of agonist and antagonist substances, pD2 and pA2 respectively, can also be calculated by the experiments on isolated guinea pig ileum.3

The usual submaximal concentrations of certain agonists and antagonists drugs are

given in Table 1.4

Table 1: Submaximal concentrations of some agonists and antagonists

Name Conc (µg/ml)

Acetylcholine 0.05 Histamine 0.05 Serotonin 2.0 Nicotine 1.0 Atropine 0.002-0.008 Mepyramine 0.001-0.006 Hexamethonium 1.0 Barium chloride 200.0 Papaverine 1.0-3.0

Antispasmodic drugs are either the receptor antagonists of the endogenous active

substances causing a physiologic or pathologic tonic contractile response in the intestine or the non-specific spasmolytic drugs interfering with the downstream contraction mechanisms of smooth muscles. A drug of potential antispasmodic action of the former type may be tested in this preparation against endogenous agonists and of the latter type by using non-specific spasmodics like barium chloride. Papaverine, an opium alkaloid, abolishes the spasms by a non-specific spasmolytic action at postreceptor level. A potential papaverine-like antispasmodic drug may be tested in this preparation by challenging the barium chloride-induced contractile response. In conclusion, isolated guinea pig ileum preparation is a versatile simple preparation to test drugs for spasmodic and antispasmodic actions on the gastrointestinal system.

Several modifications of the isolated guinea pig ileum preparation are addressed

below. Rat isolated ileum preparation may also be used as a test organ with a different pattern of sensitivity to agonistic substances.2 6.2.2 Electrically Stimulated Guinea Pig Ileum Preparation

Depending on the position of the stimulating electrodes, a variety of electrically

stimulated guinea pig ileum preparation is described. A field stimulation approach is more commonly used in which the tissue is positioned in isolated organ bath between two platinum or preferably silver chloride electrodes with at least one electrode away from the tissue. The ileum may be stimulated directly by two electrodes such as clip or needle electrodes placed on


54

or within it. The clip and needle electrodes should be insulated appropriately except the small hole of the needle of the electrode contacting the tissue to reduce the current bypassing the tissue.

During field stimulation most of the electrical current flows through the bathing

solution because the resistance of the gut tissue (about 10 kOhm/cm2) is higher than the resistance of the solution (about 1 kOhm/cm2).4 Because of the dispersion of the current, a high intensity stimulus should be used from a high output (100 mA or 400 V) stimulator. Square wave stimuli of 1 millisecond duration may be applied with a frequency of 0.1 Hz. Higher frequency stimuli applied for a long period accelerate the polarization of the electrodes causing progressively reduced stimulus intensity and tissue response. Furthermore the bath solution may be heated by the current flowing through it. The intensity of the stimulus is supramaximal and usually adjusted to a level twice that inducing maximal response.

The ileum is prepared and mounted in the isolated organ bath filled with Tyrode`s

solution as described above and the electrodes are placed according to the stimulation required (direct or field). The contractions are recorded on a polygraph.

The electrical stimulation activates the cholinergic neurons in the Auerbach`s

plexus, functionally a parasympathetic ganglion equivalent lying between circular and longitudinal smooth muscle layers, and their axon endings releasing acetylcholine at muscle junctions. Given that the smooth muscle cells are less sensitive to electrical stimuli than the neural tissue they are not directly stimulated. The contractile response is produced by activated longitudinal smooth muscles. It may be blocked by antimuscarinic drugs such as atropine. Given that the stimulation of the preganglionic nerve endings at the Auerbach’s plexus also contribute in part to contractile response, hexamethonium and related ganglion blocking nicotinic receptor antagonists may partially inhibit the response to electrical stimulation. Furthermore opioid agonists acting on the inhibitory opioid mu receptors at prejunctional cholinergic nerve endings may also block the contractile response in a dose dependent manner.5 The electrically stimulated ileum preparations are used to test potential antimuscarinic or antinicotinic drugs and opioid mu receptor agonists and their antagonists. The inhibitory action on the acetylcholine release at cholinergic nerve endings may also be tested.

One of the various types of the electrically stimulated guinea pig ileum preparations is the co-axially stimulated ileum preparation described by Paton.6 A platinum wire electrode of 4 to 5 cm length protruding from the tip of the glass tube holding ileum in the bath and an indifferent platinum electrode are immersed into the bath fluid. The former wire electrode is passed through inside the lumen while mounting the organ into the bath. The ileum is stimulated by square-wave stimuli of 1 millisecond duration with an intensity of 40 mA at a frequency of 0.1 Hz. The contractions are recorded on a polygraph.

6.2.3 Guinea Pig Mesenteric Plexus-longitudinal Muscle Preparation

It is an improved version of the electrically stimulated guinea pig ileum preparation

which in turn was a modification of a rabbit ileum preparation.7,8 Guinea pig ileum is isolated and a piece of 5 to 6 cm is cut. A glass rod of a diameter just big enough to cause slight stretching is inserted into the lumen. The longitudinal shallow fissure produced along the ileum when the mesentery was cut out, is identified. A wooden stick of about 10 cm long, 2 to 3 mm in diameter, wrapped in a thin layer of cotton wool and soaked in Krebs’ solution, is applied with gentle pushes along the fissure at a right angle to its lip to strip away the outer longitudinal smooth muscle layer of the ileum together with its serous cover. While stripping progresses, the glass rod inside the ileum is slowly rotated to keep the stripping line up from one end to the other. A full rotation is needed to strip the layer completely. The longitudinal muscle layer is removed from the Auerbach’s plexus which tightly adheres to it. Therefore the preparation is a nerve-smooth muscle preparation. It is mounted in an isolated organ bath


55

filled with Krebs’ solution and oxygenated continuously by a mixture of oxygen and carbon dioxide. Stimulated platinum wire electrodes are placed in parallel to vertically suspended tissue. The electrical stimulation causes contractions by activating the cholinergic neurons in the plexus. The contractions are recorded on a polygraph.

The mesenteric plexus-longitudinal muscle preparation is used to test the drugs with

a potential mu opioid agonist and antagonistic activities. The agonist inhibits the contractile response dose-dependently and the inhibitory action is reversed by opioid mu receptor antagonists such as naloxone.

6.2.4 Guinea Pig Ileum Preparation (Modified Method of Trendelenburg)

The method was described by Trendelenburg9 and a modification allowing

administration of drugs from the inside and outside of the ileum was later described.10 This preparation has the purpose of studying drug actions on the pressure-induced peristaltic movements.

A 6 cm piece of ileum is prepared for mounting in the isolated organ bath. The

caecal end of the ileum is tied to the tip of a tube connected to a reservoir containing Tyrode’s solution on a platform which may be elevated and lowered to vary hydrostatic pressure inside the lumen of the ileum. The other end is tied with a thread and is attached to a force displacement transducer to record peristaltic movements on a polygraph. The ileum is mounted in the isolated organ bath of 30 to 50 ml capacity containing Tyrode's solution. When the reservoir is elevated, the pressure inside the lumen increases and induces peristaltic reflex movements. The pressure is increased for 15 to 30 seconds every two minutes. The peristaltic movements cause a to and fro movement of Tyrode's solution within the ileum. These movements change the pressure in the air pocket over the fluid in the reservoir which is recorded via a tube connecting the air pocket to a volume transducer. Therefore, both the rhythmic contractions and relaxations of the ileum and changes in volume of the air pocket in the reservoir are recorded simultaneously on a polygraph. The former (length) response represents the contractions and relaxations of the longitudinal smooth muscles and the latter (volume response) records the contractions and relaxations of both circular and longitudinal muscles.

The height of the reservoir is raised to an adequate level to induce regular peristaltic

response. The height may be measured by a ruler. This preparation is used for testing atropine-like and ganglion blocking

anticholinergic drugs and local anaesthetics on peristaltic movements induced by internal pressure in the guinea pig ileum. However, since the peristaltic response is not stable and fluctuates during the experiment, it is suited only to observe qualitative response.

6.2.5 Rabbit Periarterial Sympathetic Nerve Jejunum Preparation (Finkelman

Method)

The preparation is a sympathetic nerve-smooth muscle preparation aimed at testing the drugs on postganglionic noradrenergic transmission.11

A piece of rabbit jejunum 2 to 3 cm long in which mesenteric arterial supply may be

clearly seen, is removed and mesenterial tissue is dissected out without damage to the arterial branches. The postganglionic sympathetic nerve supply from the lumbar ganglia to the jejunum travels along the branches of the mesenteric artery. The periarterial nerves may be dissected out or retained intact given that they make up a plexus around the arterial branches. The piece of jejunum is mounted in an isolated organ bath of at least 25 ml capacity containing Tyrode’s solution. The periarterial nerves are either dissected or all of the


56

mesenteric arteries are placed on a bipolar platinum electrode. Square-wave-stimuli of 0.5 millisecond duration at a frequency of 30 to 50 Hz are applied for up to 30 seconds every 2 minutes. Lower frequency stimulation does not activate the sympathetic nerves but activates parasympathetic nerves.12

The rabbit jejunum exerts regular peristaltic movements under resting conditions

and periarterial nerve stimulation stops the peristalsis as long as stimulation continues. The inhibition of peristalsis is caused by noradrenaline released at postganglionic nerve endings making junctions with smooth muscle cells. A similar inhibitory action is produced by application of noradrenaline-like adrenergic agonists. The preparation is used to study the actions of potential adrenergic neurone blockers inhibiting noradrenaline release from adrenergic nerve endings. 6.2.6 Rat Gastric Fundus Preparation (Vane Method13)

This method is a bioassay to quantify serotonin in biological fluids. A rat which has fasted overnight is sacrificed by a blow on the head and cutting the

throat. The abdomen is opened and the stomach is removed and cut along the lesser curvature up to gastroesophageal junction. After the contents have been cleaned, the fundus which may be easily distinguished by its grey colour from the pink coloured antral portion is cut out by a transverse incision. It is stretched flat on a cork plate. Strips 3 to 4 mm wide and 4 to 5 cm long are cut along the longitudinal axis of the stomach parallel to curvatures as shown in Figure 1. The strip is mounted in a narrow isolated organ bath of 8 to 10 cm long and less than 10 ml capacity containing Krebs’s solution at 37°C to avoid excessive dilution of the serotonin containing biological fluid in serotonin bioassay tests. Such a volume limitation is not necessary when the preparation is used for other purposes. The contractions are recorded via a force displacement transducer on a polygraph. A resting tension of 1 g is applied and the preparation contracts and relaxes slowly. Therefore drugs should be added to the bath not more frequently than once every 6 and sometimes 10 minutes. Fundus strips are very sensitive to serotonin and may respond to a concentration as low as 0.4 to 0.8 ng/ml.12 It is used as a bioassay organ for serotonin in the presence of atropine or l-hyoscine (2 x 10-6 M). The drugs can also be tested for their potential serotonin agonist and antagonist actions in this preparation. Not only the agonistic actions but also the antagonistic actions develop slowly. Therefore adequate time should be allowed between two doses.

Figure 1: Schematic illustration of cutting smooth muscle strips from rat fundus

6.2.7 Isolated Guinea Pig Oddisphincter and Gall Bladder Preparation

In vitro guinea pig choledocus-Oddi sphincter and gall bladder preparations are

used to test drugs on the tone and motility of smooth muscles.14, 15


57

Guinea pigs of 300 to 500 g are sacrificed by stunning and then exsanguinating. The abdomen is opened. The choledocus with choledocoduodenal junction and duodenal wall surrounding it are removed in toto and dissected to isolate the choledocus together with ampulla and papilla. The tube-like tissue is opened longitudinally and the resulting rectangular strip of circular smooth muscle of 6 to 8 mm long and 2 to 3 mm wide is mounted in an isolated organ bath of 10 ml containing Krebs-Henseleit solution bubbled with a mixture of 95 % oxygen and 5 % carbon dioxide. The strip is tied to a force displacement transducer to record isometric contractions on a polygraph. The resting tension is kept at 1 g. The experiment is started following an equilibration period of 60 to 90 minutes. The drugs are tested for their potential relaxing effect in this preparation. Contractions are induced by potassium chloride (150 mM). Indomethacin (10 µM) induces sustained rhythmic contractions which may be abolished by prostaglandin E2.

To set up gall bladder preparation, the bladder is removed by cutting the cystic duct

and extrahepatic biliary tree.15 Longitudinal strips, 5 to 6 mm long and 2 to 3 mm wide, are cut out and are mounted in an isolated organ bath to record the tone and motility as described above. Drugs may be tested for relaxing effect in this preparation against contractions induced by high concentration of potassium chloride in the medium (80 and 100 mM). The method is a modification of an earlier method.16 A method for cat gall bladder strip preparation has also been described.17 6.2.8 Test for Histamine H2 Receptor Antagonist on Mucosal Adenylate Cyclase

Endogenous histamine released from histaminocytes in gastric mucosa is one of the

three major physiological stimulator of gastric acid secretion (the other two are acetylcholine and gastrin). It activates the H2 receptors on parietal cell membrane. Histamine H2 receptor antagonists have a potent inhibitory action on gastric acid secretion. Currently these are the most efficacious antiulcer agents together with proton pump inhibitors.

The drugs may be evaluated for their antagonist potency on the basis of their

inhibitory action on adenylate cyclase in membrane vesicles isolated from guinea pig gastric mucosa.18

Guinea pigs (400 to 600 g) are sacrificed by a blow on the head. The fundic portion

of the stomach is removed, opened flat and rinsed with ice-cold 50 mM Tris buffer at pH 7.4 with 4mM EDTA and 0.25 M sucrose. It is then stretched on a petri dish filled with ice-cold buffer to strip off the mucosa from the underlying muscle layer. The stripped material is suspended in 10 ml ice-cold buffer and homogenized first in an Ultra-turrax homogenizer and then in a Potter-Elvehjem teflon-glass homogenizer. The resulting suspension is centrifuged at 700 G to eliminate the cell debris and nuclear fraction. The supernatant is removed and spun at higher speed to obtain membrane fraction. The pellet is resuspended in the buffer to give a protein concentration of 2.4 mg/ml as measured by the method of Lowry et al.19

The activity of adenylate cyclase in homogenate incubated in the buffer at 30°C in

the presence of ATP for 10 minutes is measured while stimulating with histamine at increasing concentrations from 0.1 to 100 µM. The amount of cyclic AMP produced is measured by a method using ³H-cyclic AMP in a competitive protein binding assay described by Brown et al.20 The inhibitory action of drugs is evaluated by comparing the amount of cyclic AMP formed in their presence and absence. The IC50 values are estimated from the response-concentration curves.


58

6.2.9 Rat Uterus Preparation

Isolated rat uterus preparation may be used as a simple test to screen drugs for histamine H2 receptor antagonist activity. Drugs which are found to have an activity in this preparation may be further tested in the assay described in the preceding method.

The original method was described by Eltze.21 Young female rats in oestrous weighing 120 to 150 g are used. Oestrous may be induced by injecting 24 hours before a subcutaneous dose of stilbestrol (0.1 mg/kg of 20 µg/ml solution in ethanol).12 Animals are sacrificed by a blow on the head and the abdomen is opened to remove two horns of the uterus. Each horn is freed from the surrounding tissue and is cut open longitudinally. Pieces of 2 to 3 cm long are mounted in an isolated organ bath (5 to 8 cm long of 10 ml capacity) containing de Jalon’s solution at 30 to 32°C bubbled with a mixture of 95 % oxygen and 5 % carbon dioxide. This solution contains less calcium than the other physiological mediums to suppress the spontaneous rhythmic activity. The uterus strip is tied to a force displacement transducer to record isometric contractions in a polygraph. A resting tension of 0.5 to 1 g is applied and the experiment starts after an equilibration period of 15 to 30 minutes. Histamine applied at an interval of 15 to 20 minutes causes H2 mediated concentration-dependent transient contractions reaching peak in about 30 seconds. The H2 receptor antagonists selectively block this response.

A version using superfusion technique is also available.18 Spontaneously beating

guinea pig a trial preparation may also be used to test drugs for histamine H2 receptor activity, but the responses may be complicated by co-localized H1 receptors.

6.2.10 Test for Proton Pump Inhibition

Protons produced by carbonic anhydrase reaction inside parietal cells are secreted

as hydrochloric acid by cell membrane H+/K+-ATPase called proton pump which exchanges K+ from outside with H+. The proton pump inhibitors are more potent inhibitors of gastric acid secretion than histamine H2-receptor antagonists. They are used for the treatment of gastroduodenal ulcers, gastroesophageal reflux disease and Zollinger-Ellison syndrome.

Currently used pump inhibitors are prodrugs converted to active sulphenic acid and

sulphenamide derivatives in the highly acid medium of parietal cell canaliculi. A method was described by Ljungstrom et al.22 to test drugs for proton pump

inhibiting activity in H+/K+-ATPase-containing membrane vesicle suspensions prepared from the gastric mucosa scrapped from pig stomachs obtained from a slaughter house.

Ten g portions of the scrapped material are rinsed thoroughly with cold saturated

sodium chloride solution and homogenized in 0.25M sucrose solution with seven strokes in a Potter-Elvehjem teflon-glass homogenizer. The homogenized portions are combined and centrifuged at 20,000 G for 40 minutes. The pellet is discarded and the supernatant is centrifuged at 75,000 G for 1 hour. The pellet is resuspended in 30 ml of 0.25M sucrose solution. Aliquots of 15 ml are centrifuged against gradient by a complex process.18 Membrane vesicles from one of the fractions are collected and frozen at -70°C under nitrogen for later use. During the test, ATPase activity is measured at 37°C on the basis of inorganic phosphate released from ATP. The test drug and omeprazole (standard) are pre-incubated in concentrations from 0.01 to 100.0 µM in ATPase-containing vesicle suspension in parallel at pH 6.0 and 7.4 for 30 minutes. The reaction is started by addition of nigericin and stopped after 4 minutes by addition of 50 % trichloroacetic acid. The precipitated protein is centrifuged and inorganic phosphate content of the supernatant is determined.


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6.3 Methods to Evaluate Antiulcer Drugs

Peptic ulcer affects gastric mucosa and first few centimetres of duodenum. The size varies from several millimetres to several centimetres. It penetrates at least the muscularis mucosal layer. Several factors such as hypersecretion of hydrochloric acid, disruption of mucus layer, which protects underlying epithelial cells from acid insult, reduced generation of local healing factors such as growth factors and prostaglandins, reduced blood flow and colonization of Helicobacter pylori within mucosa. The major symptom of the disease is epigastric pain, which may be relieved by appropriate food or antacids. The major complications are haemorrhage, perforation and penetration of the surrounding tissues.23 Although the hypersecretion of acid may not be a primary factor, the mainstream treatment is aimed at reducing the generation or activity of the acid by pump inhibitors, histamine H2 receptor blockers or antacid compounds and eradicating H. pylori with antibiotics. 6.3.1 In Vivo Evaluation in Gastric Ulcers

Several methods are described to induce gastric ulcerations in rats. In all of them

the rats weighing 150 to 200 g are starved up to 48 hours depending on the method, before the insult leading to ulcer development in gastric mucous membrane is applied. Access to water is allowed ad libitum. Since the starved animals tend to coprophagy (eat stools) and cannibalism, they should be housed singly in perforated cages to let fecal beads out of the cage. Each group comprises eight to ten animals. After the animals are exposed to ulcerogenic insult for specified periods of time, they are sacrificed by carbon dioxide anaesthesia or by inhalation of an overdose of ether. A long incision is made along midline and the abdomen is opened. A ligature is placed at the gastroesophageal junction just under the diaphragm and then the stomach is removed. It is opened along the greater curvature and stretched and pinned on a cork or foam plate with opened surface up after gently washing with warm water at a temperature not exceeding 37°C. The sites of ulceration and other damaged sites on the mucosa are examined by overhead magnifier (3 x magnification) or under stereomicroscope.

The rat stomach has three different anatomical parts. The upper part close to the

oesophageal junction is called fundus which is a grey colour and separated by a ridge from the remaining part of a pink colour. The fundus and the antrum, the lowest part close to the pylorus, are the most vulnerable areas to ulcer development. The medium part making up about 40 % of the stomach is relatively resistant to ulceration because of a well developed protective mechanism. Therefore sites of ulceration occur mostly in the fundal and the antral part of the gastric mucosa.

The test agents are usually administered either by oral route through gastric canula

(gavage) or subcutaneously within 10 to 30 minutes prior to administration of ulcerogenic insult. Control animals receive only the vehicle.

The protective effect of the tested drugs is usually evaluated by comparing percent

group mean intensity of ulceration and other lesions in the control and test groups. A dose response curve may be plotted according to percent response by administering a series of increasing doses of protective or inhibitory agents. ID50 (dose of protective agent providing 50% inhibition) may be calculated from this curve. The following specific methods are used to evaluate protective activity in experimentally induced gastric ulceration.

6.3.2 Method Using Pylorus Ligation Ulcers

The original method was described by Shay et al.24 In addition to ulcer development

it measures the acidity of gastric content.


