The efficacy of Arnica montana 30CH and 200CH to thrombolise a

blood clot in an in-vitro sample.

A dissertation submitted to the Faculty of Health Sciences at the Technikon Witwatersrand for the partial fulfilment Degree of Master of Technology in the program Homoeopathy by

Jarne Stefan van Tonder (Student number: 9476443)

Supervisor: ______Date ______

Dr. N. Wolf

Co-Supervisor: ______Date ______

Dr. K.E. Sinclaire

Johannesburg, 2005

DECLARATION I declare that this dissertation is my own, unaided work. It is being submitted for the Degree of Master of Technology in Homoeopathy at the Technikon Witwatersrand, Johannesburg. It has not been submitted before for any other degree or examination in any other Technikon or University.

______

Jarne Stefan van Tonder ____ Day of ______

ii ABSTRACT

Coagulation or the formation of blood clots is the result of several complex interactions between humoral coagulation factors, platelets, and fibrin. Unnatural or excessive coagulation is inhibited by the fibrinolytic system. In normal homeostasis, there is a dynamic state in which thrombi are constantly being formed and removed from the circulatory system. Fibrin being the main component of a blood clot is formed by the activation of the clotting cascade. Its production is followed by the fibrinolytic system, resulting in plasmin generation and subsequent fibrin degradation (Soria et al., 1983). Plasmin is the enzyme responsible for fibrin degradation. It is derived from its inactive precursor, plasminogen, by the action of thrombin and plasminogen activators.

Homoeopathic Arnica montana is prescribed for pathological conditions that have a sudden onset, are traumatic in nature, and result from the complications of the initial trauma (Vermeulen, 1997). It has been prescribed for various thrombotic disorders and it is known that it speeds up healing and revascularisation of the surrounding tissue (Savage and Roe, 1977). It is a short acting homoeopathic medication, and needs to be repeated often, but it is quick in its actions (Gordon Ross, 1977).

This study aimed to establish if Arnica montana 30CH and 200CH potencies caused thrombolysis of a blood clot within an in-vitro sample. It was hypothesised that Arnica montana in 30CH and 200CH potencies would cause thrombolysis in- vitro.

The research sample group consisted of fifteen male participants, between eighteen and thirty years of age. Only male participants were selected to prevent any gender variables that may influence the study. Four blood samples each consisting of five milliliters was taken from each participant. Current and efficient phlebotomy techniques were used and

iii the samples were placed in non-treated plastic phlebotomy containers to allow speedy clot formation. One sample was treated with one drop of 30CH Arnica montana and the other with one drop of 200CH Arnica montana. The third sample for each subject was used as a control where one drop of 0.9% sterile saline was added. Thus, consistency concerning diluting effects was maintained. D-dimer levels were measured using the D- DI Test from Diagnostica Stago, which is a rapid latex agglutination slide test. A semi- quantitative testing mode was used to gather the relevant research information.

The results of the study showed that in all the samples tested, no agglutination of D- dimers occurred. This indicated that if thrombolysis occurred in the samples, a D-dimer level well below 0.5 micrograms per milliliter of FEU occurred.

It has been established that as separate entities homoeopathic Arnica montana 30CH and 200CH have little or no direct effect on a clotted in-vitro sample. This indicates that if the medicine has thrombolytic properties, it has to utilize or activate other endogenous factors in-vivo. As these factors require the presence of vascular endothelium, it makes it potentially difficult to conduct studies due to ethical reasons.

iv DEDICATION

This dissertation is dedicated to all my loved ones that motivated and supported me through this process. To my Angel, that guided and supported me in times of need and despair.

v ACKNOLEDGEMENTS

I would like to express my sincere gratitude to the following individuals: Dr. N. Wolf: Supervisor Dr. K. E. Sinclaire: Co-supervisor Dr. G. Ferguson and W. Last Homoeopathics for the preparation and donation of medicine Ms. E. Repenseck and Ampath Laboratories

vi TABLE OF CONTENTS

DECLARATION ii

ABSTRACT iii

DEDICATION v

ACKNOLEDGEMENT vi

TABLE OF CONTENTS vii

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF GRAPHS xiii

1. INTRODUCTION 1 1.1. Problem statement 1 1.2. The aim of the study 1 1.2.1. Objectives 1 1.2.2. Outcomes 2 1.3. Importance of the study 2 1.4. Hypothesis 2 1.5. Null-hypothesis 3 1.6. Possible methodological limitations 3

vii 2. LITERATURE REVIEW 4 2.1. Introduction 4 2.2. Blood and its functions 4 2.2.1. Introduction 5 2.2.2. Blood functions 5 2.2.3. Plasma constituents 6 2.2.4. Plasma proteins 7 2.3. Coagulation 7 2.3.1. Haemostasis 8 2.3.2. The dynamics of haemostasis 8 2.3.3. Blood vessels 9 2.3.4. The platelet plug 11 2.3.5. The coagulation pathways 13 2.3.5.1.The intrinsic pathway 14 2.3.5.2.The extrinsic pathway 16 2.3.5.3.The common pathway 17 2.4. Fibrinolysis 20 2.4.1. Fibrinogen to fibrin 21 2.4.2. Plasminogen to plasmin 21 2.5. The immune systems inflammatory response during blood coagulation 22 2.6. Coagulopathies 23 2.7. Homoeopathy 25 2.7.1. Principles of homoeopathy 25 2.7.1.1.The vital force 25 2.7.1.2.The law of similars 26 2.7.1.3.Minimum dose 26

viii 2.7.1.4.Homoeopathic potencies 27 2.7.2. The science behind homoeopathy 28 2.7.2.1.The cluster theory 29 2.7.2.2.Homoeopathic human studies: The proving 30 2.7.2.3.The coherent frequencies in living systems and homoeopathic medication. 31 2.7.2.4.The chaos theory and homoeopathy 31 2.8. Arnica montana 32 2.8.1. Classification 32 2.8.2. Description 33 2.8.3. Origin of the herb 34 2.8.4. Parts of the herb used 34 2.8.5. Herbal therapeutic category 34 2.8.6. Uses and properties 35 2.8.7. Active ingredients 35 2.8.8. Pharmacological effects 35 2.9. Homoeopathic Arnica montana 36 2.9.1. The prescription of Arnica montana 36 2.9.2. Indications for homoeopathic Arnica montana 37 2.9.2.1.Angina 37 2.9.2.2.Bruises and blunt trauma 37 3. METHODOLOGY 39 3.1. Sample group 39 3.1.1. Inclusion criteria 39 3.1.2. Exclusion criteria 39 3.2. Research procedure 39

ix 3.3. Analysis of data 42 4. RESULTS 43 4.1. Control Sample 44 4.2. Arnica montana 30CH Sample 45 4.3. Arnica montana 200CH Sample 46 5. DISCUSSION 47 5.1. Control samples 47 5.2. Arnica montana 30CH samples 47 5.3. Arnica montana 200CH samples 48 5.4. The importance of this study 49 6. CONCLUSION AND RECOMMENDATIONS 50 6.1. Conclusion 50 6.2. Recommendations 51 7. REFERENCES 53 8. APPENDICES 8.1. APPENDIX A 59 8.2. APPENDIX B 63 8.3. APPENDIX C 65

x LIST OF TABLES

Table 2.1: The functions of blood 5 Table 2.2: The molecular weights of plasma proteins 6 Table 2.3: Coagulation factors 15-16

xi LIST OF FIGURES

Figure 2.1 33

Figure 2.2 34

xii LIST OF GRAPHS

Graph 4.1: Control samples 44 Graph 4.2: Arnica montana 30CH samples 45 Graph 4.3: Arnica montana 200CH samples 46

xiii CHAPTER 1

INTRODUCTION

1.1 Problem statement

Homoeopathic Arnica montana is prescribed for pathological conditions that have a sudden onset, are traumatic in nature, and result from the complications of the initial trauma (Vermeulen, 1997). It has been prescribed for various thrombotic disorders, and it is known that it speeds up healing and revascularisation of the surrounding tissue (Savage and Roe, 1977). Very little is known of the actual homoeopathic pathophysiological pathways involved in resulting in cure in patients. No study has been published either locally or internationally that attempts to establish the thrombolytic properties of Arnica montana 30CH and 200CH.

1.2 The aim of the study

The aim of the study is to establish the efficacy of homeopathic Arnica montana in 30CH (Centesimal Hahnemanian) and 200CH to thrombolise a blood clot in an in- vitro sample.

1.2.1 Objectives

The study aims to determine if Arnica montana 3OCH and 200CH acts directly on the available plasma plasminogen in-vitro, resulting in thrombolysis. This study will asses this by measuring the D-dimer concentration available post treatment of in-vitro blood samples with Arnica montana 30CH and 200CH.