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Rats are starved for 48 hours before the experiment. The abdomen is opened by a midline incision and the pylorus is ligated. Care should be taken not to damage the pylorus and stomach. Any damage to them such as squeezing with forceps causes ulcer development in the underlying mucosa. The abdomen is closed by suturing and the animal is placed in a plexiglass tube of 45 mm diameter with both ends closed by a wire mesh. The animal is allowed to survive for the next 19 hours and then sacrificed under anaesthesia. The abdomen is opened to remove the stomach and its contents are completely drained into a centrifuge tube to measure the volume and acidity. After opening the stomach along the greater curvature and fixing it on a plate its mucosa is examined preferably under a stereomicroscope.

The magnitude of ulceration is calculated on the basis of number and severity of

ulcers. The following score method is employed to assess the severity of ulcers.18 No ulceration, 0; superficial ulcers, 1; deep ulcers, 2; and perforation, 3.

An overall ulcer index (Ui) is calculated from the following equation:

Ui = Un + Us + Up x 10-1

Un = average number of ulcers in individual animals; Us = average of severity scores; Up = percent of animals with ulcers. Volume of gastric content is measured and after centrifugation the acidity is

measured by titration against 0.1N sodium hydroxide solution.

6.3.3 Method Using Immobilization Induced Stress Ulcers

Immobilization for a long time is a stress factor. This method may be regarded as a model for stress ulcers caused by various stressors. The original method was described by Hanson and Brodie.25

Rats are starved for 24 hours and completely immobilized by gently tying both upper

and two lower limbs together. They are placed in a plexiglass immobilization tube or wrapped in a wire mesh cage and are kept in the dark in a horizontal position for 24 hours. Thereafter, they are sacrificed and their stomachs are removed and opened to measure the number and intensity of ulcers using a magnifier or preferably a strereomicroscope. Ulcer index is calculated as described above.

6.3.4 Method Using Cold Exposure Induced Stress Ulcers

The original method was described by Takagi et al.26 and was applied by West27 to

test the antiulcer agents. The method combines cold stress with immobilization stress which are synergistic.

In the original method rats were placed singly in the restraint cage immersed vertically up to the tip of the xifoid bone in water at 22°C for 1 hour. The test drugs are given 10 to 30 minutes before the exposure to cold. In a modified version of the method, the rats are placed in a cold room at 4°C. They are removed from the cold environment and dried if immersed in water. In order to make ulceration sites more visible animals are injected with Evans blue, 30 mg/kg, i.v. 10 minutes prior to sacrifice. The stomach is removed and ligated at both the pylorus and gastroesophageal junction after being filled with tissue fixing solution (2 % v/v formol in saline). It is kept overnight and opened the next day to examine the ulceration.

The length in millimetres of each ulcer at its longest diameter is measured and

summed up to obtain total lesion score for individual rats. The effect of the protective drug is assessed on the basis of the average score for each group expressed as a percent of the average in the respective control group.


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In experiments carried out with these two stress methods, not only the acid neutralizing and acid inhibiting agents, but also neuroleptic drugs reducing psychogenic stress were found to inhibit ulcer formation by stressors. Interestingly the cold stress ulcer is relatively resistant to the healing action of histamine H2-receptor blockers in comparison with ulcers induced by other methods.27 6.3.5 Method Using Indomethacin or Like Substances Induced Ulcers

Indomethacin is a potent non-steroidal anti-inflammatory drug (NSAID). It is one of

the most noxious drugs to the gastrointestinal system even when used at therapeutic dose level. It acts by inhibiting cyclooxygenase-1 (COX-1) enzyme in gastric mucosa, which is involved in the production of protective prostacyclin. At high single doses it produces gastric ulcer rapidly within hours after its intragastric or parenteral administration. The original method was described by Djahanguiri.28

Indomethacin is not sufficiently soluble in water and its 4 mg/ml concentration is

prepared using 0.1 % Tween 80 solution before the experiment. It is administered by a gastric canula at a dose of 20 mg/kg to fasted rats which are killed 6 hours later under anaesthesia. The drug is administered orally or parenterally 10 minutes prior to indomethacin. The stomach is removed; both orifices are tied up and filled with diluted formol solution as described above to fix the tissue. The next day the stomach is opened along the greater curvature, rinsed and the mucosa is examined under a magnifier.

The size of each lesioned area is measured as the length in millimetres at its

longest diameter. The lengths of the individual lesions on the mucosal surface of the stomach are summed up to obtain an individual total ulcer score. A group mean is calculated and inhibitory response is expressed in the protective drug-treated group as a percent of the score in the respective control group. The mean lesion count for control groups is found to be consistently around 30 (range 25 to 32).27

Several modifications to this method were done by changing the ulcerogenic

substance. Acetylsalicylic acid27, 29 may also be used to induce ulcers. It is administered as a solution, 80 mg/ml in 0.1 % v/v Tween 80, via gastric canula.27 The test drug is administered intragastrically or parenterally 10 minutes prior to the challenge.

6.3.6 Method Using Ethanol Induced Mucosal Lesions

Intragastric administration of ethanol damages cells by precipitating cell proteins. It

mostly induces haemorrhagic lesions in gastric mucosa. The action is proportional to the concentration of alcohol in the solution applied. Absolute alcohol (at least 99.4 % v/v or 99 % w/w of ethanol) may be used to produce gastric lesions and cytoprotective agents may be tested against this insult.

One millilitre of absolute alcohol is administered by a gastric canula into the

stomach of 24 hour fasted rats. The drug to be tested should be given 30 minutes before by the same route or parenterally. One hour after the administration of absolute alcohol the animals are sacrificed under anesthesia. The stomach is removed, opened along the greater curvature, rinsed and the lesions are graded from score 0 to 3 by an approach essentially similar to that described under pylorus ligation.

This method may be combined with the ligation of the pylorus.30 The pylorus is

ligated under ether anaesthesia. The drug to be tested is given orally 2 minutes later and 1 ml of absolute alcohol is given 10 minutes after the oral administration of test drug. Animals are sacrificed one hour after alcohol administration.


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6.3.7 In Vitro Evaluation of Antacids

Antacid drugs such as aluminium hydroxide, magnesium oxide and carbonate, calcium carbonate and sodium bicarbonate are used for the treatment of gastroduodenal ulcers, gastroesophageal reflux disease and heartburn because of their capacity to chemically neutralize hydrochloric acid secreted by parietal cells of gastric mucosal glands. Their use has reduced after the introduction of more potent drugs inhibiting acid formation and secretion such as proton pump inhibitors and histamine H2 receptor antagonists. Secondary actions of antacid drugs such as adsorption of pepsine and bile acids and possibly increased formation of prostaglandins and nitric oxide in gastric mucosa are also involved in their anti-ulcer activity.31

The chemical reaction between antacids and hydrochloric acid present in gastric

fluid within the stomach also occurs in the test tube to a similar extent. Therefore, the neutralizing capacity may be measured in a test tube by titration.

An in vitro method was described by Fordtran et al.32 together with an in vivo

method. The tablets of the antacids are crushed and mixed with distilled water under constant stirring in a test tube or a glass beaker. The antacid mixture is slowly titrated against 0.1N hydrochloric acid. The standard acid neutralizing capacity of the tablet is expressed as the volume of 0.1N hydrochloric acid required to maintain the solution at pH 3. Aluminium compounds are weaker and less rapidly reacting antacids than magnesium and calcium compounds and sodium bicarbonate. Therefore they require a longer titration time. Antacids are shown to protect the gastric mucosa against damage when tested in stress-, NSAID- and ethanol-induced ulcer models.18

6.3.8 In Vivo Evaluation in Duodenal Ulcers

Various methods have been described to produce experimental duodenal ulcers by

giving irritant substances by different routes. In the model described originally by Dzan et al.33 cysteamine hydrochloride is given

to rats weighing 200 g three times at 280 mg/kg dose by oral route on day 1. Propionitrile may be used instead of cysteamine. The drugs to be tested for their potential protective actions are given 30 minutes before cysteamine administration. Rats are sacrificed on day 3 and stomach and duodenum are removed and fixed in 10 % buffered formol solution overnight. The tissue is embedded into paraffin and sections are stained by haematoxylin-eosin. Ulcers develop mostly in the mesenteric and antimesenteric part of the duodenum in an area up to 4 mm from the pylorus.

The severity of the ulcers was rated by scores from 0 to 3. Score 0 indicates no

ulcer; 1, superficial mucosal erosion; 2, deep ulcer usually with transmural necrosis; 3, perforated or penetrated (into pancreas) ulcer. In a modified method mepirizole is used as insulting agent in rats.34 This substance was given subcutaneously at a dose of 200 mg/kg and the drugs with a potential protective action 30 minutes in advance via oral route. Control rats are given mepirizole and the vehicle and test group receives mepirizole and the test drug. In order to make the mucosal lesions more visible, 1 ml of 0.5 % Evans blue solution is injected intravenously 24 hours later and the rats are sacrificed under carbon dioxide asphyxiation in 10 minutes. Stomach and duodenum are removed for the evaluation of the lesions by the method of scoring.

6.4 Methods for Screening Antidiarrhoeal Activity

Diarrhoeal diseases are observed frequently in developing countries because of the

deficiency of sanitary measures. They are mostly caused by bacterial, viral and parasitic


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infections and treated with specific antimicrobial drugs and rehydration solutions. Diarrhoea is usually caused by inhibition of the absorption of water and electrolytes and/or increased secretion of them in the intestine (secretory diarrhoeas) leading to a significant loss of water and electrolytes, and consequently dehydration. Increased motility and resulting accelerated gastrointestinal transit are also involved in the pathogenesis of diarrhoea. The antidiarrhoeal drugs are used for the symptomatic treatment of diarrhoeal diseases in appropriate cases. They have an antisecretory action and may increase water and electrolyte absorption mainly by prolonging the intestinal transit time. Several models have been developed to test the drugs for potential antidiarrhoeal action. Most models use 8 to 10 animals in each group. Some of the models are described below. 6.4.1 Castor Oil Induced Diarrhoeal Model

Castor oil, which is an oil extracted from Ricinus communis seeds has a powerful

purgative action. When given orally it is hydrolyzed in the gut to ricinoleic acid which has an irritant action on gut mucosa. It also increases water and electrolyte secretion from intestinal glands and stimulates intestinal motility. Consequently, it produces secretory diarrhoea combined with hypermotility.

The method was described by Niemegeers et al.35 Rats weighing 180 to 200 g are

starved for 24 hours before the experiment with free access to water. They are placed singly in individual cages with no access to water. The test substance is given orally 1 hour prior to the castor oil and the control rats receive only the vehicle. One hour later 1 ml of castor oil is administered orally to each rat. The rats are observed for the next 8 hours at hourly intervals for the presence or absence of diarrhoea. Dose response curves are plotted each hour on the basis of the percentage of rats with diarrhoea in each test group. The time to the first diarrhoeal bout may also be recorded.

6.4.2 Enteropooling Assay in Rats

The rats in groups of 10 to 12 are fasted overnight and administered a diarrhoeal

agent, magnesium sulphate (2 ml of 10 % solution per rat) via a gastric canula.36 A 2 % solution of gum acacia is used as a vehicle to dispense the dose of test substance and control. Animals are sacrificed 30 minutes later. The drug to be tested for potential antidiarrhoeal action is administered orally (intragastrically) one hour before the magnesium sulfate. The small intestine is removed completely after ligating it at the pyloric and distal (ileocaecal) end and is then weighed. The weight of the intestine is standardized as weight per 100 g of body weight. The means ± SEM of the groups treated with the various doses of the test drug are compared on the basis of the differences from the mean of the control group by using Student’s t test.

6.4.3 Diarrhoea Induced by Surgical Removal of Caecum in Rats

Caecum is a significantly voluminous part of the intestine in rats. It is a relatively high capacity reservoir for the watery intestinal content coming from the last part of small intestine where the content is further digested by bacterial flora and its water and electrolytes are absorbed. For this reason removal of caecum (caecectomy) causes an increased sensitivity to the diarrhoeal agents such as prostaglandins, cholera toxin and carbachol producing a secretory type of diarrhoea. The potential antidiarrhoeal action may be tested in secretory diarrhoea induced by these agents. The original method was described by Fondacaro et al.37 Non-starved rats are anaesthetized with 60 mg/kg methohexital injected intraperitoneally for caecectomy. Thiopental may also be used instead of methohexital.

A midline incision of 2 to 3 cm long is made in the middle part of the abdominal

wall. Caecum is taken out on a sterile gauze drape. It is dissected out from the mesocaecum


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and a silk suture is placed around its junction with the intestine. Extreme care is taken to secure ileocolonic patency. Then the suture is tied off and the caecum is resected. After the remnant caecum tissue is cleaned with saline and cauterized, the intestine is replaced into the abdominal cavity. Abdominal wall facia is closed with silk sutures and the skin incision with metal wound clips. Animals are allowed a recovery period of at least 48 hours before the experiments.

The animals are placed singly in individual cages with a wire mesh bottom to let out

the faecal beads down onto the clean sheets of paper put under the cage. The animals are deprived of food and water for the duration of the experiment. In order to avoid anxiety-induced defaecation, a 2 hour period of familiarization with the new environment is allowed during which the animal is observed for the pattern of stool. Rats producing only pelleted stools are included in the experiments and those with loose stool or spontaneous diarrhoea are excluded. Diarrhoea is induced by oral administration of the secretagogue agents such as 0.3 mg/kg of 16,16-dimethylprostaglandin E2 in 3.5 % ethanol solution or 0.5 mg/kg of cholera toxin in a solution of 2% each of sodium bicarbonate and casamino acids or 15 mg/kg of carbachol in aqueous solution.

The test drug is given orally as soon as the diarrhoea starts. The paper placed under

the cages are removed and examined at intervals of 15 minutes for carbachol-induced diarrhoea, 30 minutes for prostaglandin-induced diarrhoea and 1 hour for cholera toxin-induced diarrhoea. The number of defecation episodes during each interval is recorded and the consistency of stools is scored as follows: 1, normal pelleted stools; 2, soft-formed stools; 3, watery stools and/or diarrhoea.

Faecal output index is calculated for each interval by summing up the number of

defecation episodes rated according to their consistency scores. A mean index ± SEM is calculated for each group.

6.4.4 Diarrhoea Induced by Cold Restrain in Rats

Stress caused by restraining rats in a cold environment increases the faecal output.

This kind of diarrhoea resembles the one occurring in humans, especially patients with irritable colon, under stressful conditions.

The original method was described by Barone et al.38 Rats of 250 to 300 g

maintained on a standard laboratory diet are put into wire mesh restraining tubes allowing the stool to fall down. The control animals are placed in regular living cages. Food is not withdrawn before experiments. The test drug is administered orally. The cages are placed in a cold room at 4°C. The number of pellets discharged by each rat is measured at 1 and 3 hours. The faecal pellets are modified usually in their water content rather than completely loosing their consistency. Therefore, the pellets are weighed as such and then after drying them in an oven at 37°C. The difference is considered as fluid content. The response is evaluated on the basis of total pellet output and total fluid content for each group of rats and is expressed as a percent of the control group. The ID50 (dose inhibiting the maximal response by 50 %) is estimated from the dose-response curve of each drug.

Several modifications of this method have been published.18

6.5 Method of Evaluation of Effect on Propulsive Intestinal Motility

Laxative drugs accelerate and constipating drugs slow down the propulsive

intestinal motility. Increased or decreased intestinal motility is partially responsible for laxative and constipating actions respectively.


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One of the commonly used assays to test drugs for these actions is charcoal meal test in which transit time related to strength of the propulsive intestinal motility is evaluated. The original method was described by Macht and Barba-Gose39 and later modified by Janssen and Jageneau40 and Sanvordeker and Dajani.41

Groups of 6 to 10 mice weighing 20 to 25 g are fasted with free access to water for

18 to 24 hours and pretreated with the test drug intragastrically or subcutaneously. Thirty minutes later the animals are administered intragastrically a single dose of charcoal (0.2 ml of 10 % charcoal suspension in 1 % methylcellulose per mouse). Three and a half hours after the administration of charcoal, the mice are sacrificed and the caecum is examined for the presence or absence of charcoal on an all or none basis. Dose response curves are plotted for test compounds and their ED50 values are estimated.

In another version of the method, groups of ten mice are used for each dose of test

drug and one group serves as a control. Animals are sacrificed at 20, 40, 60 and 120 minutes after charcoal administration. The whole intestine is immediately removed and fixed in a 5 % formaldehyde solution. The distance from the pylorus travelled by the charcoal is measured and expressed as a percent of the distance between the pylorus and caecum. The difference from the control expresses the action of drug.

The test is used for the evaluation of laxative or antispasmodic actions and also for

inhibitory action on intestinal motility or constipating action. In the modified versions, rats are used with charcoal meals or charcoal is replaced

by other stained substances such as carmine red suspended in 1 % tragacanth suspension or by radioactive chromium solution.

6.6 Anti-inflammatory Evaluation 6.6.1 Anti-inflammatory Evaluation in Ileitis

Several models are developed which differ from one another by the inflammation-

inducing agent applied to the ileum. These models may be used to predict the therapeutic potential of drugs in the treatment of Crohn’s disease (granulomatous ileitis or ileocolitis, terminal ileitis). This is a disease associated with a non-specific chronic transmural inflammation that most commonly affects the distal ileum and colon but may occur in any part of the gastrointestinal tract.23 A genetic predisposition resulting in an unregulated intestinal immune response to environmental, dietary and infectious factors is involved in its pathogenesis. When localized to colon it imitates the clinical features of ulcerative colitis. Experimental ileitis may also be used to test drugs with therapeutic potential for treatment of ulcerative colitis.

One of these models was described by Sjogren et al.42 In this model, ricin, a

cytotoxic lectin isolated from castor beans (seeds of Ricinus communis), is applied into a transiently in situ isolated segment of albino rabbit ileum. In this experiment rabbits are anaesthetized by intramuscular injection of a mixture of ketamine (50 mg/kg) and xylazine (9 mg/kg), a veterinary sedative analgesic muscle relaxant drug, and anaesthesia is maintained with intravenous injection of pentobarbital (15 mg/kg). The abdomen is opened by a midline incision and a short segment (about 10 cm long) of the terminal part of ileum is ligated at both ends without compromising the blood circulation. Ricin, 1 mg in 1ml vehicle, is injected into the lumen. Control animals receive only the vehicle. Test substance is given before or at the beginning of the ricin injection by an appropriate route or intraluminally. After 6 hours the segment is cut out and the animal is sacrificed by an overdose of pentobarbital. A local acute ileitis develops and is manifested by mucosal damage, polymorphonuclear leucocyte infiltration, increased leukotriene and prostaglandin E2 generation and an abnormal


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myoelectric spike activity. An increased response to electrical field stimulation and also to substance P and other stimulant tachykinins is observed in the in vitro preparation of the ileal segment exposed to ricin. Increased myoelectric activity is suppressed in part by the leukotriene antagonist. A similar phenomenon is produced in this experiment by the intraileal administration of hapten, 2,4,6-trinitrobenzene sulfonic acid (TNBC), producing cell-mediated immune response, instead of ricin.

The isolated ileal segment is opened along the longitudinal axis and lumen is

flushed gently with cold oxygenated incubation solution. Muscle strips of about 1 x 0.4 cm with mucosa scrapped away are cut out along the circular muscle axis and mounted into an isolated organ bath of 10 ml containing oxygenated Krebs-bicarbonate-saline solution at 37°C. Strips from the ileum segments exposed only to vehicle solution are used as controls.

Strips are connected to a force displacement transducer to record their contractions

isometrically on a polygraph paper. Strips are allowed to equilibrate with the incubation solution under zero tension for 20 minutes. Then a resting tension allowing maximal response to acetylcholine administration at 0.5 to 1 µM concentration is applied and maintained. Strips are allowed to equilibrate under this condition for another 20 minutes.

Strips are passed through a pair of ring electrodes mounted in the isolation organ

bath for electrical field stimulation experiments. In the bathing solution, atropine (1 mM) and N-nitro-L-arginine methyl ester (L-NAME) are added to abolish interfering cholinergic and nitrergic responses respectively to electrical stimulation. The stimulus parameters are of 0.5 millisecond duration, at frequency levels ranging from 1 to 10 Hz at supramaximal voltage for 10 seconds. The contractile responses to electrical responses are expressed as a percent of the maximal response to acetylcholine (1 µM) to reduce the variation of individual responses. Responses to increasing concentrations of tachykinins are expressed as absolute tension development measured on polygraph paper calibrated at the beginning of the experiment. Drugs with potential anti-inflammatory action reduce the responses including the maximal response to electrical field stimulation and tachykinins.

The average responses ± SEM at each concentration (tachykinin experiments) and

frequency level are plotted to obtain log concentration-response and frequency response curves.

The differences in curves with and without (control) the tested drug are evaluated

statistically by multivariate analysis of variance. When a significant difference is found between relevant curves, a final comparison is made on the basis of the maximal responses using Student’s t test.18

In the modified versions of the method iodoacetamide and trinitrobenzene sulfonic

acid with or without absolute alcohol are applied in the ileal lumen.18 Subcutaneous injection of two doses of 7.5 mg/kg of indomethacin at an interval of 24 hours is reported to produce ileitis in rats.43

The ileal inflammation produced by these agents is an acute reaction. Therefore the

strength of data obtained with tested drugs in this model may be weak in predicting the potential effects in chronic ileal inflammatory diseases. 6.6.2 Anti-inflammatory Evaluation in Colitis

Ulcerative and granulomatous colitis are inflammatory diseases of the colon with pathogenesis similar to that of Crohn’s disease. Several experimental colitis models are described to test the drugs for their therapeutic potential for the treatment of inflammatory colon diseases.