1 1.2.2 Outcomes

• There may be a change in D-dimer level of the samples medicated with Arnica montana 30CH and 200CH. This could either indicate that the Arnica montana acted on the available plasminogen to convert it to plasmin directly or by activating tissue plasminogen activator (tPA) to cause fibrinolysis.

• No change may occur to the D-dimer levels.

1.3 Importance of the study

The study will enhance our understanding of the action of homoeopathic Arnica montana 30CH and 200CH, and the pathophysiological processes involved in thrombolysis. In creating this study, a new methodology has been designed to isolate the factors that influence thrombolysis in-vitro. This will ascertain if homoeopathic Arnica montana acts directly on the fibrin structure or indirectly by activating the plasminogen in-vitro. The variables obtained through the study will also provide other potential researchers with insight and understanding into the topic of homoeopathic thrombolysis. Potential areas of research and study regarding this topic will be opened.

1.4 Hypothesis

Arnica montana 30CH and 200CH will cause thrombolysis of a clot in-vitro, by either acting on fibrin directly, or by converting plasminogen into plasmin.

2 1.5 Null-hypothesis

Arnica montana 30CH and 200CH will not cause thrombolysis of a clot in-vitro, and therefore will not significantly change the D-dimer concentration of the in-vitro samples.

1.6 Possible methodological limitations

Using the methodology for this study, no measurable levels of D-dimers may be produced. This does not exclude that no D-dimers were formed in-vitro; it merely indicates that no D-dimer levels could be measured above 0.5 micrograms per millilitre of FEU.

3 CHAPTER 2

LITERATURE REVIEW

2.1 Introduction

The following chapter explains the different aspects of coagulation, fibrinolysis, and the potential mechanisms involved in the thrombolysis of blood clots with the use of Arnica montana 30CH and 200CH. The basic principles of homoeopathy in this study explained. Arnica montana in both herbal and homoeopathic forms will be discussed demonstrating the rationale for using it in thrombolysis.

2.2 Blood and its functions

2.2.1 Introduction

Blood circulates in a virtually closed system within blood vessels. Blood consists of red blood cells, white blood cells and platelets, suspended in a liquid medium, namely the plasma. Blood and plasma in particular perform many functions critical for the maintenance of life. Once, the blood has coagulated, the remaining liquid phase is termed serum. Serum lacks the clotting factors (including fibrinogen) that are normally present in plasma, which have been consumed during the process of coagulation. Serum does contain some degradation products of clotting factors — products that have been generated during the coagulation phase and are thus not present in normal plasma. Fibrinolytic enzymes are however present in this solution although in an inactive state (Murray and Harenist, 1990).

4 2.2.2 Blood functions

The functions of blood (Table 2.1), all apart from specific cellular ones such as oxygen transport and cell mediated immunity, are carried out by plasma and its constituents. Plasma consists of water, electrolytes, metabolites, nutrients, proteins and hormones. The water and electrolyte composition of plasma is practically the same as that of all extra-cellular fluids (Murray and Harenist, 1990).

Table 2.1: The functions of blood (Murray and Harenist, 1990).

Respiration Transport of oxygen and carbon dioxide from the tissue to the lungs.

Nutrition Transport of absorbed food materials.

Transport of metabolic waste to the kidneys, lungs, skin, and intestines Excretion for removal.

Acid-base Maintenance of normal acid base balance in the body. balance

Regulation of water balance in the body through the effects of blood on Water balance the exchange of water between circulating fluid and tissue fluid.

Body The regulation of body temperature and the distribution of body heat. temperature

Defense against infection by the white blood cells and circulating Defense antibodies.

Hormones Transport of hormones and the regulation of metabolism.

Metabolites Transport of metabolites.

Coagulation Facilitates blood coagulation

5 2.2.3 Plasma constituents

The concentration of total protein in plasma is approximately 7-7.5 g/dL consisting mainly of solid plasma proteins. These plasma proteins form a very complex mixture consisting of both simple proteins and conjugated proteins (glycoproteins and various types of lipoproteins). Table 2.2 gives a comprehensive breakdown of the molecular weights of the individual plasma proteins (Murray and Harenist, 1990).

Table 2.2: The molecular weights of plasma proteins (Murray and Harenist, 1990).

Albumin 69,000 Daltons Haemoglobin 64,450 Daltons ß1 globulin 90,000 Daltons γ globulin 156,000 Daltons α1 lipoprotein 200,000 Daltons ß1 lipoprotein 130,000 Daltons Fibrinogen 340,000 Daltons Factor IX 55,000 Daltons Factor X 56,000 Daltons Factor VIII 330,000 Daltons Factor VII 53,000 Daltons Factor V 330,000 Daltons Prothrombin 72,000 Daltons Thrombin 34,000 Daltons Plasminogen 90,000 Daltons

6 2.2.4 Plasma proteins

Plasma proteins can be separated into three major groups; fibrinogen, albumen and globulins. The concentration of these proteins in plasma is important in determining the distribution of fluid between the blood and tissue. Blood plasma by definition is an intravascular fluid. Although the liver synthesizes most of the plasma proteins, some are synthesized by the blood vessel endothelium (Murray and Harenist, 1990).

Plasma proteins are generally synthesized on membrane bound polyribosomes. Thus, most are synthesized as pre-proteins and initially contain N-terminal signal peptides. They are usually subjected to various post-translational modifications (proteolysis, glycosalysion, and phosphorylation) as they travel through the cell. Transit times through the hepatocytes from the site of synthesis to the plasma vary for individual proteins from thirty minutes to several hours or more. Almost all plasma proteins are glycoproteins. They contain sugar residues, either N or O-linked oligosaccharide chains or both. Albumin, the major protein present in plasma, is the exception in that it does not contain any sugar residues (Murray and Harenist, 1990).

The levels of circulating plasma proteins increase during acute inflammatory states and certain types of tissue damage. These proteins are called acute phase proteins and include C-reactive protein (CRP) and fibrinogen (Murray and Harenist, 1990).

2.3 Coagulation

Coagulation or the formation of blood clots is the result of several complex interactions between humoral coagulation factors, platelets, and fibrin. Unnatural or excessive

7 coagulation is inhibited by the fibrinolytic system; here coagulation is counterbalanced to protect the organism from widespread coagulopathy.

Worldwide, arterial and venous thromboses are leading causes of patient morbidity and mortality. Pathological thromboemboli result in disorders such as; myocardial infarction, stroke, and pulmonary embolism (Djulbegovic, 1992).

2.3.1 Haemostasis

Haemostasis is defined as the impedance of bleeding from an injured blood vessel. This is achieved through the combined activity of vascular, platelet, and plasma factors. This process is maintained through a feedback mechanism, maintaining a balanced state in the vascular system. The feedback mechanism prevents the pathological accumulation of platelets and fibrin in the injured area (Berkow, Fletcher, and Beers, 1992).

2.3.2 The dynamics of haemostasis

In normal haemostasis, there is a dynamic state in which thrombi are constantly being formed and removed from the circulatory system. This haemostatic system consists of four dynamic phases. The first phase consists of the vasoconstriction of the injured blood vessel. In an attempt to decrease blood loss, the flow to the injury site is restricted. The second phase consists of the formation of a temporary platelet plug at the site of vessel injury. It consists of platelet adhesion, activation, and the aggregation of circulating platelets. The tertiary phase is comprised of the formation of a fibrin mesh or thrombus. This mesh entraps the platelet plug, allowing for the formation of both white and red thrombi. The mesh also serves as a trap for other plasma compounds increasing both thrombus size and density. The final and fourth phase consists of the partial or the

8 complete dissolution of the blood clot by plasmin. This is known as the fibrinolytic phase and is essential for the resolution of formed thrombi (Murray and Harenist, 1990).

2.3.3 Blood vessels

Immediately after a blood vessel is ruptured or cut, local humoral factors reduce the blood flow from the traumatized area. This is achieved by local vasoconstriction and compression of the injured blood vessel via extravasated blood in the interstitial tissue (Berkow et al., 1992). The vasoconstriction results from sympathetic nervous stimulation, local myogenic vasospasm, local humoral factors released from the traumatised tissues, and the platelets active in coagulation (Guyton and Hall, 1996).

The greatest amount of vasoconstriction results from a local myogenic contraction of the smooth muscular wall of the blood vessel. This is initiated by direct trauma to the vascular wall destroying its integrity (Guyton and Hall, 1996).

For smaller blood vessels, the platelets are responsible to a greater extent for most of the vasoconstriction by releasing thromboxane A2, a vasoconstrictor enzyme. The more the vessel is traumatized the greater the degree of spasm. This local vascular spasm usually lasts for about thirty minutes, during which platelet plugging and blood coagulation can occur. The blood vessel endothelial cells contract to expose the underlying basement membrane to the blood stream. These endothelial cells then secrete local factors and hormones (adenosine diphosphate, tissue factor, and prostacyclin). These endothelins and peptide hormones released stimulate smooth muscle contraction and promote local vascular spasm. Proliferation of endothelial cells, smooth muscle fibers, and fibroblasts

9 occurs rapidly at this injury site. This accelerates the vascular repair process (Guyton and Hall, 1996).