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In order to produce a chronic colitis in experimental animals, the haptens like TNBS, producing cell-mediated immune response or irritating substances such as acetic acid are applied chronically to the colon either indirectly by a catheter placed into the distal end of the ileum or directly by intracolonic instillation.

Several methods in rats, mice, guinea pigs, rabbits and monkeys were described

using different haptens or irritants. One of these methods described by Vogel is explained below.18

The method involves an initial active immunization to produce a specific systemic

hypersensitivity followed by local ileal and colonic inflammation produced and maintained by chronic local administration of the immunogenic agent.

Rats weighing 150 to 200 g are initially sensitized by intradermal injection of TNBS

in 0.05 ml Freund incomplete adjuvant containing 0.1 % ovalbumin for 3 days. Eighteen days later, a booster injection is given and followed 14 days later by an intradermal dose of 0.08 % TNBS in saline with or without ovalbumin.

The application of local challenge to colon starts 10 days after the intradermal

challenge. For this purpose a polyethylene indwelling catheter of about 0.5 mm diameter is implanted in the lumen of the most distal portion of the ileum at a point 15 cm proximal to the caecum under anaesthesia with 100 mg/kg intraperitoneal ketamine. The other end of the catheter is tunnelled under the skin and exteriolized on the back of the rat. After a recovery period of ten days a series of daily local administration of 0.2 mg TNBS per rat as 0.08 % solution in saline is started and is continued for 3 weeks.

Rats reserved as control group are also implanted with catheter and receive saline

instead of the test drugs. Drugs to be tested are administered either locally or via catheter once a day in saline solution or by systemic routes. A suspension of drug in carboxymethyl cellulose is used for oral (intragastric) administration.

At the end of experiment the animal is sacrificed and the most distal 10 cm

segment of ileum where the catheter is placed is removed. The segment is cut open longitudinally and rinsed thoroughly. The mucosa is examined with a magnifier for lesions which are rated by the following scores.18 No visible damage at all, 0; slight inflammation, slight hyperemia, villi visible under 15-fold magnification, 1; intermediate inflammation, discontinuous hyperemic areas, intermediate redness of villi, 2; intensive inflammation, intensive hyperemia, intensive redness of villi, 3.

The effect is evaluated on the basis of differences in the mean score ± SEM of the

treated and control group. The significance of differences is evaluated by Wilcoxon-Mann-Withney U-test for unpaired data at the level of significance of 0.05.

The intensity of inflammation may also be evaluated on the basis of leukotriene C4

and prostaglandin E2 formation in incubated mucosa homogenates. The formation of these prostanoids is increased significantly.42

This method and its modification involving chronic experimental enteritis mostly

induced by immunogens have a better predictive strength to evaluate the potential anti-inflammatory action of test drugs in chronic ileitis and colitis than the method employing acute inflammation.


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6.7 Methods for Evaluation of Emetic and Antiemetic Action

Vomiting is the spontaneous discharge of the contents of the stomach and upper portion of small intestine upon reflex contraction of diaphragm and abdominal wall muscles. It is triggered by stimuli of various origins, such as the gastrointestinal tract, brain, vestibular organ in the inner ear and psychogenic stimuli. It is usually preceded by nausea. Spoiled food constituents or contaminants and chemicals, including drugs, induce nausea and vomiting (together called emesis) either by irritating the gastrointestinal tract or by stimulating a receptor site in the brainstem called chemoreceptor trigger zone (CTZ). Some endogenous substances formed under physiological or pathological conditions are also involved in induction of emesis. Emesis is a sign of several pathological conditions.

Antiemetic drugs are used to prevent or treat emesis. Several emesis models are

developed to evaluate the antiemetic action. The emetic action is important as a potential side effect. An emesis comparable to that occurring in man does not occur in rats, mice, guinea pigs or rabbits. Dogs, and less frequently ferrets and pigeon,s are used in emesis models. The house musk shrew (Suncus murinus) exposed to a linear shaking motion is used to test the drugs with a potential therapeutic efficacy against motion sickness.

A dog model of emesis which may be used to test both emetic and antiemetic drugs

involves injection of apomorphine, a potent emetic drug.18 In order to determine the threshold emetic dose, mongrel dogs weighing 15 to 20 kg are administered by intramuscular injection into the gluteal muscle. The starting dose is 22 mg/kg, which is increased or decreased and given after a 5-day interval. A standard food of 200 g is given prior to each injection of apomorphine to facilitate emesis. The steps between successive doses are kept small in order to find a threshold emetic dose as precise as possible. Injection site is alternated to avoid tissue damage. Pavlovian conditioning may occur after successive doses of emetic drugs which interfere with drug effect. An equivalent volume of non-drug vehicle is injected after every third or fourth dose to ascertain if this has occurred. Once the threshold dose is established, it usually remains relatively stable for the group of dogs.

The antiemetic drug to be tested is administered via an appropriate route at

different dose levels at a suitable time interval prior to the injection of the apomorphine. The new threshold dose of apomorphine is determined with each dose of the test drug. The initial dose of a new antiemetic drug is selected as a fraction of its acute LD50 dose (in mg/kg) in mice. A relative potency of the antiemetic drug may be calculated in comparison with chlorpromazine.

A drug with a potential emetic effect or side effect may be tested on the basis of the

threshold effect as described for apomorphine. Its potency relative to apomorphine may be calculated.

Cisplatin, an anticancer drug with a potent emetic side effect can be used in place

of apomorphine.44 However this drug is carcinogenic and should be handled with extreme care. This modified model is preferred to screen the drugs with antiemetic potential in anticancer drugs producing emesis such as serotonin 5-HT3 receptor antagonists.

Cisplatin-induced emesis in ferrets is also used to test drugs with a potential 5-HT3

antagonist activity.45

6.8 Conclusions

The first part of this chapter presents the bioassays employing gastrointestinal tissues which may be carried out in the usual laboratory conditions with relatively simple and inexpensive recording instruments. The straw levers and smoked paper kymographs were


69

used for recording in most of these bioassays until 45 years ago when the force displacement transducers and electronic recording instruments such as polygraphs were made available as recording systems. In the absence of electronic equipment the older non-electronic devices can still be used.

In the second part the in vivo methods for potential therapeutic action on

gastrointestinal disorders are addressed by giving emphasis to the most common diseases of this system such as peptic ulcers, diarrhoea and chronic enteritis. The tests for the drugs with a potential choleretic and cholagogue actions are left out because of the controversial clinical efficacy of these drugs. The assay systems such as Heidenhain pouch, a chronic gastric pouch in dogs, and Thiry-Vella fistula based on jejunum prepared in dogs and other animals are not addressed because there are simpler and more convenient substitutes for them.

References 1 Magnus, F., 2002, Versuche am uberlebenden Dünndarm von Saugetieren, Pflugers Archiv fur die Gesamte

Physiologie des Menschen und der Tiere, 102, 1904

2 Van Rossum, J. M., 1963, Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters, Archives of International Pharmacodynamics and Therapeutics, 143, 299-330

3 van den Brink, F. G. and Lien, E. J., 1977, pD2-, pA2- and pD2'-values of a series of compounds in a histaminic and a cholinergic system, European Journal of Pharmacology, 44, 251-270

4 Ustunes, L., 1993, Izole kobay ileum preparati, in Izole Organ Preparatlari 1. Duz Kas Preparatlari, Paper presented at the Turkish Pharmacological Association Training Symposia, Series No.2, Ankara, Turkey

5 Paton, W. D., 1957, The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guinea-pig ileum, British Journal of Pharmacology, 12, 119-127

6 Paton, V. D. M., 1955, The response of the guinea-pig ileum to electrical stimulation by co-axial electrodes, Journal of Physiology, 127, 40

7 Paton, W. D. and Vizi, E. S., 1969, The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip, British Journal of Pharmacology, 35, 10-28

8 Ambache, N., 1954, Separation of the longitudinal muscle of the rabbit's ileum as a broad sheet, Journal of Physiology, 125, 53-55P

9 Trendelenburg, P., 1917, Physiologische Versuche über die Dünndarmperistaltik, Archives of Experimental Pathology and Pharmacology, 81, 55-129

10 Bülbring, E., Crema, A. and Saxby, O. B., 1958, A method for recording peristalsis in isolated intestine, British Journal of Pharmacology, 13, 440- 443

11 Finkelman, B., 1930, The nature of inhibition in the intestine, Journal of Physiology, (London), 70, 145-157

12 Staff of Department of Pharmacology, University of Edinburgh, 1973, Pharmacological Experiments on Isolated Preparations (2nd ed.), E. & S. Livingstone, Edinburgh

13 Vane, J. R., 1957, A sensitive method for the assay of 5-hydroxytryptamine, British Journal of Pharmacology and Chemotherapy, 12, 344-349

14 Gocer, F., Yaris, E. and M., T., 1994, The action of amyl nitrite and isosorbide dinitrate on the contractility of sphincter of Oddi of guinea-pigs, General Pharmacology, 25, 995-999

15 Yasar, U., Erdem, S. R. and Tuncer, M., 1999, Cyclosporine a preparations and their vehicles induce contraction of the guinea pig gallbladder in vitro: the role of cyclooxygenase metabolites, Pharmacology, 58, 309-318


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16 Chowdhury, J. R., Berkowitz, J. M., Praissman, M. and Fara, J. W., 1975, Interaction between octapeptide-cholecystokinin, gastrin, and secretin on cat gallbladder in vitro, American Journal of Physiology, 229, 1311-1315

17 Fara, J. W. and Erde, S. M., 1978, Comparison of in vivo and in vitro responses to sulfated and non-sulfated ceruletide, European Journal of Pharmacology, 47, 359-363

18 Vogel, H. G. (Ed.). 2002, Drug Discovery and Evaluation. Pharmacological Assays (2nd ed.), Springer, Berlin

19 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., 1951, Protein measurement with Folin phenol reagent, Journal of Biological Chemistry, 13, 265-275

20 Brown, B. L., Albano, J. D., Ekins, R. P. and Sgherzi, A. M., 1971, A simple and sensitive saturation assay method for the measurement of adenosine 3':5'-cyclic monophosphate, Biochemical Journal, 121, 561-562

21 Eltze, M., 1979, Proestrus and metestrus rat uterus, a rapid and simple in vitro method for detecting histamine H2-receptor antagonism, Arzneimittel Forschung, 29, 1107-1112

22 Ljungstrom, M., Norberg, L., Olaisson, H., Wernstedt, C., Vega, F. V., Arvidson, G. and Mardh, S., 1984, Characterization of proton-transporting membranes from resting pig gastric mucosa, International Journal of Biochemistry, Biophysics and Molecular Biology, 769, 209-219

23 Beers, M. and Berkow, R. (Eds.), 1999, The Merck Manual (70th ed.), Merck Research Labs, Whitehouse Station, New Jersey

24 Shay, H., Komarov, S. A., Fels, S. S., Meranze, D., Gruenstein, H. and Siplet, H., 1945, A simple method for the uniform production of gastric ulceration in the rat, Gastroenterology, 5, 43-61

25 Hanson, H. M. and Brodie, D. A., 1960, Use of the restrained rat technique for study of the antiulcer effect of drugs, Journal of Applied Physiology, 15, 291-294

26 Takagi, K., Kasuya, Y. and Watanabe, K., 1964, Studies on the Drugs for Peptic Ulcer. A Reliable Method for Producing Stress Ulcer in Rats, Chemical and Pharmaceutical Bulletin, 12, 465-472

27 West, G. B., 1982, Testing for drugs inhibiting the formation of gastric ulcers, Journal of Pharmacological Methods, 8, 33-37

28 Djahanguiri, B., 1969, The production of acute gastric ulceration by indomethacin in the rat, Scandinavian Journal of Gastroenterology, 4, 265-267

29 Konturek, S. J., Piastucki, I., Brzozowski, T., Radecki, T., Dembinska-Kiec, A., Zmuda, A. and Gryglewski, R., 1981, Role of prostaglandins in the formation of aspirin-induced gastric ulcers, Gastroenterology, 80, 4-9

30 Robert, A., Nezamis, J. E., Lancaster, C. and Hanchar, A. J., 1979, Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury, Gastroenterology, 77, 433-443

31 Hoogerwerf, W. A. and Pasricha, P. J., 2001, Agents used for control of gastric acidity and treatment of peptic ulcers and gastroesophageal reflux disease, In: Hardman, J. G., Limbird, L. E. and Goodman, A. G. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, pp. 1005-1020

32 Fordtran, J. S., Morawski, S. G. and Richardson, C. T., 1973, In vivo and in vitro evaluation of liquid antacids, New England Journal of Medicine, 288, 923-928

33 Dzan, V. J., Haith Jr, L. R., Szabo, S. and Reynolds, E. S., 1975, Effect of metiamide on the development of duodenal ulcers produced by cysteamine or propionitrile in rats, Clinical Research, 23, 576A

34 Okabe, S., Ishihara, Y., Inoo, H. and Tanaka, H., 1982, Mepirizole-induced duodenal ulcers in rats and their pathogenesis, Digestive Diseases and Sciences, 27, 242-249

35 Niemegeers, C. J., Lenaerts, F. M. and Janssen, P. A., 1972, Difenoxine (R 15403), the active metabolite of diphenoxylate (R 1132). 2. Difneozine, a potent, orally active and safe antidiarrheal agent in rats, Arzneimittel Forschung, 22, 516-518

36 Pillai, N. R., 1992, Anti-diarrheal activity of Punica granatum in experimental animals, International Journal of Pharmacognosy, 30, 201-204

37 Fondacaro, J. D., Kolpak, D. C., Burnham, D. B. and McCafferty, G. P., 1990, Cecectomized rat. A model of experimental secretory diarrhea in conscious animals, Journal of Pharmacological Methods, 24, 59-71


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38 Barone, F. C., Deegan, J. F., Price, W. J., Fowler, P. J., Fondacaro, J. D. and Ormsbee, H. S., 1990, Cold-restraint stress increases rat fecal pellet output and colonic transit, American Journal of Physiology, 258, G329-337

39 Macht, D. I. and Barba-Gose, J., 1931, Two methods for pharmacological comparison on insoluble purgatives, Journal of the American Pharmaceutical Association, 20, 558-564

40 Janssen, P. A. and Jageneau, A. H., 1957, A new series of potent analgesics: dextro 2:2-diphenyl-3-methyl-4-morpholino-butyrylpyrrolidine and related amides. I. Chemical structure and pharmacological activity, Journal of Pharmacy and Pharmacology, 9, 381-400

41 Sanvordeker, D. R. and Dajani, E. Z., 1975, In vitro absorption of diphenoxylate hydrochloride on activated charcoal and its relationship to pharmacological effects of drug in vivo, Journal of Pharmaceutical Sciences, 64, 1877-1879

42 Sjogren, R. W., Colleton, C. and Shea-Donohue, T., 1994, Intestinal myoelectric response in two different models of acute enteric inflammation, American Journal of Physiology, 267, G329-337

43 Sukumar, P., Loo, A., Magur, E., Nandi, J., Oler, A. and Levine, R. A., 1997, Dietary supplementation of nucleotides and arginine promotes healing of small bowel ulcers in experimental ulcerative ileitis, Digestive Diseases and Sciences, 42, 1530-1536

44 Gylys, J. A., Doran, K. M. and Buyniski, J. P., 1979, Antagonism of cisplatin induced emesis in the dog, Research Communications in Chemical Pathology and Pharmacology, 23, 61-68

45 Florczyk, A. P., Schurig, J. E. and Bradner, W. T., 1982, Cisplatin-induced emesis in the Ferret: a new animal model, Cancer Treatment Reports, 66, 187-189


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7 Bioassay Models for Evaluation of Antihepatotoxic Activity

Maninder Karan, Jaswant Singh and Karan Vasisht 7.1 Introduction

The liver is the largest and most complex organ in the human body and has a unique and remarkable power to regenerate. It carries out a wide range of exocrine and endocrine functions such as maintenance of carbohydrate homeostasis, storage of nutrients, secretory and excretory functions, protein synthesis and a couple of vital metabolic processes including hormone, lipid and protein metabolism and detoxification of xenobiotics mediated by cytochrome P450-dependent oxidases and conjugates.1 The wide range of its functions makes the liver vulnerable to a variety of disorders which can be broadly classified into acute or chronic hepatitis (inflammatory disease), hepatosis (non-inflammatory disease) and liver cirrhosis.2 Although viruses are the leading cause of liver diseases, liver lesions arising from therapeutic agents, uncontrolled environmental pollutants and excessive drug therapy - exposing humans to a vast array of chemicals and pharmaceuticals - are not uncommon.3 Viral hepatitis has become an alarming public health hazard in both developing and developed nations. The vaccines available are used for preventive measures. A cure for viral hepatitis is not available.

The drugs available in the modern system of medicine are corticosteroids and

immunosuppressive agents which bring about symptomatic relief without cure and their use is associated with the risk of relapses and dangerous side effects. Whereas, a large number of plant derived products which either support or promote the process of healing or regulation of the liver hepatocytes, have been used in various traditional systems of medicine for centuries in the treatment of liver diseases,. Pharmacological and clinical studies have shown them to be beneficial in combating liver toxicity. The most successful example is silymarin, a mixture of flavanolignans derived from milk thistle Silybum marianum (fam. Asteraceae), effective in the treatment of liver damage. During early investigations of silymarin it was shown to be a potent and promising agent against the deadly liver toxins, phalloidin and α-amanitin present in the fungus Amanita phalloides.4 Treatment with silymarin, or its principal component silibinin, shows satisfactory clinical outcome if the treatment is started within 48 hours of ingestion of the fungus.

Other plants with potential hepatoprotective activity are Andrographis

paniculata,5,5a Glycyrrhiza glabra,6 Picrorhiza kurrooa,7 Phyllanthus amarus,8 Schizandra chinensis,6 Swertia chirata9, 10 and Uncaria gambier.6

Lack of therapeutic agents in modern medicine and the use of a vast array of plants

in traditional practices warrant investigation of these plants using modern techniques to validate hepatoprotective claims and to search for novel molecules for use against liver toxicity. A review of the literature shows that plants will prove to be the most fertile ground to look for useful liver drugs10a.

BIOASSAY MODELS FOR EVALUATION OF ANTIHEPATOTOXIC ACTIVITY

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7.2 Bioassay Screening Techniques

Diseases associated with the liver are so complex that finding a system for testing plants for the treatment of such diseases is not simple. Testing is carried out by first producing liver damage in experimental animals followed by estimation of recovery after treatment. Damage and restoration are measured by liver function tests (usually liver specific enzymes) or following an indirect route to assess liver activity (quantifying response of drugs metabolized by the liver e.g. hypnosis following barbiturate administration) or histopathological studies to directly and conclusively know the rate and extent of regeneration of hepatocytes.

In vivo testing of samples involves inducing liver damage in experimental animals to

mimic natural disease state. Animals are then treated or pretreated with plant extracts or compounds and the activity is judged from the results of liver function tests and histopathological studies.

In vitro testing employs determining drug metabolizing enzymes in cultured

hepatocytes. For screening large number of samples, the in vitro technique using cultured hepatocytes is the method of choice. The hepatocytes act as representatives of an intact liver capable of metabolically activating the drugs. The method is more convenient, less expensive and gives more reproducible results. In this technique the hepatocytes are isolated using collagenase and cell viability is determined. The cells are cultured for a specific period of time before adding hepatotoxin and the test sample. They are further incubated together for a suitable period and the activity is determined from the results of biochemical estimations. 7.3 In Vivo Bioassay Methods

The in vivo models can be broadly grouped into two categories. The first group is

represented by a 'barbiturate-induced sleep model'. This model is based on the fact that certain drugs are metabolized by the enzymes present in the liver. The level of these enzymes diminishes following liver toxicity or damage, which results in prolongation of drug action. The difference in drug action is related to level of liver toxicity and can be quantified. For example, the barbiturate-induced hypnosis is extended in liver damage and length of extension of hypnosis quantifies the extent of liver damage in live animals. Liver toxicity is induced by using one of the several known liver toxins. The extent of normalization of hypnosis following administration of a test substance is the measure of its activity.

The second group has several models named after the hepatotoxin used in the

investigations. A number of liver toxins are known which produce assessable liver damage. All models share the common principle of inducing liver toxicity using one of the several toxins and treating the animals with the test substance. The biochemical estimations are carried out at the end of the study. The results of the control group (receiving only the vehicles used for dispensing toxin and test substance), toxin group (receiving toxin and vehicle of test substance) and test group (receiving toxin and test substance) are compared to evaluate the activity of the test substance.

The substance being tested is considered to be effective if it blocks or helps to

recuperate the toxic effect of the hepatotoxin. The magnitude of the toxic effect can be measured by estimating certain suitable liver-function parameters and through direct histological examination of liver tissues. For convenience, a few specific markers of liver injury can be monitored to know the extent and recovery of liver toxicity in experimental animals.

Several toxins are used to induce liver toxicity in animals. Different models using

these toxins in a predetermined schedule with test drugs have been developed and established. A brief account of common toxins, their effects and description of important biochemical parameters used in evaluating liver state is given below.


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7.3.1 Liver Toxins 7.3.1.1 Carbon Tetrachloride (CCl4)

It is the most investigated of all the hepatotoxins and frequently used in screening tests.11 It is low cost and small volumes are required to induce liver damage. It causes acute liver cell damage in a dose dependent manner. The liver toxicity is mediated through enzymatic activation of CCl4 by cytochrome P450 to free radical •CCl3 (trichloromethyl radical) within membranes of endoplasmic reticulum. The free radicals induce microsomal lipid peroxidation, disrupt the structure and functions of lipid and protein macromolecules in the membranes of cell organelles and inhibit the calcium pump of microsomes leading to centrilobular liver cell necrosis. The hepatocytes thus show fatty change and/or are irreversibly injured. 7.3.1.2 Paracetamol (Pcml)

Paracetamol also causes centrilobular liver damage in a dose dependent way. It is one of the most frequently consumed drugs and responsible for many cases of poisoning.12 In larger doses paracetamol induces liver injury both in human and experimental animals. The hepatotoxicity is mediated through a reactive toxic metabolite, N-acetyl-p-benzoquinone imine generated by cytochrome P450. This electrophilic toxic metabolite covalently binds to thiol groups of cysteine residues in proteins and other macromolecules with consequent stimulation of lipid peroxidation and depletion of the cellular reservoirs of glutathione leading to hepatocellular toxicity. 7.3.1.3 D-Galactosamine (GalN)

Administration of galactosamine produces dose dependent peripheral liver cell necrosis in rats which closely resembles acute viral hepatitis in humans from a morphological and functional viewpoint.13 The response to galactosamine administration is a multi-step process which starts with the accumulation of high concentration of its metabolites followed by trapping of uracil nucleotides leading to decreased synthesis of RNA, glycoproteins and glycolipids, followed by cell death. 7.3.1.4 Ethanol

Chronic use of alcohol even in small doses acts as an intoxicating agent for hepatic tissue and produces known biochemical and chemical alterations of liver membrane.14 The deleterious effect of ethanol is attributed to the accumulation of acetaldehyde in the liver during catabolic conversion of ethanol to acetic acid. Acetaldehyde is a toxic intermediate of bioconversion of alcohol and is responsible for alcohol toxicity. 7.3.1.5 Thioacetamide

It acts both as a hepatocarcinogen and a hepatotoxicant. An acute administration produces centrilobular hepatic necrosis while chronic administration causes liver cirrhosis and nodal tumors.7 The hepatotoxicity is mediated by the formation of thioacetamide-S-oxide which covalently binds to liver cell macromolecules such as proteins, nucleic acids and lipids. Inhibition of the calcium pump of microsomes leads to liver cell necrosis. 7.3.2 Biochemical Parameters for Evaluating Hepatic Functions

A number of enzymes are biosynthesized and stored in liver cells and their level increases in the serum following liver cell damage except choline esterases which diminish following impaired protein synthesis or reduced liver cell mass or both. The increased serum


76

enzyme level is a reflection of their release from the damaged cells or enhanced production by abnormal cells and seepage to blood. The location of the enzyme within the cell, on the cell membrane, in cytoplasm or cell organelle is an important consideration. In reversible inflammatory processes, which increase membrane permeability, blood levels are more likely to be raised for enzymes located on the cell membrane and cytosol than those present in mitochondria.The following enzymes or biochemical markers are specifically important in liver damage. 7.3.2.1 Transaminases (AST/ALT or GOT/GPT)

Aspartate transaminase (AST) [glutamate oxaloacetate transaminase (GOT)] and alanine transaminase (ALT) [glutamate pyruvate transaminase (GPT)] are the most widely employed enzymes in monitoring liver diseases. These are also present in heart, skeletal muscles and kidneys. Both are cytosolic enzymes, and GOT is also present in mitochondria. Although the absolute amount of GPT is less than GOT a greater proportion is present in liver which makes GPT more liver-specific. GOT/GPT ratio is useful in differential diagnosis of liver diseases.

7.3.2.2 Alkaline Phosphatase (ALP)

It is a membrane enzyme and is present in a number of tissues and organs. Three forms of ALP are recognized: intestinal, placental and bone/liver/kidney. ALP shows marked elevation, following biliary obstruction. The level of ALP rises considerably in cholestasis and to a lesser extent in hepatocytic damage. 7.3.2.3 Gamma-Glutamyl Transpeptidase (γ-GT)

It is widely distributed in different tissues and is present in the outer surface of plasma membrane. It is most abundant in cells with secretory and absorption functions which make it highly liver specific for studying liver disorders. 7.3.2.4 Sorbitol Dehydrogenase (SDH)

It is a cytoplasmic and mitochondrial enzyme mainly present in liver and catalyses conversion of glucose to sorbitol. The importance of the estimation of SDH is based on the fact that it is virtually absent in the serum of healthy subjects and is detectable during infections, toxic or hypoxic liver damage. Thus, SDH activity in the serum is an organ specific indicator of liver cell damage. 7.3.2.5 Glutamate Dehydrogenase (GIDH)

It is a mitochondrial enzyme with greatest activity in the liver. It is also present in the kidney and brain. Only trace amounts occur in the serum of healthy subjects and a rise of GIDH levels signifies hepatic cellular necrosis due to acute viral hepatitis or hepatotoxicity. 7.3.2.6 Bilirubin (BRN)

It is the end product of degradation of haem, carried out extra-hepatically in the reticulo-endothelial system. In blood, the bile is bound to albumin and is cleared from the blood only by the liver. High bilirubin content in serum results following impairment in its hepatic uptake, glucuronidation and excretion in bile. A typical pattern of bilirubin content therefore, reflects the pathophysiology of the liver.

7.3.2.7 Hepatic Triglycerides (HTG)

Triglycerides act as a store of energy and also as a means for transport of energy


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from the gut and liver to peripheral tissues. Increased HTG level in live tissue is indicative of disturbance in lipid metabolism arising out of liver injury or obstructive jaundice. 7.3.2.8 Hepatic Glycogen (HGN)

Glycogen is exclusively present in the liver and maintains blood glucose levels through reversible interconversion. In damaged liver cells, the metabolism is disturbed which results in a decrease in glycogen level. 7.3.3 Test Protocols 7.3.3.1 Animals

Barbiturate-induced sleep model uses mice of either sex in a weight range of 20 to 25 g. Other models use rats of 150 to 175 g of either sex. Each group consists of six or more animals. The control group is given only vehicle(s). The toxin group receives vehicle and hepatotoxin. The test group is administered hepatotoxin plus plant extract or a test compound. The standard reference group (optional) is given any known liver protectant such as silymarin plus hepatotoxin. Animals are allowed free access to standard rat diet and water.

7.3.3.2 Equipment

Spectrophotometer, centrifuge, tissue homogenizer, serological water bath, wax

oven, microtome, microscope and weighing balance are some of the specific instruments required in the bioassay.

7.3.3.3 Chemicals, Solvents and Reagents

Chemicals, solvents and reagents of a high level of purity or analytical grade are used. Their requirement depends on the liver parameters to be included in the experimentation. The following list is provided. Hepatotoxins: the commonly used hepatotoxins are carbon tetrachloride diluted 1:1 in olive oil (1.4 ml/kg, i.p. equivalent to 0.7 ml/kg of CCl4), paracetamol (2 g/kg, p.o.) in normal saline or 50 % sucrose solution, and galactosamine (800 mg/kg, i.p.) in normal saline.

Pentobarbitone sodium: dose of 35 mg/kg, i.p. is used in barbiturate-induced sleep model.

SDH: β-NADH disodium salt, tris HCl buffer, β-D-(-)-fructose.

GlDH: ADP, EDTA, LDH, β-NADH disodium salt, triethanolamine, ammonium acetate, 2-oxoglutarate.

GOT (AST): DL-aspartic acid, α-ketoglutaric acid, phosphate buffer, 2,4dinitrophenylhydrazine (DNPH), sodium pyruvate, sodium hydroxide.

GPT (ALT): same as GOT except aspartic acid, which is replaced with alanine.

ALP: disodium phenyl phosphate, sodium carbonate-sodium bicarbonate buffer, sodium hydroxide, sodium bicarbonate, 4-aminoantipyrine, potassium ferricyanide and phenol.

HTG: sodium chloride, triolein, isopropyl alcohol, heptane, sulphuric acid, potassium hydroxide, sodium metaperiodate and acetylacetone.

HGN: potassium hydroxide, alcohol, sulphuric acid, phenol red indicator, sodium hydroxide and standard glucose test kit.

Histopathological studies: formaldehyde, sodium chloride, alcohol, paraffin, alum-haematoxylin, eosin, ammonia, xylene and D.P.X.


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Test solutions: plant extract(s) are dissolved or suspended in 50 % sucrose solution or 2 % gum acacia for oral administration or in sterile normal saline (0.9 % sodium chloride) for intraperitoneal administration.

7.3.3.4 Procedure

The methodology of the three most commonly used in vivo models i.e. carbon

tetrachloride, paracetamol and galactosamine besides barbiturate-induced sleep model is described below. 7.3.3.4.1 Carbon Tetrachloride Model

The animals of the control and toxin groups are given the vehicle on four consecutive days while animals of the test group receive test substance on the same four days. The animals of control group are also given olive oil (vehicle) while those of the toxin and test groups on day 2 and 3 receive carbon tetrachloride diluted 1:1 in olive oil (1.4 ml/kg, i.p. equivalent to 0.7 ml/kg of CCl4). On day 5, the animals’ blood is taken for biochemical analysis. The schedule for the carbon tetrachloride model is given in Table 1.

Table 1: Schedule of treatment for carbon tetrachloride model

Group Days

1 2 3 4 5

Control V V, OO V, OO V CCl4 V V, CCl4 V, CCl4 V

Test TS TS, CCl4 TS, CCl4 TS

Animals sacrificed for biochemical and histopathological examination

V = vehicle; OO = olive oil; TS = test substance; CCl4 = carbon tetrachloride in olive oil (1:1)

7.3.3.4.2 D-Galactosamine Model

The animals of the control and toxin groups are given vehicle and the material under test is given to the test group of animals 48 hours, 24 hours and 2 hours prior to the administration of D-galactosamine (800 mg/kg, i.p.) in normal saline. Concurrent with the administration of D-galactosamine to the animals of the test and toxin groups, the animals in the control group receive normal saline. The biochemical and histopathological studies are done 48 hours after administering the toxin. The schedule for D-galactosamine model is given in Table 2.

Table 2: Schedule of treatment for GalN/PcmL model

Group -48 h -24 h -2 h 0 h +48 h

Control V V V NS/V GalN/PcmL V V V Gal/PcmL Test TS TS TS Gal/PcmL

Animals sacrificed for biochemical and histopathological examination

V = vehicle; NS = normal saline; TS = test substance. The '-' sign indicates that the treatment was given prior to the administration of the challenger while '+' sign indicates the time after the challenger

7.3.3.4.3 Paracetamol Model

The schedule of treatment given to various groups of animals under this model is the same as D-galactosamine model (Table 2) except for the substitution of D-galactosamine with paracetamol (2g/kg, p.o.).


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7.3.3.5 Estimation of Biochemical Parameters

The animals are anaesthetized with urethane (1.2 g/kg, i.p. of 25 % w/v aqueous solution). The blood is collected from the carotid artery and is allowed to coagulate at 37°C for 30 minutes. The serum is separated by centrifugation at 2,500 rpm. The liver is removed and processed separately for hepatic triglycerides, hepatic glycogen and histopathological examination. 7.3.3.5.1 Sorbitol Dehydrogenase (SDH)

SDH is estimated by Rose and Henderson procedure.15 Add 200 µl of serum to 1.4

m1 of tris HCl buffer (100 mM/l, pH 6.6, 37°C) containing 355 µM/l β-NADH disodium salt. Mix well and incubate exactly for 5 minutes at 37°C. Add 0.4 ml of β-D-(-)-fructose substrate (2.5 M/l dissolved in 100 mM/l tris HCl, pH 6.6, 37°C) and note the absorbance change at 340 nm over a period of 3 minutes at 30 second intervals and calculate absorbance change per minute.

Calculation of enzyme activity: One unit of SDH activity is equal to the reduction of one µMof fructose/l/min. This is stoichiometrically related to the oxidation of NADH in the system as follows:

total volume 103 U/l = -------------------------- x ------ x ∆A/min

sample volume 6.22 7.3.3.5.2 Glutamate Dehydrogenase (GIDH)

GlDH is determined by the method of Schmidt and Schmidt.16 Add 500 µl of serum to 2.5 ml of the reaction mixture containing 25 parts of TEA buffer (TEA, 64.5 mM/l; EDTA, 3.22 mM/l; CH3COONH4, 129 mM/l; pH 7.8 at 30°C) and 1 part of NADH solution (NADH, 6.45 mM/l; ADP, 32.3 mM/l; LDH 65 kU/l). Mix thoroughly and incubate for 5 minutes at 30°C. Record absorbance change exactly for next 5 minutes at 1 minute intervals (∆A1/∆t) for non-specific interference. Immediately, thereafter, add 0.1 ml of 2-oxoglutarate solution (217 mM/l, pH 6.8-7.0), mix thoroughly and again note absorbance change for next 5 minutes at 1 minute intervals (∆A2/∆t).

Calculation of enzyme activity: The mean value of (∆A1/∆t) is subtracted from the mean value of (∆A2/∆t) and the difference (∆A/∆t) is used for calculation of GlDH activity. The catalytic activity in the sample is: U/l = (∆A/∆t) x (V/v) [158.73].

7.3.3.5.3 Glutamic Oxaloacetate Transaminase (GOT)

GOT (AST) is determined by the method of Reitman and Franke.17 Take 0.5 ml of the substrate (200 mM DL-aspartic acid, 2 mM α-ketoglutaric acid in phosphate buffer, pH 7.4, 37°C), add 100 µl of serum and incubate the test mixture exactly for 30 minutes in a water bath at 37°C. After incubation, remove the mixture from the water bath and add 0.5 ml of 1 mM 2,4-dinitrophenylhydrazine (DNPH). The blank samples are prepared and treated exactly as the test samples except using water in place of serum. In the control samples add 0.1 ml serum to a mixture of 0.5 ml substrate and 0.5 ml of DNPH. The standard samples, in triplicate, are prepared by mixing 0.1 ml of the standard working pyruvate (4 mM sodium pyruvate), 0.4 ml of substrate, 0.1 ml of water and 0.5 ml of DNPH. After allowing the DNPH to react in all the tubes for 20 minutes at room temperature, add 5 ml of 0.4 N sodium hydroxide and leave for another 10 minutes. Compare the colour in all tubes at 510 nm against water. The activity of GOT is calculated and expressed in Karmen units (KU).


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Calculation of enzyme activity: The pyruvate formed per minute per litre of serum is: (T-C/S-B) x 0.4 x (1/30) x (1000/0.1) = (T-C/S-B) x 67 µM. 7.3.3.5.4 Glutamic Pyruvic Transaminase (GPT)

The method for the determination of GPT (ALT) is same as for GOT except for the use of GPT substrate (200 mM alanine, 2 mM α-ketoglutaric acid in phosphate buffer, pH 7.4) in place of GOT substrate and decreasing the incubation time to 15 minutes. The activity of GPT is expressed as Karmen units and enzymatic activity is calculated as described for GOT. 7.3.3.5.5 Alkaline Phosphatase (ALP)

ALP is estimated by the method of Kind and King.18 Add 2.0 ml of the buffer substrate, (1:1 mixture of 10 mM disodium phenyl phosphate and 100 mM sodium carbonate - sodium bicarbonate buffer, pH 10) to each of the two sets of test tubes labelled as test and control, and warm in a water bath at 37°C for a few minutes. Add 30 µl of the serum to one set of the tubes (test) and incubate exactly for 10 minutes at 37°C. Remove both sets of tubes from the water bath and add 0.8 ml of 0.5 N sodium hydroxide and 1.2 ml of 0.5 M sodium bicarbonate to all the tubes followed by 30 µl of serum to the second set of tubes (control). Add 1 ml of 0.6 % w/v solution of 4-aminoantipyrine and 1 ml of 2.4 % w/v solution of potassium ferricyanide to all tubes of both the sets and mix well. The standard sample is prepared by mixing 1.1 ml of the buffer-substrate with 1 ml of the working phenol standard solution (0.001 % w/v of phenol), and the blank sample is prepared by mixing 1.1 ml of the buffer substrate with 1 ml of water. Add sodium hydroxide, sodium bicarbonate, 4-aminoantipyrine and potassium ferricyanide to the standard and the blank samples in the sequence and volumes as mentioned above.

The colour in all the tubes is compared at 520 nm against air. Activity of ALP is calculated and expressed in King-Armstrong Units per 100 ml. Calculation of enzyme activity: ALP (in King-Armstrong Units) per 100 ml = (T-C/S-C) x 0.01 x (100/0.1)

7.3.3.5.6 Hepatic Triglycerides (HTG)

Homogenize 0.5 to 1.0 g of accurately weighed fresh liver with 5 ml of normal saline and estimate the triglycerides in the homogenate by the method of Gottfried and Rosenberg.19 Add liver homogenate (0.5 ml), standard triolein solution (0.5 ml containing 2 mg triolein/ml of isopropyl alcohol) and water (0.5 ml) respectively to three stoppered test tubes labelled as test, standard and blank. Add 0.5 ml water and 3.0 ml isopropyl alcohol to the standard tube and only 3.5 ml of isopropyl alcohol to each of the test and blank tubes. To each tube add 2.0 ml of heptane and 1.0 ml of conc sulphuric acid and vortex the mixture for 30 seconds. Allow the two phases to separate and transfer 0.4 ml of the heptane (upper) layer from each tube into a stoppered test tube. Add further 2 ml of isopropyl alcohol and one drop of 6.25 M potassium hydroxide to each test tube and incubate for 10 minutes at 70°C. After incubation add 0.2 ml of sodium metaperiodate reagent and 1 ml of the acetylacetone reagent to each of the tubes and again incubate at 70°C for 10 minutes. The colour in the test and standard is compared at 425 nm against the blank (note the absorbance while the mixture is warm). Calculate the triglyceride content in the liver and express as mg/g (wet weight) of the liver.

7.3.3.5.7 Hepatic Glycogen (HGN)

Glycogen is estimated in the fresh liver by the method of Hawk et al..20 Mince 0.5 to 1.0 g of accurately weighed fresh liver with 6 ml of 30 % w/v potassium hydroxide solution.


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Digest the tissue by heating in boiling water bath for 15 to 20 minutes, agitating the solution occasionally to ensure thorough disintegration. Add 6 ml of 95 % alcohol to each tube and immerse in the water bath until boiling just begins. Cool the tubes at room temperature for 2 hours. Separate the precipitated glycogen by centrifugation and wash the precipitate with 5 ml of 60 % alcohol followed again by centrifugation and decantation. Remove the last traces of alcohol by immersing the tubes in boiling water for just long enough to dry the precipitate. Dissolve the precipitate in 5 ml of water with gentle warming. Transfer 1 ml of this solution to a calibrated test tube and hydrolyse by heating in a boiling water bath for 2 hours with 1 ml of 2 N sulphuric acid. Add one drop of phenol red indicator to each of the tubes and neutralize the excess acid cautiously using 1 N sodium hydroxide with constant stirring. Dilute the contents of the tube with 10 ml water and estimate the glucose formed by the hydrolysis of glycogen using the standard glucose test kit. Calculate the amount of glycogen in the liver and express in mg/g (wet weight) of the liver. 7.3.3.6 Histopathological Examination of the Liver

Cut and fix the liver pieces of about 2 to 4 mm thickness of the experimental animals in 10 % formal saline (10 % v/v formaldehyde in normal saline) for 24 hours and process for paraffin embedding following the standard microtechniques.21 Cut 5 µ thick section of the paraffin-embedded liver using a microtome. Alum-haematoxylin and eosin may be used for staining the liver sections in the following order: deparaffinized sections (slides) are downgraded through various grades of alcohol to water. Stain the sections in alum-haematoxylin for 30 minutes, rinse in water and differentiate in acid water until the nuclei turn pink. At this stage, the slides are dipped in the ammonia water to stain the nuclei blue. The slides are then upgraded until 90 % alcohol and counterstain with eosin (2 to 4 minutes), differentiate in 90 % alcohol, dehydrate in absolute alcohol and clear in xylene before mounting in D.P.X. The stained sections are examined under the light microscope and photographs of the sections may be taken using suitable macro-lens for histopathological examination. Representative sections of normal, toxic and treated rat liver are shown in Figure 1. 7.3.3.7 Barbiturate-induced Sleep Model

A suitable protocol is similar to that of carbon tetrachloride model. The treatment for

the first four days is the same as given in Table 1. However, on the fifth day the mice are treated with 35 mg/kg, i.p., of pentobarbitone sodium (8.75 mg/ml solution in normal saline) or hexobarbital or thiopental and the sleep time i.e. the duration between loss of righting reflex and regaining it, of the individual mouse is recorded. 7.3.3.8 Estimation of Liver Protective Activity

The results of various biochemical estimations are used to assess the activity of the test substances. The substance is considered active if it blocks the toxic effect of a toxin and normalizes the enzyme levels towards control values. The percent protection can be calculated from the following expression:

(Toxin treated) – (Toxin + Test substance) Percent protection = ------------------------------------------------------------------------ (Toxin treated) – (Control)


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Figure 1: Liver sections of rat. a, normal liver; b, CCl4 damaged liver; c, paracetamol damaged liver; d, galactosamine damaged liver; e, treated liver showing partial recovery from galactosamine toxicity; f, treated liver showing nearly complete recovery from CCl4 toxicity. 7.3.3.9 Statistical Evaluation

Results of the biological experiments are expressed as mean ± S.D. (standard deviation). In order to know the overall response of experimental animals to a particular type of treatment, the variations in the levels of various biochemical parameters estimated in the animals are represented by a single value indicating the proportion of individual abnormal results obtained during their determination.22 This proportion, expressed as a percentage, is calculated as the ratio of total number of abnormal individual values for the biochemical parameters to the total number of estimations carried out (number of animals in a group x number of biochemical parameters estimated). The value is considered abnormal if it falls outside the limits formed by the mean value for the control group of animals ± 3 S.D. (mean ±


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3 S.D.) as this range covers up to 99.7 % of the values of the individuals in the control group provided. Of course, the individual values are normally distributed across the mean value. Thus, percentage proportion of abnormal value:

Total no. of abnormal values in a group PAV percent = ----------------------------------------------------------------------------------------------------------------------- x 100 No. of animals in the group x no. of biochemical parameters estimated

A 100 % proportion of abnormal value indicates that there is no overall inhibition of

the toxin-induced increase in the levels of the biochemical parameters and its low values, reflects higher inhibitory activity of the test substance with respect to the biochemical parameters that are estimated to assess the antihepatotoxic activity.