The endothelial cell membranes become adherent. In small capillaries, the endothelial cell membranes on opposing sides of the capillary may adhere and close off the blood vessel (Martini, Ober, Garrison, Welch, and Hutchings, 1995).

Blood vessels produce prostacyclins and other compounds that affect clotting. The endothelial cells in the blood vessel walls regulate clotting, and the synthesis of prostacyclins, specifically PGI2. PGI2 is a potent inhibitor of platelet aggregation. These oppose the actions of the thromboxanes, preventing further clotting (Murray and Harenist, 1990).

Endothelial cells play other roles in the regulation of thrombosis. For instance, these cells metabolise adenosine diphosphate (ADP), thus opposing its aggregating effect on the platelets. In addition, these cells appear to synthesise heparin sulphate and plasminogen activators, which help bind some clotting factors and dissolve formed blood clots (Murray and Harenist, 1990).

Haemostatic plugs consist mainly of adherent platelets accumulated at the site of vessel wall injury. Platelets also release factors to augment vasoconstriction (e.g. serotonin, thromboxane A2) and initiate vessel wall repair (platelet derived growth enzyme or cofactor complexes) (Murray and Harenist, 1990).

10 2.3.4 The platelet plug

Platelets are anuclear cells that originate from the cytoplasm of megakaryocytes. These cells circulate in-vivo within blood as lens-shaped pieces of membrane-bound cytoplasm (Gresele, Page, and Fuster, 2002). These platelets continually monitor the integrity of the blood vessel endothelium, thereby playing a key role in the promotion of vascular repair and the formation of arterial thrombi (Patel and Moliterno, 2000).

Trauma exposes the underlying vessel endothelial matrix to circulating blood, resulting in platelet plug formation (Fuster, Badimon, Badimon, and Chesebro, 1992). This process in thrombus formation follows a chain of events. It begins with the injury to the vessel wall, followed by platelet adhesion, activation and finally aggregation of circulating platelets (Wiggins, Wittkowsky, and Nappi, 2001, Fuster et al., 1992).

Platelet adhesion is the next step in haemostatic plug formation. Under normal conditions, the circulating platelets do not adhere to the vascular endothelium or to one another. Adhesion only occurs once damage has occurred to the blood vessel endothelial wall, exposing the sub-endothelium, containing collagen fibres. Adhesion requires the participation of a protein called Von Willebrand’s factor. Damaged endothelial cells secrete this factor. Von Willebrand’s factor lines the vessel endothelium like a blanket (Tsikouris and Tsikouris, 2001), acting as a ligand in an attempt to seal the injured vessel site during platelet adhesion, via a glycoprotein receptor (GP Ib) on the platelet surface membrane (Wiggins et al., 2001).

Over ninety stimuli of platelet aggregation have been identified. The most significant contributors include: collagen, thrombin, nor-adrenalin, thromboxane A2, serotonin, and

11 adenosine diphosphate (ADP) (Wiggins et al., 2001; Dobesh and Latham, 1998; Chong, 1998). These platelet aggregators induce changes of the platelet receptor glycoprotein (GP) IIb - IIIa (Tsikouris and Tsikouris, 2001).

Collagen and the primary thrombin formed at the site of injury, in turn activate the surrounding platelets. These reactions activate phospholipase C, an enzyme that hydrolyses specific membrane phospholipids called inositiol phospholipids. A product of this reaction activates protein kinase C and increases the calcium concentration within the platelet cytosol (Wiggins et al., 2001).

A series of progressive overlapping steps occur in platelets during thrombus formation:

• The platelets change structure and develop pseudopods. • From circulating GP IIb and GP IIIa, glycoprotein receptors are assembled on the platelet’s surface membrane. • Fibrinogen and other circulating proteins bind to these receptors, resulting in the adhesion of the available platelets to one another. • Arachodonic acid is released from the platelet cell membrane, where it is oxidized to prostaglandin 112 (an important co-factor for collagen induced platelet activation), and thromboxane A2. • The platelet contents are secreted into the blood plasma. • ADP also activates platelets and recruits new platelets into the growing haemostatic plug. • The platelet plasma membrane contains phospholipids necessary for the formation of enzyme-cofactor complexes. Secretion of platelet factor V from platelet alpha granules provides another key component for one of these enzyme-

12 cofactor complexes. It increases the amount of thrombin that is generated to clot surrounding fibrinogen, with the formation of fibrin strands. These strands then radiate outward from aggregated platelets, thus securing the plug in place. • Platelet aggregation provides the surface for consolidation of fibrin on the platelet plug. This process lays the foundation for the coagulation cascade (Chong 1998, Fuster et al., 1992).