Estimate total variation present in a set of data by performing one-way analysis of

variance (ANOVA). The F ratio obtained by ANOVA should be tabulated along with the critical value of F ratio reported in literature.22, 23 A value of F ratio greater than the critical value indicates that significant variation exists among the groups under analysis. In order to check the pairs of means which are different, the data for every biochemical parameter in each set of experiment is analyzed by Least Significant Difference (LSD) procedure24 at 95 % (p = 0.05) confidence level. Significant difference between means of various groups in a particular biochemical parameter should be tabulated appropriately. 7.4 In Vitro Bioassay Method Using Rat Hepatocyte Primary Monolayer

Cell Culture

The model permits the demonstration of both anti-lipoperoxidant and antihepatotoxic activities of plant products. Primary cultures of rat hepatocytes express differentiated functions of liver and are metabolically competent in terms of expression of various drug-metabolizing enzymes over a longer period of time. The expression of liver specific cytochrome P450 is essential for activation of certain chemical hepatotoxicants to reactive metabolites which consequently produce liver injury. The hepatocytes are isolated from rat liver, grown in monolayer cultures and treated with carbon tetrachloride (or paracetamol) in the presence and absence of test substance. The biological end points of toxicity of CCl4 viz. leakage of lactic acid dehydrogenase (LDH) and glutamate-pyruvate transaminase (GPT), and reduced glutathione (GSH) contents in hepatocytes are measured. The magnitude of reduction in enzyme leakage and restoration of GSH contents are taken as the index of antihepatotoxic activity of the test substance.

Primary culture can also be used as a simple and sensitive method to check the

hepatotoxicity of the plant extracts/isolates.25 7.4.1 Biochemical Parameters for Evaluating Hepatic Functions in Cell Culture 7.4.1.1 Lactate Dehydrogenase (LDH)

Leakage of LDH along with ALT is a common manifestation of cell injury. Hepatic injury due to drugs or chemicals is quite prominent which is reflected in leakage of LDH with the progress of cell toxicity.

The enzyme catalyzes the oxidation of L-lactate to pyruvate with the mediation of

NAD as a hydrogen acceptor. LDH is primarily used for diagnosing and evaluating the tissue damage. It is present in many body tissues such as heart, liver, kidney, skeletal muscle, brain, blood cells and lungs. Low levels of the enzyme are normally present in the blood stream but on cell injury the enzyme leaks out of the damaged cells and its level in the blood rises. The reaction is reversible and strongly favours the conversion of pyruvate to lactate. The rate of


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increase in absorbance of the reaction mixture due to the formation of NADH is proportional to the LDH concentration present in the biological sample.

Since the assay involves spectrophotometric measurement of NADH oxidation

mediated by LDH, the enzyme activity can be measured with great sensitivity from minor to major injury to cells. This is a commonly employed method of assessing cell toxicity, which indicates damage to plasma membrane.

7.4.1.2 Glutathione (GSH)

Glutathione offers protection by scavenging reactive oxygen species and electrophiles formed within the cells. This process is mediated by a family of glutathione-S-transferases which utilize GSH as cofactor. It is present intracellularly in millimolar concentrations, and is a key compound in maintenance of reduction potential in cells. Within the cells, GSH is maintained in a redox couple with its oxidized form GSSG and is regenerated by GSH-reductase, a cytosolic NADPH- dependent enzyme. Acute cellular toxicity by chemicals or drugs is commonly associated with depletion of GSH. Pretreatment of cells to deplete intracellular GSH enhances the toxicity of drugs. Thus depletion of GSH contents is an important indicator of predisposition leading to cellular injury. Loss of mitochondrial functions as a result of GSH depletion may affect the energy status of the cells and therefore is a critical event in the induction of cell injury. 7.4.2 Requirements

7.4.2.1 Animals

Adult male rat (200 g), fasted overnight is anaesthetized with sterile sodium

pentobarbitone, 0.4 ml (60 mg/ml water) i.p. 7.4.2.2 Equipment and Apparatus

Sterile silicon tubing, sterile media filtration assembly, peristaltic pump, perfusion equipment, water bath, bubble trap, intravenous plastic canulae gauze, glass pipettes, automatic pipettes, pipette aid, surgical instruments, sterile nylon cloth (mesh width, 0.3 mm), tissue culture dishes, sterilized centrifuge cups, sterile air flow hood, U.V. and gamma rays sources, analytical weighing balance, refrigerated low speed centrifuge, refrigerator, liquid nitrogen, deep freezer, spectrofluorometer, UV/visible spectrophotometer and glass wares. 7.4.2.3 Chemicals, Solvents, Reagents and Test Solutions

All media after sterile filtration should be stored in a refrigerator. The preparation of various media and solutions is as follows:

Ca2+ free perfusion medium A - dissolve 7.88 g NaCl (final concentration 135 mM), 1.26 g NaHCO3 (15 mM), 1.0 g glucose (5 mM), 0.15 g MgSO4 (0.61mM), 0.44g KCl (5.9 mM) and 0.10 g KH2PO4 (0.74 mM) in double distilled water and make up the volume to one litre. Bubble with oxygen to bring pH to about 7.4. Divide medium A in two parts. To one part add EDTA to a final concentration of 0.1 mM. Enzyme perfusion medium B - dissolve 30 mg collagenase A, 22 mg pyruvate and 16.6 mg (3 mM) CaCl2 in medium A and make up the volume to 50 ml. Sterile and filter the collagenase medium a through sterile filter.

Cell washing medium C - dissolve 7.7 g NaCl (131.7 mM), 1.0 g glucose (5.0 mM), 0.0176 g CaCl2 (0.12 mM), 0.209 g MgSO4 (0.85 mM), 0.387 g KCl (5.2 mM), 3.57 g Hepes (15 mM), phosphate buffer (3 mM), and 6.5 ml of 1 N NaOH (6.5 mM) in double distilled water and make up the volume to 1 litre. Adjust the pH to 7.4 and sterile filter.


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Tissue culture medium - dissolve 11.0 g William medium E (Sigma), 2.2 g NaHCO3, 2.38 g Hepes, 0.1 g pencillin, 0.1 g streptomycin sulphate in double distilled water. Make up the volume to one litre, adjust pH to 7.4, and sterile filter.

Preparation of hormones and cofactors solutions - prepare 1000-fold concentrated stock, sterile filter and store at -20°C. The stock solutions are prepared by dissolving 0.039 mg of dexamethasone (100 µM) per ml DMSO; 6 mg of insulin (100 µM) per 2 ml of 0.01 N HCl diluted to 10 ml with 0.15 M NaCl; 0.035 mg of glucagon (1.0 µM) in 10 ml of 0.15 M NaCl and 0.3 mg of epidermal growth factor (5 µM) in 10 ml of 0.15 M NaCl. Use 0.1 ml per 100 ml of William medium E.

Test substance: plant extracts are dissolved in DMSO or water at a concentration of 20 mg/ml and 15 µl aliquots are added to 3 ml of medium per plate to obtain a final concentration of 100 µg/ml. DMSO is used as a vehicle in control cultures or cultures that receive water extracts. In general for a 95 % methanol/ethanol extract use DMSO; for 50 % aqueous MeOH/EtOH extract use 50 % aqueous DMSO and for aqueous extracts use water, close the cap and place in boiling water bath for 15 minutes before use under sterile hood or sterile filter.

Hepatotoxicants: the EC50 concentrations are 2.5 mM CCl4, 10 mM paracetamol and 1.0 mM D-galactosamine-HCl. The stock solutions of CCl4 and paracetamol are made in DMSO and diluted with medium to obtain working solution; and of D-galactosamine in water. In case of 3 M CCl4 in DMSO, use 2.5 µl/3 ml tissue culture medium; for paracetamol 4.2 M in DMSO, use 7 µl/3 ml tissue culture medium and for D-galactosamine 100 mM in water (sterile), use 30 µl/ 3 ml of tissue culture medium. 7.4.3 Method 7.4.3.1 Preparation of Collagen and Collagen-coated Plates

Preserve rats’ tails at -20°C. Thaw out tails, clean with cotton swab soaked in 70 % ethanol. Pull out tendons and wash with 3 x 400 ml of 10 % NaCl solution followed similarly with 70 mM K2HPO4 solution. Extract lipids with 100 ml of ether. Decant off ether and keep the collagen fibres under UV light overnight. Dissolve collagen fibres in 0.3 % sterile acetic acid (250 mg collagen in 250 ml) stirring continuously for 2 to 3 days in the refrigerator. Centrifuge the collagen solution in sterile cups at 10,000 x G for 1 hour, dilute the supernatant (1:1) with sterile water to obtain absorption of about 1.0 at 280 nm and expose to γ-radiations (8000 rads) before use. Store the solution in the refrigerator.

Deliver 2.5 ml of collagen solution to each 60 mm tissue culture plate and incubate

the plates in CO2 incubator in humidified atmosphere at 37°C overnight. Remove the supernatant and wash with 3 ml PBS buffer containing 10 mM Hepes and 1 mM EDTA at pH 7.4 followed by 3 ml PBS buffer containing 10 mM Hepes. 7.4.3.2 Isolation of Hepatocytes

Sterile all the media, warm at 37°C and aerate with carbogen or oxygen before perfusion. Spray the rat fur with 70 % alcohol, cover with saran wrap and open the abdomen. Perfuse the liver through portal vein with medium A for about 5 minutes followed by medium A containing EDTA for another 5 minutes at a flow rate of 35 ml/min. Thereafter perfuse the liver with medium A continuously at a flow rate of approximately 25 ml/min. Additional canula is inserted into the thoracic part of the inferior vena cava to ensure the flow through the liver. Close the ligation of the inferior vena cava. Extreme caution is now required to liberate the liver very gently from the body without causing any mechanical damage to the lobes. Place the liver gently on the platform under the flow hood. This is followed by perfusion of collagenase medium B for about 15 minutes at 35 to 40 ml/min until the liver is ready. Place the liver into ice-cold medium C and open the Glissons capsule to disperse cells. Filter the cells through a


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nylon cloth and centrifuge three times at 50 x G for 2 minutes each. Disperse the cell pellet gently in medium C at protein concentration of 40 to 60 mg/ml on ice. (1 mg protein ≅ 0.6 x 106 cells).

Cell viability is determined by staining cells with 0.05 % trypan blue and preparation

viability of greater than 85 % is used for experimental studies. The same batch of collagenase should be used to avoid batch-to-batch variations for reproducible results.

7.4.3.3 Primary Culture of Adult Rat Hepatocyte and Treatment with Toxicants

Suspend the hepatocytes in Williams medium E containing 10 % fetal calf serum and 0.1 µM insulin and seed at a density of 2 to 3 x 106 cells/3 ml/60 mm collagen precoated plates. Incubate the cells at 37°C in humidified atmosphere of 5 % CO2 in a CO2-incubator. Cells usually adhere to the surface exhibiting epithelial morphology within 3 hours. Siphon off the medium to remove the dead cells and wash the plates twice with PBS.

Add to cultures fresh William medium E containing 0.1 µM insulin, 0.1 µM

dexamethasone, 5 nM epidermal growth factor and 1 nM glucagon without FCS. Add 15 µl DMSO to control plates as a vehicle. Treat the monolayer cultures immediately with the medium containing 2.5 mM carbon tetrachloride in DMSO with and without test sample for 18 to 22 hours. Positive and negative controls are included for comparative evaluation.

Collect the medium and centrifuge at 3,000 rpm for 10 minutes to spin down any

cell debris and transfer the supernatant to separate tubes (1.5 ml ependorf) and store at -20°C. Wash the cells twice with cold PBS and collect in 1 ml of PBS. Immediately use aliquots of cell suspensions for extraction of glutathione contents and store the remaining portions in liquid nitrogen for enzyme assays.

7.4.3.4 Estimation of Biochemical Parameters 7.4.3.4.1 Lactate Dehydrogenase (LDH)

LDH is determined by the method of Moldeus et al.26 Add 500 µl of phosphate buffer (pH 7.4), 40 µl of 35 mM sodium pyruvate and 40 µl of 10 mM NADH to 10 to 100 µl of the biological sample and make up the volume to 1 ml with water. Record the optical density change per minute at 25ºC at 340 nm and calculate nanomole of NADH oxidized per minute using 6.22 x 103 mol-1 cm-1 as the molar extinction coefficient of NADH. Calculate the antihepatotoxic potential of the test compound by measuring the extent of LDH leakage compared to the toxicant induced leakage alone.

LDH in medium Percent leakage = --------------------------------------- x 100 Total LDH (medium + cells) 7.4.3.4.2 Glutamic Pyruvic Transaminase (GPT)

GPT is estimated according to the method of Reitman and Franke17 as described under in vivo technique and percent leakage is calculated as shown in the formula under LDH above. 7.4.3.4.3 Glutathione (GSH)

Extract GSH from 500 µl of cell suspension at 4ºC after precipitating proteins with perchloric acid containing 10 mM EDTA following the method of Tietze.27 Neutralize 300 µl of


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the supernatant to pH not exceeding 7.0. Use the extract on the same day or store it at -70°C for not more than 7 days.

GSH is estimated in the extract spectrofluorometrically by orthophthaldehyde under

subdued light using the method of Hissn and Hilff.28 An aliquot of the extract is made up to 1.9 ml with 0.1 M phosphate buffer (pH 8.0), containing 5 mM EDTA. Add 100 µl of orthophthaldehyde, (1 mg/ml in methanol) in the dark. Measure relative fluorescence of the fluorescent product against the blank, prepared similarly, at 350 nm excitation, 420 nm emission, wavelengths. Express the values as µg GSH/plate.

100 x (Control - CCl4 and drug combine)

Percent GSH restoration = 100 - ----------------------------------------------------------------------- (Control - CCl4) 7.5 Conclusions

The most common method of evaluating liver damage is based on estimating liver specific enzyme levels in the serum. Several enzymes can be studied, however most commonly employed are the aminotansferases (transaminases), aspartate transaminase (AST) also known as glutamate oxaloacetate transaminase (GOT) and alanine transaminase (ALT) also called glutamate pyruvate transaminase (GPT). Necrosis or membrane damage releases the enzymes into circulation that are measured in serum. Increased level of enzymes over control indicates liver damage. ALT which is more specific to the liver catalyses the conversion of alanine to pyruvate and glutamate and is released in a similar manner to that of AST. A high level of these enzymes indicates acute liver damage. A number of other liver-specific enzymes such as, glutamyl transpeptidase (γ-GT), sorbitol dehydrogenase (SDH) and glutamate dehydrogenase (GlDH) are employed but in conjunction with AST and ALT. A general precaution should be observed to use non-haemolysed serum for enzyme estimations. The barbiturate-induced sleep model is fairly simple, easy to conduct and produces good results in live animals. It needs larger population size of animals in each group to produce valid results. Mice with sleep time ranging between 8 to 20 minutes are to be preselected for the experiment by giving the sedative drug to a large group of mice and recording sleep time. The animals falling in this range are separated and used in experimentation after a period of time. As a consequence this method requires a larger number of animals.

The histopathological examination provides a direct and visual examination of liver

damage but is not suitable for screening a large number of samples. The efficacy of the test substance can be observed easily and accurately by examination of liver sections under a microscope but the effect cannot be quantified.

In vitro technique using cultured hepatocytes can scan a large number of test

samples in a much shorter time frame, test parameters can be better controlled to get more reproducible results. References

1 Guyton, A. C., 1981, Textbook of Medical Physiology (6th ed.), WB Saunders Company, Philadelphia, pp. 869-

876

2 Wagner, H., 1980, Plant constituents with antihepatotoxic activity, In: Beal, J. L. and Reinharde, E. (Eds.), Natural Products as Medicinal Agents, Hippokrates Verlag Stuttgart, Germany, pp. 217-241


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3 Passmore, R. and Robinson, J. S., 1973, A Companion to Medical Studies, Blackwell Scientific Publications, Oxford, England, pp. 32.1-32.12

4 Morazzoni, P. and Bombardelli, E., 1995, Silybum marianum (Carduus marianus), Fitoterapia, 66, 33-49

5 Choudhury, B. R., Haque, S. J. and Poddar, M. K., 1987, In vivo and in vitro effects of Kalmegh (Andrographis paniculata) extract and andrographolide on hepatic microsomal drug metabolozing enzymes, Planta Medica, 53, 135-140

5a Handa, S.S. and Sharma A., 1990, Hepatoprotective activity of andrographolide from Andrographis paniculata against galactosamine and paracetamol intoxication in rats, Indian Journal Medical Research, 91B, 284-292

6 Hikino, H. and Kiso, Y., 1988, Natural products for liver diseases, Economic and Medicinal Plant Research, Vol. 2, Academic Press Ltd, U.S.A, pp. 39-72

7 Dwivedi, Y., Rastogi, R., Sharma, S. K., Garg, N. K. and Dhawan, B. N., 1991, Picroliv affords protection against thioacetamide-induced hepatic damage in rats, Planta Medica, 57, 25-28

8 Thyagarajan, S. P., Subramanian, S., Thirunalasundari, T., Venkateswaran, P. S. and Blumberg, B. S., 1988, Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus, Lancet II, 764-766

9 Karan, M., Vasisht, K. and Handa, S. S., 1999, Antihepatotoxic activity of Swertia chirata on paracetamol and galactosamine induced hepatotoxicity in rats, Phytotherapy Research, 13, 95-101

10 Karan, M., Vasisht, K. and Handa, S. S., 1999, Antihepatotoxic activity of Swertia chirata on carbon tetrachloride induced hepatotoxicity in rats, Phytotherapy Research, 13, 24-30

10a Handa, S.S., Sharma A and Chakraborti K.K., 1986, Natural products and plants as liver protecting drugs, Fitoterapia, 57, 307-351

11 Recknagel, R. O., 1983, Carbon tetrachloride hepatotoxicity, Trends in Pharmacological Sciences, 4, 129-131

12 Dixon, M. F., Dixon, B., Aparcio, A. R. and Loney, D. P., 1975, Experimental paracetamol-induced hepatic necrosis: A light and electron microscope and histochemical study, Journal of Pathology, 116, 17-29

13 Reutter, W., Hossels, B. and Lesch, R., 1975, Induction and prevention of galactosamine hepatitis, In: Bertelli, A. (Ed.), New Trends in the Therapy of Liver Diseases, S Karger, Basel, Switzerland, pp. 121-127

14 Van, W. L. and Lieber, C. S., 1980, Mechanisms whereby ethanol exerts its adverse effects on the liver, In: Farber, E. and Fisher, M. M. (Eds.), Toxic Injury of the Liver, Marcel Dekker Inc, New York, pp. 632-653

15 Rose, C. I. and Henderson, A. R., 1975, Reaction-rate assay of serum sorbitol dehydrogenase activity at 37°C, Clinical Chemistry, 21, 1619-1626

16 Schmidt, E. and Schmidt, F. W., 1983, Glutamate dehydrogenase, In: Bergmeyer, H.-U. (Ed.), Methods of Enzymatic Analysis, 3rd ed., pp. 216-227

17 Reitman, S. and Franke, A. S., 1957, A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases., American Journal of Clinical Pathology, 28, 56-63

18 Kind, P. R. N. and King, E. J., 1954, Estimation of plasma phosphatase by determination of hydrolysed phenol with antipyrine, Journal of Clinical Pathology, 7, 322-326

19 Gottfried, S. P. and Rosenberg, B., 1973, Improved manual spectrophotometric procedure for determination of serum triglycerides, Clinical Chemistry, 19, 1077-1078

20 Hawk, P. B., Oser, B. L. and Summerson, W. H., 1978, Practical Physiological Chemistry (13th ed.), McGraw-Hill Book Company Inc, UK, p. 1071

21 Galigher, A. E. and Kozloff, E. N., 1971, Essentials of Practical Microtechnique (2nd ed.), Lea and Febiger, Philadelphia, pp. 77 & 210

22 Bliss, C. I., 1967, Statistics in biology, Statistical Methods for Research in the Natural Science, Vol. 1, McGraw-Hill Book Company, New York

23 Perrissoud, D. and Weibel, I., 1980, Protective effect of (+)-cyanidol 3 in acute liver injury induced by galactosamine or carbon tetrachloride in the rat, Naunyn-Schmiedeberg's Archives of Pharmacology, 312, 285-291

24 Osol, A., 1980, Remington's Pharmaceutical Sciences (16th ed.), Mack Publishing Company, Easton, Pennsylvania, p. 118


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25 Reen, R. K., Karan, M., Singh, K., Karan, V., Johry, R. K. and Singh, J., 2001, Screening of various Swertia species extracts in primary monolayer cultures of rat hepatocytes against carbon tetrachloride and paracetamol induced toxicity, Journal of Ethnopharmacology, 75, 239-247

26 Moldeus, P., Hogberg, J. and Orrenius, S., 1978, Isolation and use of liver cells, Methods in Enzymology, 52, 60-71

27 Tietze, F., 1969, Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione applications to mammalian blood and other tissues, Analytical Biochemistry, 27, 502-522

28 Hissn, P. J. and Hilff, R., 1976, A fluorometric method for determination of oxidized and reduced glutathione in tissues, Analytical Biochemistry, 74, 214-226


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8 A Practical Approach for Assessing Cardiovascular Activity of Natural Products

Raimo Hiltunen, H.J. Damien Dorman and Yvonne Holm 8.1 Introduction

Cardiovascular diseases (CVD) may be divided into diseases associated with the heart muscle and its function and diseases associated with the blood vessels. Heart diseases include cardiomyopathy (leading to heart failure or insufficiency), coronary heart disease (CHD), arrhythmias and infections in the heart muscle (endocarditis, myocarditis and pericardi-tis).