Platelets also play a fundamental role in many non-cardiovascular disorders. Abnormalities in platelet function have been described in infections, malignancies, and psychological disorders. Increased platelet reactivity has been observed in depressed and hostile subjects (Daichi and Pickering, 2004).

2.3.5 The coagulation pathways

Both the intrinsic and extrinsic coagulation pathways result in the formation of fibrin. The initiation of thrombus formation in a blood vessel with restricted blood flow due to arteriosclerosis or scarring, without direct endothelial injury occurs via the intrinsic pathway. Formation of a fibrin clot in response to tissue injury occurs through the extrinsic coagulation pathway. These pathways converge into a final common pathway involving the activation of prothrombin to thrombin as well as the thrombin catalysed cleavage of fibrinogen to fibrin blood clot. The coagulation pathways consist of the conversion of inactive proteins into active enzymes by specific activators (Murray and Harenist, 1990).

13 Coagulation proteins are categorised into four types:

• Zymogens derived from serine dependant proteases. These activate during the coagulation process. • Cofactors • Fibrinogen • Transglutaminase, acts by stabilising the fibrin clot (Murray and Harenist, 1990).

2.3.5.1 The intrinsic pathway

The intrinsic pathway leads to the activation of Factor X. It involves factors XII, XI, IX, VIII and X as well as pre-kallikrein, high molecular weight kinogen, calcium, and platelet phospholipids (Murray and Harenist, 1990).

This pathway commences with the “contact phase” in which pre-kallikrein, factor XII, and factor XI are exposed to the negatively charged blood vessel surface. Collagen on the exposed surface of the blood vessel provides the coagulation site in-vivo, whereas glass or kaolin surfaces are used for in-vitro coagulation (Murray and Harenist, 1990).

When the components of the contact phase assemble on the activated endothelial surface, factor XII is converted to factor XIIa through proteolysis by kallikrein. Factor XIIa continues to act on pre-kallikrein to produce more kallikrein, setting up further activation. Factor XIIa once formed, activates factor XI to XIa, and releases bradykinin. Bradykinin is an inflammatory mediator acting as a potent vasodilator. Factor XIa in conjunction with calcium acts on factor IX to form factor IXa. This in turn cleaves an

14 Arg-Il bond in factor X to produce the 2-chain serine protease, factor Xa. The former reaction requires the formation of a tenase complex on the surface of the activated platelets. This consists of: calcium, factor VIIa, as well as factors IXa and X. The assembly of the tenase complex requires platelets to be activated and expose their acidic (anionic) phospholipids; phosphatidyl serine and phosphatidyl inositiol. Within inactive platelets these phospholipids are usually located on the internal surface of the plasma membrane (Murray and Harenist, 1990).

Factor VIII a glycoprotein is not a protease precursor, but a cofactor that serves as a receptor for factors IXa and X on the platelet surface membrane. Minute quantities of thrombin activate factor VIII to form factor Villa, which in turn inactivates thrombin (Murray and Harenist, 1990).

Table 2.3 discusses the various coagulation factors and their common names they tend to be referred to.

Table 2.3: Coagulation factors (Murray and Harenist, 1990).

Factor Common names

I Fibrinogen

II Prothrombin

III Platelet phospholipids

15 IV Calcium

V Proaccelerin

Proconvertin, serum prothrombin conversion accelerator (SPCA), co- VII thromboplastin

VIII Antihaemophiliac factor A, antihaemophiliac globulin (AHG)

Antihaemophiliac factor B, Christmas factor, plasma thromboplastin IX component (PTC)

X Stuart-Prower factor

XI Plasma thromboplastin antecedent

XII Hageman factor

XIII Fibrin stabilising factor (FSF), fibrinoligase

2.3.5.2 The extrinsic pathway

The extrinsic pathway also leads to the activation of factor X, but through a different mechanism. Factor Xa is formed where the intrinsic and extrinsic pathways converge, leading to the final, common pathway of coagulation. The extrinsic pathway involves tissue factor, factor VII, factor X, and calcium, resulting in the production of factor Xa. This is initiated at the site of tissue injury with the release of tissue factor (abundant in the placenta, lung and brain tissues), which acts as a cofactor in the factor Vila-catalysed activation of factor X. (Murray and Harenist, 1990).

16 Factor Vila cleaves the same Arg-Il bond in factor X that is cleaved by the tenase complex of the intrinsic pathway. Factor VII, a circulating glycoprotein (GP Ia) is synthesised in the liver. Its activity is increased by its conversion to factor Vila, via thrombin (factor Xa). The activation of factor X provides an important link between both the intrinsic and extrinsic pathways (Murray and Harenist, 1990).

2.3.5.3 The common pathway

The final common pathway consists of the conversion of prothrombin to thrombin. Factor Xa is produced via the intrinsic or extrinsic coagulation pathways. It converts prothrombin (factor II) to thrombin (factor ha), which in turn converts fibrinogen to fibrin. The activation of prothrombin, like that of factor X occurs on the surface of activated platelets, and requires the assembly of a prothrombinase complex, consisting of platelet anionic phospholipids, calcium, factor Va, factor Xa, and prothrombin. Factor V, a glycoprotein similar to factor VIII and ceruloplasmin, is synthesised in the liver, spleen, and kidneys. It is found on the platelet surface as well as in the blood plasma. It functions as a cofactor in a manner similar to factor Viii in the tenase of thrombin. It binds to specific receptors on the platelet cell membranes, and forms a complex with factor Xa and prothrombin. In turn thrombin inactivates factor V, thereby limiting the conversion of prothrombin to thrombin (Murray and Harenist, 1990).

Prothrombin can also be activated by staphylocoagulase. This occurs through a simple conformational change not through the cleavage process of the coagulation cascade. This explains the general thrombotic tendency during sepsis (Terres, Kümmel, Sudrow, Meinertz, Hamm, and Reuter, 1998).

17 The conversion of fibrinogen to fibrin is mediated by thrombin. Fibrinogen (factor I) is a soluble glycoprotein consisting of three different pairs of polypeptide chains (Aa, B3, and y) covertly linked by disulphide bonds. All three of these fibrinogen chains are synthesized within the liver. The amino terminal regions of the three chains are held in close proximity by a number of disulphide bonds, while the carboxyl-terminal regions are spread apart, giving rise to a highly asymmetrical, elongated molecule. Each α and ß chain consists of A and B portions, referred to as fibrinopeptides A (FPA) and B (FPB). These portions bear excess negative charges, which contribute to the solubility of fibrinogen in plasma and serve to prevent aggregation by causing electrostatic repulsion between fibrinogen molecules. Fibrin monomers in plasma thus allow a relatively stable estimation of in-vivo coagulant activity (Terres et al., 1998).

Thrombin is a protein present in blood plasma. It breaks down the amino acid bonds between the fibrinopeptides of fibrinogen. The release of fibrinopeptides by thrombin generates fibrin monomers, which have the subunit structure (α, ß, γ). The removal of the fibrinopeptides exposes binding sites that allow the molecules of fibrin monomers to aggregate spontaneously in a regular, staggered array, forming an insoluble fibrin clot. This fibrin clot functions as a trap for platelets, red blood cells, and other components to form white and red thrombi. The initial fibrin clot is rather weak, bound by the non- covalent association of fibrin monomers Factor VIII a glycoprotein is not a protease precursor, but a cofactor that serves as a receptor for factors IX and X on the platelet surface membrane. Minute quantities of thrombin activate factor VIII to form factor Villa, which in turn inactivates thrombin (Murray and Harenist, 1990).

Thrombin in addition to converting fibrinogen to fibrin, also converts factor XIII to factor XIIIa. This factor is a highly specific transglutaminase that covertly cross-links

18 fibrin molecules, yielding a more stable fibrin clot with increased resistance to proteolysis (Murray and Harenist, 1990).

The concentration of circulating thrombin is carefully controlled to prevent disastrous clot formation. This is achieved in two ways: Firstly, thrombin circulates as its inactive precursor. This in turn is activated through a cascade of enzymatic reactions, each converting an inactive protein into an active enzyme, finally converting prothrombin to thrombin. At each point in the cascade a feedback mechanism produces a delicate balance of activation and inhibition. The second means of controlling thrombin formation is via circulating inhibitors. Antithrombin acts as the most crucial inhibitor. The activity of antithrombin is increased by the action of heparin as a blood anticoagulant. Antithrombin also inhibits the activities of factors IXa, Xa, XIa, and XIIa. α2- macroglobulin acts as the second biggest contributor to the prevention of thrombin formation. Heparin, cofactor II, and α1-antitrypsin act as minor coagulation inhibitors Factor VIII a glycoprotein is not a protease precursor, but a cofactor that serves as a receptor for factors IXa and X on the platelet surface membrane. Minute quantities of thrombin activate factor VIII to form factor Villa, which in turn inactivates thrombin (Murray and Harenist, 1990).