Vascular diseases include hyper-/hypotension, deep vein thrombosis, atherosclero-sis and chronic venous insufficiency. Migraine and diabetes are often included among vascu-lar diseases.1, 2 These will not, however, be discussed in this context, as migraine can be re-garded as a type of headache, although the cause remains unclear. In the case of diabetes, vascular diseases are a complication that occurs if the underlying disease is either mistreated or untreated for a long period of time. 8.1.1 Cardiomyopathy and Heart Failure

There are various types of cardiomyopathy, the two major categories being (i) ischemic and (ii) non-ischemic cardiomyopathy. The ischemic type refers to heart muscle dam-age resulting from coronary artery disease, e.g. heart attack. The non-ischemic types include several forms, which are dilated, hypertrophic and restrictive. Of these forms, the dilated (con-gestive) cardiomyopathy is by far the most common type. Both other forms tend to be rare, at least in industrialized countries. Dilated cardiomyopathy occurs most often in middle-aged people and more often in men than in women. The specific cause of dilated cardiomyopathy is unknown, but some factors have been linked to its occurrence. For instance, the excessive consumption of alcohol, various infections, toxins, drugs (doxorubicin and daunorubicin),2 and extensive malnutrition, resulting in deficiency of vitamin B1.3

Dilated cardiomyopathy is the cause of heart insufficiency and heart failure. It is treated with diuretics, vasodilators (ACE-inhibitors), digitalis and other cardiac glycosides, cal-cium channel blockers as well as beta-blockers.2 In some countries hawthorn preparations (herbal remedies) are popular in the treatment of mild to moderate heart insufficiency.1 See Table 1. 8.1.2 Arrhythmias

Arrhythmias may be associated with heart disease but most of them occur in people who do not have underlying heart disease. They occur commonly in middle-age adults. Other causes than heart disease include: stress, caffeine, tobacco, alcohol, diet pills and cough and cold medicines. Arrhythmias can originate in the atria or in the ventricles of the heart and are called atrial (supraventricular) or ventricular arrhythmias, respectively. In general, ventricular arrhythmias caused by heart disease are the most serious.2

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Many arrhythmias require no treatment whatsoever but if necessary may be treated by several types of drugs. Sometimes heart disease is treated to control the arrhythmia.2 Chinidin, beta-blockers and calcium channel blockers represent some of the drugs used.4 8.1.3 Coronary Heart Disease

A common symptom of coronary heart disease (CHD) or coronary artery disease is angina pectoris, which is a recurring pain in the chest. The pain occurs when a part of the heart does not receive enough blood i.e. oxygen, because the coronaries have become nar-rowed and blocked due to atherosclerosis. An episode of angina is often triggered by physical exertion. Other causes can be emotional stress, extreme cold or heat, heavy meals, alcohol, and cigarette smoking.

The underlying coronary artery disease that causes angina should be treated by con-trolling existing risk factors. These include high blood pressure, high blood cholesterol levels, overweight/obesity, diabetes, physical inactivity and cigarette smoking.3 Angina is often con-trolled by drugs. The most commonly prescribed drug for angina is nitroglycerine, which re-lieves pain by widening the coronaries. Beta-blockers and calcium channel blockers are also used to slow the heart rate and reduce the frequency and severity of angina attacks.2 In some countries hawthorn (Crataegus sp.) preparations are quite popular in treating stable angina pectoris.1

Drugs used to treat CHD and its risk factors include aspirin and thrombolytic agents, digitalis, ACE inhibitors, diuretics and cholesterol-lowering agents as well as drugs used for treating angina.2 See Table 1. 8.1.4 Atherosclerosis

Atherosclerosis is the hardening and narrowing of the arteries caused by the slow build up of plaque on the endothelium of the inner wall. Plaque is made up of fat, cholesterol, calcium and other substances found in the blood. Plaque can be hard and stable or soft and unstable. Soft plaque is more likely to break apart from the walls, enter the bloodstream and cause thrombosis. Atherosclerosis can affect the arteries of the brain, heart, kidneys, arms or legs, and may cause serious diseases and complications. These include coronary artery dis-ease (CAD), such as angina, heart attack and sudden death, cerebrovascular disease (CVD), such as stroke and transient ischemic attack (TIA), and peripheral arterial disease.2

Risk factors enhancing the development of atherosclerosis are high cholesterol lev-els, high blood pressure, smoking, overweight/obesity and physical inactivity. The prevention and treatment of atherosclerosis start with life style changes, a healthy diet and exercise. If this is not effective drugs are used to lower cholesterol levels, to lower blood pressure or to prevent the blood from coagulating.2 There are also several herbal remedies and functional foods available for the prevention and treatment of atherosclerosis. These include garlic (Al-lium sativum L.), soy (Glycine max (L.) Merr.), beans, fish oil, oat (Avena sativa L.) bran, pectins, guar gum, red yeast rice (Monascus purpureus Went) and β-sitosterol (1, 5). Antioxi-dants may also be useful since they prevent the oxidation of LDL.5 See Table 1.

For the treatment of peripheral arterial occlusive disease (intermittent claudication) there is the well documented special extract of ginkgo (Gingko biloba L.) leaves, which is able to improve the pain-free walking distance. It is also used in the symptomatic treatment of cog-nitive deficits, so called ‘cerebral insufficiency’, which include memory loss, concentration dif-ficulties, and lessened social skills.6 Promising results have been achieved with ginkgo ex-tracts in the treatment of Alzheimer’s disease.7


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8.1.5 Deep Vein Thrombosis and Chronic Venous Insufficiency

Most deep vein clots occur in the leg or hip veins. Those in the thigh or hip veins are usually more serious than blood clots in the lower leg. If such a clot breaks off into the blood-stream it may cause pulmonary embolism. Deep vein thrombosis is treated or prevented by anticoagulants and thrombolytics.2

Chronic venous insufficiency is a syndrome resulting from the obstruction or persis-tent incompetence of deep veins or perforating veins in the lower legs. It is divided into three stages based on its degree of severity. The therapy consists of vascular surgery (possible in only a small percentage of patients), support stockings and symptomatic treatment with ve-nous remedies, often containing heparin. Herbal remedies containing horse chestnut (Aescu-lus hippocastanum L.), butcher’s broom (Ruscus aculeatus L.) and flavonol glycosides (rutin, hesperidin and diosmin) are popular and of these the preparations containing horse chestnut are the most studied.2, 6 See Table 1.

8.1.6 Hypertension

Blood pressure of 140/90 mmHg or higher is considered to be high. In persons with diabetes and chronic kidney disease 130/80 mmHg is considered to be high. It usually has no symptoms and may not be found until complications, such as heart failure, kidney failure or heart attack, occur. In many cases, a single specific cause for high blood pressure is not known but in other cases it may be the result of another medical problem or medication.

Hypertension is treated by lifestyle changes (i.e. healthy diet, physical activity, weight loss) and medication (often two or more drugs). Medicines used are diuretics, beta blockers, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBS), calcium channel blockers, alpha blockers, alpha-beta blockers and vasodilators.2 Herbal remedies used are the extracts of Indian snakeroot (Rauvolfia serpentina Benth. et Kurz.), mistletoe (Viscum album L.), garlic (Allium sativum L.) and olive (Olea europaea L.) leaves.6 See Table 1.

Table 1: Plants used in traditional and herbal medicine for the treatment of cardiovascular diseases*

Plant name Scientific name Active constituents Effect Use

Foxglove, purple Foxglove, Grecian

Digitalis purpurea L. D. lanata Ehrh.

Cardiac glycosides digitoxin, digoxin, etc.

Positive inotropic, negative chronotropic, negative dromotropic, positive bathmotropic

Heart failure

Spring adonis Adonis vernalis L. Cardiac glycosides adonitoxin, cymarin

As foxglove Heart failure

Lily of the valley Convallaria majalis L. Cardiac glycoside convallatoxin


Squill Urginea maritime (L.) Baker

Cardiac glycoside scillarigenin


Strophanthus

Strophanthus gratus (Wall. & Hook.) Baillon

Cardiac glycoside ouabain


Christmas rose

Helleborus niger L. Cardiac glycoside helliborin

Oleander Nerium oleander L. Cardiac glycoside oleandrin


Yellow oleander Thevetia neriifolia Juss. Cardiac glycosides thevetosides


Hawthorn Crataegus sp. Oligomeric proantho-cyanidins, flavonoids

Positive inotropic, negative chronotropic

Heart failure, angina pectoris

Continued


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Plant name Scientific name Active constituents Effect Use

Motherwort Leonurus cardiaca L. Alkaloids, flavonoids Cardioactive in vitro ‘Nervous heart’, tachycardia, effort syndrome

Motherwort Leonurus cardiaca L. Alkaloids, flavonoids Cardioactive in vitro ‘Nervous heart’, tachycardia, effort syndrome

Khella Ammi visnaga Lam. Furanochromones khellin, visnagin

Coronary vasodilation, positive ino-tropic, bradycardic, calcium channel blocking

Angina pectoris

Snakewood Rauwolfia serpentina (L.) Benth. ex Kurz.

Alkaloids reserpine, ajmalicine ajmaline

Reserpine = antihyperten-sive Ajmalicine = spasmolytic Ajmaline = antiarrhythmic

Hypertension (reserpine)

Garlic Allium sativum L. Alliin and other sulphur-containing compounds

Antihypertensive, inhibits platelet aggregation, decreases cholesterol levels

Minor vascular disorders

Olive leaves Olea europaea L. Secoiridiods, oleuropein

Hypotensive, coronary vasodilatation, antiar-rhythmic

Mild hypertension Traditionally used as diuretic

Mistletoe Viscum album L. Viscotoxins, lectin Hypotensive Traditionally used in hypertension

Scotch broom Cytisus scoparius (L.) Link.

Alkaloids, sparteine Ganglioplegic Cardiac and circulatory disorders, tachycardia

White hellebore Veratrum album L. Alkaloids, jerveratrum and cerveratrum types

Hypotensive, at high doses cardiotoxic

Hypertension, cardiac insufficiency Not in use any more (toxic!)

Ginkgo, maidenhair tree

Ginkgo biloba L. Flavonoids, terpenes, ginkgolides

Increases blood flow in the brain, PAF-inhibition, radi-cal scavenging, arterial vasodilation, venous vasoconstriction

Cerebral insufficiency, tinnitus, claudicatio intermittens

Horse chestnut

Aesculus hippocastanum L.

Triterpene saponins, aescin

Anti-inflammatory, antioe-demic, anti-exudative, increase of vascular tone

Venous insufficiency

Butcher’s broom Ruscus aculeatus L. Steroidal saponins, ruscogenin

Prevents dilatation of ve-nous vessels

Venous insufficiency

*: Various sources

In any programme investigating natural products as remedies for cardiovascular drugs,

the following properties should be included: (i) inotropic (syn. cardiotonic), (ii) chronotropic, (iii) dromotropic and (iv) antiarrhythmic activities, (v) effects upon coronary dilatation and (vi) inhi-bition of thrombocytes. In addition, closely associated activities would include antioxidant properties and aggregation and anticoagulation. Inotropic investigations deal with contractility as exemplified by cardiac glycosides, sympathomimetics (positive effect) and β-receptor


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blocker (negative effect) drugs. Chronotropic investigations deal with cardiac frequency as exemplified by sympathomimetics (positive effect) and cardiac glycosides and β-receptor blocker (negative effect) drugs. Dromotropic effects concern flow rate as exemplified by sym-pathomimetics (positive effect) and cardiac glycosides and β-receptor blocker (negative effect) drugs. 8.2 Toxicology and Safety

The determination of the toxicity and safe usage of any substance or mixture of sub-stances is of cardinal importance, particularly for administration purposes. Western medicine imposes very strict requirements before drug registration with significant legal sanctions and financial loss for those unable to demonstrate an appropriate approach to toxicology and safety assessment in their drug discovery programmes. Data requirements may vary for herbal and traditional medicines, however, the following series of assessments may very well be re-quired (Table 2).

Table 2: Potential toxicological data required for traditional and herbal medicines

Systemic toxicity Local toxicity Specialized toxicity

Acute and chronic toxicity assessment

Local sensitization/irritation Mutagenicity

Adjuvant and patch Bacterial reverse mutation Maximisation Chromosomal aberration Freund’s complete adjuvant Rodent micronucleus

Open epicutaneous Carcinogenicity Systemic absorption Preliminary study Full scale study Reproductive toxicity/teratogenicity Segment I study

Segment II study Segment III study

A step-by-step guide to the workings of such screens is beyond the scope of this par-

ticular text, however, those requiring further details should refer to appropriate texts. The WHO has published guidelines for toxicity and safety investigations with particular emphasis on herbal and traditional medicines.8 8.3 Bioassay and Evaluation 8.3.1 Synthetic Free Radical Scavenging Assessment

A negative association appears to exist between the development of cardiovascular related diseases and the consumption of antioxidant-rich foodstuffs and the endogenous anti-oxidant status.9-14 The principal mode of action of antioxidants is the ability to scavenge free radical and reactive non-radical species, as these species are believed to play a important role in the aetiology and pathogenesis of cardiovascular disease.15, 16 Thus the assessment of the antioxidant properties of a herbal or traditional medicine may well be an important index of cardiovascular protective potential. One antioxidant screening assay, the ABTS decolourization assay, described below is a commonly used assay to determine free radical scavenging poten-tial of plant extracts and isolated phytochemicals.17

8.3.1.1 Preparation of the ABTS Free Radical

Dissolve 0.0192 g 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonate) diammonium salt (ABTS) in 5 ml HPLC grade water (solution A). Dissolve 0.1892 g potassium persulphate (K2S2O8) in 5 ml HPLC grade water (solution B). To solution A, add 88 µl solution B, mix well


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and allow to stand in the dark at room temperature for 12 to 16 hours (solution C). Prior to use, dilute solution C with ethanol (EtOH) until the absorbance at λ734 nm stabilizes at 0.700 ± 0.020 AU (solution D). 8.3.1.2 Preparation of Calibration Standards

Dissolve 0.0313 g Trolox in 25 ml EtOH to give a stock solution at 5 mM/L (solution E). Dilute solution E to give a final reaction concentration range of 0.5 to 20.0 µmol/L. 8.3.1.3 Methodology

An aliquot (15 µl) of each Trolox calibration standard is added to 1.485 ml solution D (the ABTS•+ radical solution), the mixture is mixed and the absorbance at 734 nm is re-corded at t = 1 minute after initial mixing and subsequently at 1 minute intervals until no fur-ther change in absorbance is observed or for predetermined period e.g. 15 minutes in toto. From this data, a inhibition calibration curve for Trolox is constructed. Samples are diluted such that a 15 µl sample when added to 1.485 ml solution D results in a 20 to 80% inhibition of the absorbance at 734 nm. As for the Trolox calibration standards, after 15 µl extract is added to 1.485 ml solution D, the absorbance at 734 nm is recorded at t = 1 minute after mixing and subsequently at 1 minute intervals (15 minutes in toto). The percentage inhibition is plotted as a function of concentration and the Trolox equivalent antioxidant capacity (TEAC) is calculated against the Trolox calibration curve. 8.3.2 Nitric Oxide (NO) and Nitrite (NO2) Scavenging Assessment

Nitric oxide is an important biological substance capable of regulating vascular tone, modulating immune response and neurotransmission.18-20 Nitric oxide is generated from the amino acid L-arginine by nitric oxide synthases.21 It is a very reactive substance, oxidizing into nitrite and nitrate depending upon the redox conditions. Nitrite is mostly found in aqueous and plasma samples, however, in blood it reacts rapidly with oxyhaemoglobin to form methaemo-globin and nitrate. Thus, practically all blood nitric oxide appears as nitrate. The method de-scribed below enables the quantification of endogenous nitric oxide in an experimental and clinical setting. The method has been used to (i) measure nitrite in the effluate of isolated per-fused guinea pig hearts before and after infusion of bradykinin and (ii) analyse nitrite in ve-nous blood samples after forearm blood flow had been influenced with increasing doses of acetylcholine.22 8.3.2.1 Flow Injection Analysis

The system consists of an HPLC, low-pulsation pump, automatic injector and ultra-violet detector (Figure 1). All capillaries are 0.25 mm in diameter and are made of PEEK tubing (polyether ether ketone). The length of the capillary tube (the reaction loop) from injector to detector is 90 cm. An HPLC computer controller controls the system and manages the UV-data acquisition. The sample is entrained in a Griess reagent carrier solution (23.2 mM/L sulfa-nilamide and 7.72 mM/L N-(1-naphthyl)ethylenediamine dihydrochloride in 1% (v/v) hydro-chloric acid) which is degassed with helium. The solution is then pumped through the system at a constant flow rate of 1 ml/min. The samples are introduced via a 20 µl injection loop and transported to the ultraviolet detector with the carrier solution and an incomplete reaction during the trip to the detector. The extent of reaction is then measured at 545 nm at the ultra-violet detector in a 3 ml measuring cell.


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Figure 1: Schematic flow injection analysis system for determining nitrite

8.3.2.2 Treatment of the Blood Samples

A 1.5 ml blood sample is drawn from a vein into a prepared syringe containing 1.5 ml 0.1 mol/L NaOH (as a stop solution). The pH value is restored to a physiological level with 1 mol/L phosphoric acid. The sample is mixed and centrifuged for 5 minutes at 10,000 G. The clear supernatant is transferred to ultrafiltration tubes with a pore size of 10,000 Da and cen-trifuged for 1 hour at 3,000 G and 20°C.

0

2

4

6

8

10

12

14

16

18

-400 -300 -200 -100 0 100 200 300 400

Nitrite [nmol/L]

Hei

ght [

µV]

Figure 2: Principle of the standard addition method for determining nitrite

A biological sample is divided into five aliquots. Increasing concentrations of nitrite are added to samples (150, 1100, 1200, and 1400 nmol/L). The intersection of the straight lines and the X-axis determines the nitrite concentration in the plain sample. The nitrite content in the sample can then be calculated by correcting for the dilution factor. 8.3.2.3 Calculation of Nitrite Concentration

For the sample analysis, 95 µl of a sample is placed in each of five prepared reac-tion vessels to which 5 µl of nitrite at increasing concentrations (1000, 2000, 4000, and 8000 nmol/L) was added. This yields final concentrations of 50, 100, 200, and 400 nmol/L in the sample. A regression line is drawn through the data obtained. The intersection between the

HPLC Pump Griess Reagent

Autosampler

Ultraviolet detector PC with integrator

Injector valve

PEEK reaction coil


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straight lines and the X-axis at point (y = 0) is the nitrite concentration in the plain sample (Fig-ure 2). The existing nitrite in the HPLC water (20 to 100 nM/L) is subtracted from the blood samples. The initial concentration of the sample can then be deduced by correcting for the dilution factor. 8.3.3 Experimental Procedure for the Investigation of the Potential Inhibitory Effect

of a Plant Extract upon Experimental Cardiac Hypertrophy

Hypertonia is one of the principal risk factors associated with chronic heart failure (CHF). Other vascular disease is cardiac hypertrophy, particularly of the left ventricle,23 which is not only a fundamental process of adaptation to increased workload but also an important cause of increased morbidity and mortality.24 As cardiac myocytes are terminally differentiated cardiac hypertrophy occurs by an increase in myocyte size and growth of non-myocyte compo-nents. Meanwhile, the protein mass is increased in hypertrophied cardiac tissue and this is brought about, in part, by an increase in the rate of protein synthesis.25, 26 All these structural changes result in an increase in myocardial volume and mass, and impaired systolic and dia-stolic function of the heart. The methodologies described below enable these parameters to be determined.23. 8.3.3.1 Materials

Male Sprague-Dawley rats (cir. 180 to 220 g), sodium pentobarbital, penicillin G, captopril, IV cannula (Φ1.1 x 50 mm), heparin, saline, pressure transducer, formalin, hema-toxylin-eosin and Total Protein Reagent Kit (Sigma Diagnostics). 8.3.3.2 Animals

Rats are housed four to five per cage in light (12-hour light-dark cycle), temperature (24°C) and humidity (60 %) controlled room with food and water available ad libitum. 8.3.3.3 Surgery

Prior to surgery, a minimum of 3 days is required as an acclimatization period. The animals are anesthetized (sodium pentobarbital, 30 to 45 mg/kg i.p.) and a midline abdominal incision is made, exposing the suprarenal abdominal aorta. The aorta is constricted by a silk thread at 0.6 mm.27 The abdomen is closed and the animals are injected with penicillin G (10000 U/ rat) and are not allowed to access food and water ad libitum. A sham-operation (without aorta banding) is performed on age-matched control rats. 8.3.3.4 Administration of Extracts After a period of 1 month, all surgically operated animals were divided into at least three groups depending upon the number of extract concentrations to be administered: (i) aortic banding model group, (ii) extract group(s) and (iii) 50 mg/kg captopril (positive control) group. The drugs are administered orally (in water) at 1 ml/100 g body weight for 8 weeks. The aortic banding model and age-matched control animals are given the same volume of water at the same time. 8.3.3.5 Determination of Time Course of Blood Pressure Changes during Experimental

Period

Prior to surgery, the blood pressure (BP) of all anesthetized animals is recorded. The value of systolic BP and mean BP is detected from the BP curve. Four weeks after surgery, as well as 2 and 8 weeks after treatment with drugs, the rats are anesthetized and blood pres-sure is measured in the same manner.


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8.3.3.6 Assessment of Hemodynamic Parameters

After 8 weeks of drug treatment, the rats are anesthetized (30 mg/kg sodium pen-tobarbital, i.p.) and the right common carotid artery is exposed. A type IV cannula (Φ1.1 x �50 mm) filled with heparinized saline is connected to a pressure transducer and inserted into the vessel and then advanced into the left ventricle to record the left ventricular pressure. Left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), the maxi-mum rates of contraction (+dp/dtmax) and relaxation (- dp/dtmax), and the time to reach the point of maximum rate from beginning of contraction (tdp/dt) are derived from the left ven-tricular pressure pulse.

8.3.3.7 Assessment of Cardiac Hypertrophy (i) Cardiac weight. After measurement of hemodynamics the rats are sacrificed (by bleeding) and the heart is removed. The wet weight of the whole heart and left ventricle is recorded and the heart weight and left ventricular weight indices calculated as heart weight in g per kg of body weight (HW/BW) and left ventricular weight in g per kg body weight (LVW/BW), respec-tively.

(ii) Cell size. The cell size of the left ventricular myocardium is defined by counting the cross-sectional area at 400 x Tissue from the left ventricle is fixed in 10 % formalin and morphologic measurement was done in hematoxylin-eosin (HE) staining. The counting fields were selected randomly from each slice and only those cells with nuclei are counted.

(iii) Total protein. Left ventricular tissue (approximately 0.1 g) is thoroughly homogenised in 2 ml saline solution. The total protein content is then determined using a total protein reagent kit (Sigma Diagnostics). The concentration of total protein is derived from a standard curve prepared using multilevel protein standards (2.5, 5.0, 10.0, 20.0, 30.0 and 40.0 mg/ml). The total protein contents in the left ventricular tissue are expressed as mg protein per mg tissue. 8.3.3.8 Statistical Analysis

Results should be expressed as mean values ± standard deviation (SD). Differences between groups can be evaluated using a two-tailed unpaired Student-t test, while the correla-tions between groups can be estimated using the Pearson Correlation test. A level of signifi-cance of p < 0.05 is usually interpreted as statistically significant. 8.3.4 Determination of Isometric Contraction Force Using Guinea Pig Papillary

Muscles, Aortic Strips, and Terminal Ilea and Chronotropic Activity Using Guinea Pig Right Atria

The inotropic or isometric contractive force (the strength of contraction of the heart

muscle) and chronotropic (the time between p waves) parameters are important indices of cardiac function. However, in the development of CVD, they change from normally accepted physiological levels. These anomalous changes may be resisted with drugs. The methodologies described below enable a researcher to assess the effectiveness of novel natural products to influence these parameters.27 8.3.4.1 Solution

For heart muscle preparations: pH 7.2–7.4 Krebs-Henseleit solution (114.9 mM/L NaCl, 4.73 mM/L KCl, 3.2 mM/L CaCl2, 1.18 mM/L MgSO4, 24.9 mM/L NaHCO3, 1.18 mM/L KH2PO4 and 10 mM/L glucose). For aortic strips: modified Krebs-Henseleit solution (118.0 mM/L NaCl, 4.8 mM/L KCl, 1.8 mM/L CaCl2, 1.2 mM/L MgSO4, 1.2 mM/L KH2PO4, 24.0 mM/L NaHCO3 and 11 mM/L glucose). For intestine preparations: nutrient solution (136.90 mM/L NaCl, 2.7 mM/L KCl, 1.80 mM/L CaCl2, 1.05 mM/L MgCl2, 24.0 mM/L NaH2CO3, 0.43 mM/L


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NaH2PO4 and 11 mM/L glucose). All bathing solutions are continuously bubbled with 95 % O2 and 5 % CO2. 8.3.4.2 Tissue Isolation

Guinea pigs (340 to 480 g, both sexes) are killed with a blow to the neck. After exci-sion of the heart, papillary muscles are dissected from the right ventricule for contractility measurements. The Purkinje fibers are carefully removed. Only muscles with a diameter of less than 0.87 mm should be used.28 8.3.4.3 Chronotropic Activity Tested on Guinea Pig Isolated Right Atria

The isolated preparations are stored at room temperature in Krebs-Henseleit solu-tion. Experiments were performed at a temperature of 35 ± 1°C. After excision of the heart, the aorta is dissected and cleaned of loosely adhering fat and connective tissue and cut heli-cally into strips 1 cm long and 4 mm wide. These strips are placed in 35 ml nutrient solution at a temperature of 37 ± 1°C. The terminal portion of the ileum is removed and the 10 cm near-est to the caecum is discarded. The preptestine is cleaned with nutrient solution, cut into 2 to 3 cm long pieces and placed in 35 ml of nutrient solution at a temperature of 35 ± 1°C. 8.3.4.4 Experimental Set Up

Isometric contraction force of electrically stimulated papillary muscles and sponta-neous activity in right atria should be measured by the Reiter method.29 A force transducer and amplifier are used for measurements of isometric contractions. The resting tensions of the papillary muscles, right atria and aortic strips were kept constant throughout the experiments. Papillary muscles were electrically driven with a stimulator at a frequency of 1 Hz and pulse duration of 3 milliseconds. The amplitude of the stimulation pulse is adjusted to threshold level + 10 %. Aortic strips and terminal ilea are precontracted with 90 and 60 mM/L KCl, re-spectively. Signals are recorded with a dual beam storage oscilloscope and a chart recorder. Photographic documentation should be taken every 5 minutes from the screen of the oscilloscope and evaluated (magnification maybe required). 8.3.4.5 Methodology

After a control period (30 minutes for heart muscle, 60–120 minutes for aortic strips and 60 minutes for terminal ilea), different concentrations of extracts are added to the bathing solution cumulatively until steady-state is achieved. To study the calcium antagonistic effects of extracts, the extracts are investigated at different concentrations on papillary mus-cles (1 Hz). When a steady-state is achieved, CaCl2 is added in increasing concentrations to antagonize a possible negative inotropic effect caused by the extracts. Glibenclamide (potas-sium channel blocker) is added at 10, 30 and 100 µM/L concentration levels to different con-centrations of the extracts to investigate a possible effect on ATP-sensitive potassium chan-nels. When investigating the reversibility of the relaxing effects of the extract on aortic strips and terminal ilea, glibenclamide is added at a concentration of 10 µM/L. 8.3.4.6 Chronotropic Effect of Extract In Vivo after Intravenous Administration and In Vitro

on Isolated Atria of Aortic Coarctated Rat

Extract levels in arterial dialysates and changes in arterial pressure and heart rate (HR) are determined after intravenous administration of the extract. While affinity of the ex-tract is determined by means of the shifting of the noradrenaline concentration-response curve and the inverse agonist activity of the extract studied in isolated atria.30


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8.3.4.7 Induction of Hypertension

Female Wistar rats were used (250 to 300 g). The experiments were carried out ac-cording to the Rojo-Ortega and Genest method.31 Control animals are sham operated (SO). The experiments were carried out on the 7th day after the corresponding operation. 8.3.4.8 In Vivo Experiment: Extract Chronotropic Effect

On the day of the experiment the animals were anesthetized (50 mg/kg chloralose, i.p. and 500 mg/kg urethane, i.p.). A bilateral vagotomy is carried out for the study of �-blockade action on heart rate (HR). A femoral vein is cannulated for the intravenous admini-stration of the extract (in isotonic solution) at a specified dose(s). A ‘shunt’ type microdialysis probe (with 1 entrance and 2 vascular exits) is used for examining the time course of MP plasma concentrations.32, 33 The entrance and vascular exit of the heparinized (50 U/ml) probe are inserted in the left carotid artery while the remaining exit is connected to a pressure trans-ducer coupled to a polygraph. The mean arterial pressure (MAP) is calculated according to the following formula:

MAP = [diastolic pressure + (systolic pressure – diastolic pressure)] / 3

The HR is calculated by means of tachograph by counting the pulsatile waves of ar-terial pressure recording. The microdialysis probe is perfused with a 147 mM/L NaCl, 4 mM/L CaCl2, 4 mM/L KCl solution (pH 7.3) using a perfusion pump adjusted to a 2 µl/min flow rate. Samples were collected at 15 minute intervals. 8.3.4.9 Chronotropic Effect of an Extract in Isolated Atria

Animals are sacrificed by cervical dislocation and the hearts are quickly removed and placed in a Petri dish filled with physiological salt solution (118.6 mM/L NaCl, 4.83 mM/L KCl, 1.18 mM/L KH2PO4, 1.70 mM/L CaCl2.2H2O, 1.34 mM/L MgSO4.7H2O and 11.10 mM/L dextrose). The combined atria are carefully dissected free of connective tissue and the ventri-cles. The atria are then mounted in the organ bath and connected to a force transducer. The temperature of the bath was maintained at 37°C and the bathing solution was bubbled with 95 % O2 and 5 % CO2. A tension of 1 g is applied to the atria. The atria are allowed to equili-brate (at least 45 minutes) in which time the preparations are washed (15 minutes). The ex-tract(s) are added when the spontaneous rate of beating of the atria changed no more than 5 % within a 10 minute observation period. In order to determine the extract potency, a cumu-lative dose-response curve of noradrenaline is constructed. After washing the preparation until obtaining initial basal rate of spontaneous beating, the extract(s) are added (e.g. 0.05, 0.50 or 5.0 µM/L). The cumulative dose-response curve for noradrenaline is made after 30 minutes of incubation with the extract(s). In order to determine the inverse agonistic activity of the extract, a cumulative extract dose-response curve is constructed with the range of concentrations (e.g. 1 x 10-8, 1 x 10-7, 3 × 10-6, 1 x 10-6, 3 × 10-5, 1 x 10-5, 3 × 10-4 M/L). 8.3.4.10 Data and Statistical Analysis

In the study of in vivo chronotropic effect of extract, the data of the extract concen-tration in serum dialysate and the data of HR change are used. A non-linear regression of the data is carried out and adjusted to the equation:

Y = Emax [X / (IC50 + X)]

where Y is the HR change, expressed as percentage of basal value, Emax is the maximal re-sponse, IC50 is the extract concentration corresponding to half maximal response and X is the extract concentration in the serum dialysate. In the study of extract potency in isolated atria, the pEC50 and Emax values of the noradrenaline control curve and noradrenaline dose-response


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curve after adding the different concentrations of the extract are calculated using a non-linear regression algorithm. Then the data from the pEC50 versus extract concentration plot is ad-justed according to the following equation:34

pEC50 = – log (X + 10log Kb) – P

where pEC50 is the negative logarithm of EC50, X is the concentration of the extract, Kb is the constant of dissociation equilibrium of the extract and P is a constant. In order to determine if the estimation of log Kb may be quoted, the data of pEC50 versus the extract concentration was fitted simultaneously to the following equations:

pEC50 = – log (X + 10logKb) – P

pEC50 = – log (Xslope + 10logKb) – P and allowed the programme to decide, via the F-test, which one is the more appropriate equa-tion.

If the simpler equation is the better equation, then the logKb estimate may be quoted. This non-linear regression method for pKb determination has several advantages over the analysis of the affinity by Schild’s plot.34 Normal distribution of the data and the variables of the study were verified using the Kolmogorov–Smirnov test. Data was expressed as mean ± S.E.M. Statistical analysis was performed by Student’s t-test or by ANOVA and the Bonferroni test as a post hoc test. References 1 Wagner, H. and Wiesenauer, M., 1995, Phytotherapie. Phytopharmaka und Pflanzliche Homöopathika Gustav

Fischer Verlag, Stuttgart-Jena, pp. 35-92

2 National Heart, Lung and Blood Institute, National Institutes of Health, Home page. Available at: www.nhlbi.nih.gov

3 Harman, R. J. (Ed.). 1990, Handbook of Pharmacy Health-care: Diseases and Patient Advice, The Pharmaceuti-cal Press, London

4 Paakkari, I., 1996, Rytmihäiriölääkkeet, In: Koulu, M., Tuomisto, J. and Paasonen, M. K. (Eds.), Farmakologia ja toksikologia, Kustannusosakeyhtiö Medicina, Finland, pp. 531-548

5 Dorman, H. J. D., Peltoketo, A., Hiltunen, R. and Tikkanen, M. J., 2003, Characterisation of the antioxidant properties of de-odourised aqueous extracts from selected Lamiaceae herbs, Food Chemistry, 83, 255-262

6 Schulz, V., Hänsel, R. and Tyler, V. E., 2001, Rational Phytotherapy: A Physicians' Guide to Herbal Medicine (4th ed.), Springer Verlag, Berlin, pp. 128-148 & 158-168

7 Oken, B. S., Storzbach, D. M. and Kaye, J. A., 1998, The efficacy of Ginkgo biloba on cognitive function in Alz-heimer disease, Archives of Neurology, 55, 1409-1415

8 Anonymous, 1993, Research guidelines for evaluating the safety and efficacy of herbal medicines, World Health Organization, Regional Office for the Western Pacific, Manila, pp. 35-57

9 Barbaste, M., Berke, B., Dumas, M., Soulet, S., Delaunay, J. C., Castagnino, C., Arnaudinaud, V., Cheze, C. and Vercauteren, J., 2002, Dietary antioxidants, peroxidation and cardiovascular risks, Journal of Nutrition, Health and Aging, 6, 209-223

10 Fattman, C. L., Schaefer, L. M. and Oury, T. D., 2003, Extracellular superoxide dismutase in biology and medi-cine, Free Radical Biology and Medicine, 35, 236-256

11 Hu, F. B., 2003, Plant-based foods and prevention of cardiovascular disease: an overview, American Journal of Clinical Nutrition, 78, 544S-551S


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12 Keen, C. L., Holt, R. R., Polagruto, J. A., Wang, J. F. and Schmitz, H. H., 2003, Cocoa flavanols and cardiovascu-lar health, Phytochemistry Reviews, 1, 231-240

13 Touyz, R. M., 2003, Reactive oxygen species in vascular biology: role in arterial hypertension, Expert Review of Cardiovascular Therapy, 1, 91-106

14 Tracy, R. P., 2003, Inflammation, the metabolic syndrome and cardiovascular risk, International Journal of Clinical Practice, 10-17

15 Maxwell, S. R., 2000, Coronary artery disease--free radical damage, antioxidant protection and the role of homocysteine, Basic Research in Cardiology, 95 Suppl 1, I65-71

16 Podrez, E. A., Abu-Soud, H. M. and Hazen, S. L., 2000, Myeloperoxidase-generated oxidants and atherosclero-sis, Free Radical Biology and Medicine, 28, 1717-1725

17 Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C., 1999, Antioxidant activity apply-ing an improved ABTS radical cation decolorization assay, Free Radical Biology and Medicine, 26, 1231-1237

18 Busse, R. and Fleming, I., 1995, Regulation and functional consequences of endothelial nitric oxide formation, Annals of Medicine, 27, 331-340

19 Ignarro, L. J., 1990, Biosynthesis and metabolism of endothelium-derived nitric oxide, Annual Review of Phar-macology and Toxicology, 30, 535-560

20 Moncada, S. and Higgs, A., 1993, The L-arginine-nitric oxide pathway, New England Journal of Medicine, 329, 2002-2012

21 Forstermann, U. and Kleinert, H., 1995, Nitric oxide synthase: expression and expressional control of the three isoforms, Naunyn-Schmiedebergs Archives of Pharmacology, 352, 351-364

22 Schulz, K., Kerber, S. and Kelm, M., 1999, Reevaluation of the Griess method for determining NO/NO2- in aqueous and protein-containing samples, Nitric Oxide, 3, 225-234

23 Hong, Y., Hui, S. C., Chan, T. Y. and Hou, J. Y., 2002, Effect of berberine on regression of pressure-overload induced cardiac hypertrophy in rats, American Journal of Chinese Medicine, 30, 589-599

24 Dostal, D. E. and Baker, K. M., 1992, Angiotensin II stimulation of left ventricular hypertrophy in adult rat heart. Mediation by the AT1 receptor, Amercan Journal of Hypertension, 5, 276-280

25 Guo, W., Kamiya, K., Yasui, K., Kodama, I. and Toyama, J., 1998, alpha1-adrenoceptor agonists and IGF-1, myocardial hypertrophic factors, regulate the Kv1.5 K+ channel expression differentially in cultured newborn rat ventricular cells, Pflugers Archiv. European Journal of Physiology, 436, 26-32

26 Sugden, P. H. and Clerk, A., 1998, Cellular mechanisms of cardiac hypertrophy, Journal of Molecular Medicine, 76, 725-746

27 Studenik, C., Lemmens-Gruber, R. and Heistracher, P., 1999, Structure-activity relationships of new thienothi-azine derivatives in isolated heart and smooth muscle preparations of guinea pigs, General Pharmacology, 33, 319-324

28 Koch-Weser, J., 1963, Effect of rate changes on strength and time course of contraction of papillary muscle, American Journal of Physiology, 204, 451-457

29 Reiter, M., 1967, Die Wertbestimmung inotrop wirkender Arzneimittel am isolierten papillarmuskel, Arzneimit-tel Forschung, 17, 1249-1253

30 Hocht, C., Di Verniero, C., Opezzo, J. A. and Taira, C. A., 2003, In vivo and in vitro pharmacodynamic properties of metoprolol in aortic coarctated rats, Pharmacological Research, 47, 181-188

31 Rojo-Ortega, J. M. and Genest, J., 1968, A method for production of experimental hypertension in rats, Cana-dian Journal of Physiology and Pharmacology, 46, 883-885

32 Hocht, C., Opezzo, J. A., Gorzalczany, S. B., Priano, R. M., Bramuglia, G. F. and Taira, C. A., 2001, Pharmacoki-netic and pharmacodynamic alterations of methyldopa in rats with aortic coarctation. A study using microdialy-sis, Pharmacological Research, 44, 377-383

33 Opezzo, J. A., Hocht, C., Taira, C. A. and Bramuglia, G. F., 2001, Pharmacokinetic study of methyldopa in aorta-coarctated rats using a microdialysis technique, Pharmacological Research, 43, 155-159

34 Lew, M. J. and Angus, J. A., 1995, Analysis of competitive agonist-antagonist interactions by nonlinear regres-sion, Trends in Pharmacological Sciences, 16, 328-337


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9 Bioassay Models for Antidiabetic Activity of Natural

Products

M. Iqbal Choudhary and Shamsun Nahar Khan

9.1 Introduction

Diabetes mellitus or diabetes is a complex metabolic disorder and is of two major types: type 1 or insulin-dependent diabetes mellitus (IDDM) and type 2 or noninsulin-dependent diabetes mellitus (NIDDM). Over 90 % diabetic patients have type 2 diabetes. It is a chronic metabolic disorder and often identifies with different syndromes. 9.1.1 Pathophysiology and Syndrome of Type 2 Diabetes

Type 2 diabetes is characterized by unregulated serum glucose levels due to a combination of factors that may include: reduced sensitivity to insulin (associated with obesity); insufficient production of insulin (β-cell deficiency, that can be exacerbated by hyperglycaemia which is toxic to β-cells) and β-cell desensitization to glucose (glucose tolerance); persisting glycogenolysis and gluconeogenesis despite high serum levels of glucose and insulin; and high levels of dietary carbohydrate and sugar.

Type 2 diabetes is known to have a strong genetic component besides contributing environmental and life style determinants. Although the disease is genetically heterogeneous, there appears to be a fairly consistent phenotype once the disease is fully manifested. Whatever the pathogenic causes are, the early stage of type 2 diabetes is characterized by insulin resistance in insulin-targeting tissues, mainly the liver, skeletal muscles and adipocytes. Insulin resistance in these tissues is associated with excessive glucose production by the liver and impaired glucose utilization by peripheral tissues especially muscles. These events undermine the metabolic homeostasis, but may not directly lead to the occurrence of diabetes in the early stage.

Blood glucose levels can be maintained within the normal range with increased insulin secretion to compensate for insulin resistance, but the patient may demonstrate impaired response to prandial carbohydrate loading and to the oral glucose tolerance test. The chronic over-stimulation of insulin secretion gradually diminishes and eventually exhausts the islet β-cell reserve. A state of absolute insulin deficiency arrives and clinical diabetes becomes full blown.1-5

The pathogenicity of type 2 diabetes can also be influenced by ethnicity, degree of obesity, distribution of body fat, sedentary lifestyle, aging and other concomitant medical conditions.6 Type 2 diabetes represents a syndrome of disordered metabolism of carbohydrate and fat. The most prominent feature is hyperglycaemia (fasting plasma level over 126 mg/dL or glycosylated Hb [A.sub.1c] exceeding 6.9 %.

BIOASSAY MODELS FOR ANTIDIABETIC ACTIVITY OF NATURAL PRODUCTS

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In most patients with type 2 diabetes, the onset of disease is in adulthood and occurs most commonly in obese people of over 40 years of age. Hypertension, hyperlipidaemia and atherosclerosis are very often associated with diabetes. 9.1.2 Complications and Risk Factors in Diabetes

The most common acute complications of diabetes are metabolic problems e.g. hyperosmolar hyperglycaemic nonketotic syndrome (HHNS) and infections. The long-term complications can be divided into two categories – macro and microvascular complications. Common macrovascular complications are hypertension, dyslipidaemia, myocardial infarction and stroke.7 Microvascular complications include retinopathy, nephropathy, diabetic neuropathy, diarrhoea, neurogenic bladder, impaired cardiovascular reflexes, sexual dysfunction and diabetic foot disorder.8 Blindness, nerve damage and increased risk of ischemic heart disease and stroke are diabetes related risk factors. 9.1.3 Treatment of Type 2 Diabetes

The drugs available for NIDDM generally deal with insulin insufficiency and insulin resistance.9 Acarbose is a recently introduced α-glucosidase inhibitor that causes delayed absorption of complex dietary carbohydrate when taken with meals, decreasing postprandial rise in blood glucose. It is a competitive inhibitor of intestinal disaccharidases.11 Its effectiveness derives from spreading out the dietary challenge to insulin.

The sulfonylurea drugs have been the primary NIDDM drugs for four decades. They stimulate pancreatic phase I output of insulin. The mechanism of action is the same for all the sulfonylureas, although the potency of the second-generation drugs is better.12 The insulin stimulatory effect of the sulfonylureas has been shown both in vitro and in vivo13, 14 and stimulation of insulin secretion immediately takes care of one of the two deficits of NIDDM. Control of the elevated blood glucose with sulfonylurea in many patients may also increase the sensitivity of target tissues to insulin by removal of glucose toxicity.15

Metformin is a biguanide that has been used worldwide for the treatment of NIDDM since 1957, but it has only recently been approved for use in the United States. It does not stimulate insulin output.16, 17 Metformin decreases blood glucose in NIDDM in several ways. It delays glucose absorption and thereby decreases the intensity of the challenge to endogenous insulin. It sensitizes the peripheral tissues to glucose-induced glucose uptake, thereby reducing blood glucose and plasma lipids and decreasing hepatic glucose production, whose quantitative contribution to lowering of blood glucose is uncertain. Most recent clinical studies suggest that decreased hepatic glucose output is the dominant effect in humans (not the enhancement in peripheral glucose uptake).16

Natural products provide a rich source of new hypoglycaemic agents.18, 19 To date over 400 traditional plant treatments for diabetes have been reported but only a small number of these have been scientifically evaluated for efficacy and safety. The hypoglycaemic effect of some natural product extracts has been demonstrated in human and animal models of type 2 diabetes and the results were fairly promising - warranting further research in this field. The World Health Organization Expert Committee on Diabetes Mellitus has recommended that traditional medicinal herbs be further investigated for drug discovery.10

9.2 Bioassay Models

Development of new antidiabetic or antihyperglycaemic agent is one of the main areas of current drug discovery regime. There is no natural diabetic animal model. The


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bioassays mainly rely on induced diabetes in experimental animals or molecular targets. A number of bioassays have been developed to facilitate antidiabetic testing. These are broadly classified into two categories: (a) in vivo antidiabetic assays in experimental animals; (b) mechanism-based assays using purified enzymes involved in glucose absorption.

First category although relatively closer to the real life situation, suffers from the requirement of large test samples and extensive animal use. On the other hand, the second category is oversimplified and is a reductionist approach to a highly complicated disease. Apart from these bioassay models, several other tissue-based (β-islet) insulin release, antioxidant and antiglycation assays have also been developed, which are fairly complex, indirect and beyond the scope of this text. 9.2.1 Antidiabetic Assay in Rats or Mice

The method uses diabetes-induced rats or mice. Streptozotocin or alloxan is used to induce diabetes in the experimental animals.20 Glibenclamide can be used as a positive control. Blood levels of glucose are determined using standard glucose kit based on glucose oxidase method. Besides normal laboratory wares the assay methods require a centrifuge, micro haematocrit capillaries and spectrophotometer.

The adult rats weighing 150 to 220 g (Charles Foster albino or Wistar albino strain) or mice weighing 20 to 25 g (Wistar albino or Theiller original strain) of both sexes and the same age group are collected randomly from the animal house. The animals are acclimatized for 2 to 7 days in the experimental laboratory, temperature 22 ± 2ºC, relative humidity 60 to 70 % and light/dark cycle of 12 hours, each one beginning at 6 in the morning. Animal are allowed free access to food and water until 30 minutes before the start of the experiment. During the experiment the animals are fed on a fixed diet, which contains fixed calories and are allowed free access to water.

The animals are divided into four groups of 5 to 6 animals each. The first group serves as a normal control, the second as a diabetic control, the third as a test group and the fourth as a standard control. Normal control is a non-diabetic group. The diabetic control group, as for the next two groups, has induced diabetes and receives no treatment but only the vehicles. The test group is of diabetic rats receiving the test samples, and the standard control group of diabetic rats receive a standard drug for comparison.

The procedure involves some important steps of selection, sampling and acclimatization of animals, induction of diabetes, preparation and administration of test drug, monitoring disease-specific determinants such as body weight on a daily basis, method of collecting blood for glucose estimation and statistical analysis of the result. 9.2.2 Induction of Diabetes in Experimental Animals

Diabetes can be induced in the animals in several ways. Two chemical substances, streptozotocin and alloxan are most commonly used for experimental induction of diabetes.

9.2.2.1 Streptozotocin-induced Diabetes Model

Streptozotocin is a valuable agent for the experimental induction of diabetes and is less toxic than other chemical agents inducing diabetes. The diabetogenic effect of streptozotocin is the direct result of irreversible damage to pancreatic β-cells, allowing degranulation and loss of insulin secretion. The characteristic symptoms of streptozotocin-induced diabetes are hyperphagia, polydipsia leading to diuresis, hyperglycaemia, hyperlipidaemia, uremia and loss of body weight. 21, 22


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The streptozotocin-induced diabetic model is a reliable animal model of human insulin dependent diabetes mellitus (IDDM). A 5 % solution of streptozotocin is freshly prepared in 0.1 mM citrate buffer (pH 4.5) before use. Diabetes is induced in overnight fasted or 18 hour fasted rats by intravenous injection of streptozotocin 55 to 60 mg/kg of the body weight. After 48 hours of the administration of the streptozotocin, determine the blood glucose levels of surviving rats by glucose oxidase peroxidase method. Only those animals which show hyperglycaemia (blood glucose levels between 200 to 500 mg/dL) are considered diabetic and taken for further experimentation. 9.2.2.2 Alloxan-induced Diabetes Model

A single intraperitoneal injection of alloxan monohydrate at a dose of 150 to 175 mg/kg body weight induces diabetes in rats. It leads to an elevation of fasting blood glucose level, which is maintained over a period of three weeks and causes a significant reduction in body weight.23,24 Alloxan is first weighed individually according to the weight of each animal and solubilized in 0.2 ml sterile saline (154 mM NaCl) immediately prior to the injection. The alloxan solution is prepared freshly in normal saline immediately before use. It should be maintained in ice for any lapsing period.

After 48 to 72 hours of the administration of alloxan, animals with blood glucose levels between 140 to 350 mg/dL are selected for the experiment and treatment with the test sample is initiated. 9.2.3 Preparation and Administration of the Test Sample

The powdered plant material and different extracts can be administered to animals with the diet in a solid dosage form. It is mixed with 3 g of powdered food and moistened with water to prepare a food which is fed to the rats in the morning. Food trays are filled only after the drug-mixed ration has been fully consumed.

The extracts can also be given to animals as a liquid or suspension. The oily extracts can be administered as an emulsion. Suspensions are prepared using gum acacia as a suspending agent. The suspension of methanol and petroleum ether extracts can be prepared using 5 % Tween 80.25 All suspensions should be thoroughly mixed to ensure homogeneity prior to administration. The test samples are administered by means of a narrow tube attached to a syringe, which is inserted into the oesophagus of the animal.

The extract dissolved in the normal saline can be injected intraperitoneally in different doses.26 The control group of animals receives only the vehicle while the test group is given the drug. The rats are treated once a day for consecutive 21 days or twice a day for 28 days. 9.2.4 Evaluation of Antidiabetic Activity

The rats are weighed everyday throughout the study to observe the effect of test drug and streptozotocin or alloxan as diabetes causes a significant loss of body weight. The blood samples are drawn under anaesthesia to determine blood glucose levels. The animals are anaesthetized by keeping them in a diethyl ether saturated chamber for a few minutes or by intraperitoneal injection of pentobarbital, 50 mg/kg in normal saline, which is prepared in deionized water.


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Blood samples are collected at various time intervals, usually according to the design of the experiment. Normally fasting blood samples for estimation of glucose are collected on day 7 and 21 of treatment. Blood is drawn from any of the following sites: puncture tail vein, retro-orbital plexus puncture, retrobulbur venous plexus with capillary tubes, retro-orbitally from the inner canthus of the eye using capillary tubes (Micro Hematocrit Capillaries, Mucaps). The blood is collected in anticoagulant MEDTA or sodium fluoride and sodium oxalate. Plasma is separated by centrifuging at 200 rpm for 2 minutes. Serum can be used for measuring blood glucose levels. 9.2.4.1 Glucose Estimation (GOPOD Method)

Glucose can be estimated in the blood samples and reducing sugar can be estimated in the urine of the animals on a daily basis. Blood glucose level is estimated by commercially available glucose kits based on glucose oxidase peroxidase method (GOPOD method)27-29 or by hexokinase/glucose 6-P dehydrogenase (G6PDH) enzymic procedure. The reagents must be essentially devoid of starch and sucrose degrading enzymes. The colour formed is stable at room temperature for at least two �hours after the test. Reagents

Glucose reagent buffer (concentrate) contains 1M potassium dihydrogen orthophosphate, 200 mM p-hydroxybenzoic acid and 0.4 % sodium azide. Glucose determination reagent (per vial) contains glucose oxidase >12,000 U, peroxidase >650 U and 0.4 mM of 4-aminoantipyrine. The two solutions are stable for 12 months at 2 to 5°C or more than 12 months if stored at -20°C.

Standard solution of glucose contains 1.0 mg/ml of glucose and 0.2 % w/v of benzoic acid. Chromogen reagent (GOPOD) is prepared by diluting content of one bottle (50.0 ml) of glucose reagent buffer to one litre with distilled water. The glucose reagent buffer stored at -20°C crystallizes out. Ensure that all crystalline material dissolves in the final volume. GOPOD reagent in an amber storage bottle is stable for 3 months at 2 to 5°C and more than 12 months in frozen state. When freshly prepared it may be light yellow or light pink and develops a stronger pink colour within 2 to 3 months at 4°C. Its absorbance should be less than 0.05 when read against distilled water. Principle

Glucose oxidase Glucose + O2 + H2O Gluconate + H2O2

Peroxidase

2H2O2 + p-Hydroxybenzoic acid + 4 Aminoantipyrine Quinoneimine dye + 4 H2O Procedure

Take 0.1 ml of serum and add 3 ml of chromogen reagent and 0.1 ml of deionized water (test sample). For blank sample take 0.2 ml of deionized water and add 3 ml of chromogen reagent. For standard sample take 0.1 ml of standard solution of glucose and add 0.1 ml of deionized water and 3 ml of chromogen reagent. To plot standard curve, take 0.1 ml of different concentrations of glucose (e.g. 1, 2, 4 mM) and add 0.1 ml deionized water and 3 ml of chromogen reagent. Deionized water can be substituted with a glucose reagent buffer. Run all samples in duplicate. Incubate all samples at 50ºC for 20 minutes and measure the absorbance at 510 nm. Estimation of Blood Glucose

Calculate blood glucose values from the standard curve of glucose using the formula: x = (y-a)/b where x = blood glucose level, y = optical density of the sample, a = linear


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regression constant and b = linear regression coefficient. If one concentration of standard solution is used in place of a standard curve, use the following formula:

O.D. Sample x 100

Glucose, g/0.1 ml = O. D. Standard (glucose, 100 µg) Calculation of Percent Reduction in Glycaemia

The percent decrease in glycaemia in test sample as a function of time is calculated by applying the formula: percent decrease in glycaemia = G0-Gx×100 where, G0 is initial glycaemia, Gx is glycaemia at different time intervals of 30, 60, 90 and 120 minutes. 9.2.4.2 Estimation of Reducing Sugar in Urine

The presence of reducing sugars in the urine of the animals is tested using Benedict's solution on a daily basis and fasting blood glucose is estimated every 5 days by the O-toluidine colour reaction.22 9.2.5 Statistical Analysis

All values of body weight, fasting blood sugar and biochemical estimations are expressed as mean ± standard error mean (S.E.M.) and analyzed for ANOVA and post hoc Dunnet’s t-test. Differences between groups are considered significant at p<0.01 levels. 9.3 Alpha Glucosidase Inhibitors (AGIs)

Alpha glucosidase inhibition is one of the possible treatment options for type 2 diabetes. AGIs are given with meals and they function by slowing the breakdown of complex sugars into glucose. This causes a delay in glucose absorption and lowers blood sugar levels following meals. The AGIs may be used alone or in combination with other medications for diabetes. α-Glucosidases are important enzymes in the dietary carbohydrate post-translational processing of glycoproteins. Small intestinal α-glucosidases, namely, maltase, hydrolyzes maltose to glucose which is absorbed through the gut wall. Given that absorption of glucose elevates the blood glucose level to cause postprandial hyperglycaemia, it is necessary for diabetic patients to decrease the hydrolytic rate of maltose.30 α-Glucosidase belongs to the class 3-hydrolases enzymes. It catalyses the selective hydrolysis of glycosidic bonds in oligosaccharides. α-Glucosidase hydrolyse both (1,4) and (1,6) linkages. This group of enzymes can be considered as a special class of the transferases in which the donor group is transferred to water.

Starch maltose + maltotriose + α−limit dextrins glucose absorption maltase α−glucosidase

Many α-glucosidases (E.C. 3.2.1.20) have been reported from micro-organisms, animal tissues, and plants. α-Glucosidases are conventionally classified into three types (I, II and III) on the basis of their substrate specificities.31 Type I includes maltase and isomaltase from Saccharomyces species and hydrolyze aryl glucosides such as phenyl glucopyranoside and p-nitrophenyl α-glucopyranoside (p-NPG) more rapidly than maltooligosaccharides such as maltose and isomaltose. Type II comprises α-glucosidase from Aspergillus niger and Saccharomyces logos which hydrolyses the maltooligosccharides. Type III includes α-


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glucosidases from pig serum, buckwheat, sugar beet and rice which hydrolyze α-glucan as well as maltooligosaccharides.

The assay based on mechanism-based inhibition of enzyme α-glucosidase of Saccharomyces origin is used. Inhibitory potential of any test compound against α-glucosidase indicates its ability to delay the hydrolysis of oligosaccharides and its potential in decreasing the rise in glucose levels after meals. This is a high throughput spectrophotometric assay which requires a very small quantity of test sample according to the molecular weight, ranging from 0.2 to 2 mg.

The assay requires an enzyme, substrate, pH meter, UV/VIS spectrophotometer, eppendorf tubes, 96-well microplates, microplate reader, multichannel and single channel micropipette, sonicator, centrifuge besides test samples and normal lab chemicals and wares. 9.3.1 Method of Assessment of the α-Glucosidase Inhibitory Activity

p-Nitrophenyl-α-D-glucopyranoside (p-NPG) is used as a substrate for the enzyme as

it has specificity towards the enzyme. The enzyme hydrolyzes p-NPG and yields D-glucose and p-nitrophenol, which is a yellow chromogenic product, detected by spectrophotometric analysis.32 Preparation of Test Sample

The test samples in various concentrations are prepared in DMSO, which does not show any inhibition of the enzyme. Blank Control

The blank control includes wells without the test sample and standard inhibitors. The reaction between the enzyme and substrate occurs without any hindrance. Positive Control

Acarbose or deoxynojirimycin is generally used as a positive control in the experiment. The positive control is prepared in the concentration of their IC50 values. It is run in triplicate for every set of experiment. Procedure

The assay can be performed in two ways: (a) continuous assays; (b) discontinuous assays. In the continuous assays, the enzymatic reaction is monitored continuously by observing the progress of the curves. Discontinuous assays use the extent of reaction after a chosen fixed time. It attempts to select a reaction time and assumes that the initial rate of reaction and conditions hold good over the assay time.33 Continuous Assay (modified by authors)

Dispense 140 µL of 50 mM phosphate buffer containing 100 mM sodium chloride in the 96-well microplate. Add 15 µl of the test sample prepared in DMSO to the wells followed by 20 µl of α-glucosidase (500 mU/ml) prepared in the phosphate saline buffer. Incubate the plates with enzyme and test sample for 15 minutes at 37°C.34 Pre read the plates before adding the substrate. After incubation add 25 µl of 0.7 mM substrate, p-nitrophenyl α-D-glucopyranoside to the wells. Continuously monitor the increase in absorption for 30 minutes at 400 nm due to hydrolysis of p-NPG by α-glucosidase using spectrophotometer. Calculate the inhibition of the test compound as percent inhibition. Discontinuous Method

Prepare 10 mM sodium phosphate buffer. Use p-nitrophenyl α-D-glucopyranoside (p-NPG) in final concentration of 80 µM and α-glucosidase (of Saccharomyces origin) in the final


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concentration of 25 mU/ml. Prepare various concentrations of test samples in 10 mM phosphate buffer, pH 7.28, containing 1 % v/v DMSO. Incubate the enzyme with p-NPG, in the above mentioned concentrations, in the presence or absence of various test samples in the reaction mixture at 37°C for exactly 10 minutes. After incubation add an equal volume of stop solution of 0.5 M sodium carbonate. Measure the hydrolyzed amount of substrate in terms of increased absorbance at 400 nm. Calculation of Percent Inhibition

The percent inhibition of a test sample against the enzyme can be calculated by using the following formula:

Percent inhibition = [Absorbance (control) - Absorbance (test sample)] x100 Absorbance (Control) Calculation of the IC50 Values

The IC50 values are calculated from the dose response curves. For the calculation, EZFIT a software program can be used which requires the data on the concentration and percent inhibition of the sample as input and provides the IC50 values.

Miglitol35,36 and acarbose are the currently available AGIs. Miglitol is indicated for

combination therapy with sulfonylureas, while acarbose may be used with sulfonylurea, metformin or insulin. References 1 DeFronzo, R. A., 1999, Pharmacologic therapy for type 2 diabetes mellitus, Annals of Internal Medicine, 131,

281-303

2 Seely, B. L. and Olefsky, J. M., 1993, Potential cellular and genetic mechanisms for insulin resistance in common disorders of obesity and diabetes, In: Moller, D. (Ed.), Insulin Resistance and its Clinical Disorders, John Wiley & Sons, London 187-252

3 Olefsky, J. M., 1999, Insulin resistance and pathogenesis of non-insulin dependent diabetes mellitus: cellular and molecular mechanisms, In: Efendic, S., Ostenson, C. G. and Vranic, M. (Eds.), New Concepts in the Pathogenesis of NIDDM, Plenum Publishing Corporation, New York

4 Bell, D. S., 2001, Importance of postprandial glucose control, Southern Medical Journal, 94, 804-809

5 Vranic, C. G. M. (Ed.). 1999, New Concepts in the Pathogenesis of NIDDM, Plenum Publishing Corporation, New York

6 Clark, C. M. J., 1998, The burden of chronic hyperglycaemia, Diabetes Care, 21, C32-C34

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