The endogenous activity of antithrombin is greatly enhanced by the presence of acidic proteoglycans such as heparin. These bind to a specific cationic site of antithrombin, causing a conformational change and promoting its binding to thrombin as well as to its other substrates. This is the basis for the use of heparin in clinical medicine to inhibit clotting. The anticoagulant effects of heparin can be antagonised by strongly cationic polypeptides such as protaimine, which binds strongly to heparin, thus inhibiting it’s binding to antithrombin. Thus it is common for individuals with inherited deficiencies of

19 antithrombin to develop frequent widespread clot formation (Murray and Harenist, 1990).

2.4 Fibrinolysis

Fibrin being the main component of a blood clot is formed by the activation of the clotting cascade. Its production is followed by the fibrinolytic system, resulting in plasmin generation and subsequent fibrin degradation (Soria, Haverrate, Henchen, Neieuwenhuizen, and Straub, 1983). In a normal homeostatic environment, there is a balance between these opposing processes.

Fibrin degradation (i.e. fibrinolysis) is the reactive mechanism by which enzymes respond to the presence of fibrin during thrombus formation (Dunn, 1984).

Plasmin is the enzyme responsible for fibrin degradation. It is derived from its inactive precursor, plasminogen, by the action of thrombin and plasminogen activators (Dunn, 1984).

There are two main plasminogen activators, tissue plasminogen activator (tPA) and pro- urokinase. tPA is released by the endothelial cells in the blood vessel wall and binds to fibrin, for which it has a strong affinity. This then leads to the conversion of inactive plasminogen into active plasmin (Dunn, 1984). Pro-urokinase, the other plasminogen activator also activates urokinase, which further aids in fibrin degradation. Plasmin is then neutralised by alpha- antiplasmin, thereby restricting its fibrinolytic activity (Bachmann et al., 1987; Emies, 1985).

Dissolution of the cross-linked fibrin strands leads to the formation of specific degradation products called D- dimers. The activity of the D-dimers is considered to reflect both the overall activity of clot formation and clot lysis. These D-dimers can be measured in both whole blood and in plasma by using monoclonal antibodies. These antibodies are directed against fragments

20 located in the D-dimer. Hence, a reaction occurs between the monoclonal antibodies and the D- dimers (Kroneman, Neieuwenhuizen, and Knot, 1990).

D-dimers are not generated in-vitro during blood collection, thus their presence consistently reflects in-vivo haemostatic activity and their levels are proportional to this activity. In addition their absence excludes the presence of intravascular thrombus, hence the importance of D-dimer measurement (Wakai A., Gleeson A., and Winter D., 2003).

2.4.1 Fibrinogen to fibrin

Fibrin monomers are products generated by thrombin’s proteolysis of fibrinogen. The concentration of fibrin monomers during ex-vivo storage was found to be stable for at least 24 hours, but the half-life in-vivo depends on the cross-linked fibrin. The in-vivo half-life was estimated to be longer than fifty minutes for fibrinopeptide A (Dempfle, Pfitzner, Doilman, Huck, Stehle, Heene, 1995).

2.4.2 Plasminogen to plasmin

Fibrin clots are dissolved by plasmin. Plasmin is a serine protease mainly responsible for degrading fibrin to fibrinogen. It circulates in its inactive form. A small amount of plasmin that is formed in the fluid phase, under normal physiological conditions, is rapidly inactivated by α2-antiplasmin. Plasminogen binds to both fibrin and fibrinogen and thus becomes incorporated in the clot as it is produced. Since plasmin is bound to fibrin is protected from α2-antiplasmin, where it remains active. Plasminogen activators of various types are found in most body tissues and all cleave the same Arg-Val bond in plasminogen to produce plasmin (Murray and Harenist, 1990).

21 Tissue plasminogen activator (tPA) is a serine protease that is released into circulation from vascular endothelium under conditions of stress or injury. It is inactive unless bound to fibrin. Neither plasmin nor tPA can remain bound to fibrin degradation products, and so they are released into the fluid phase where they are inactivated by their natural inhibitors (Murray and Harenist, 1990).

Pro-urokinase is the precursor of a second activator of plasminogen, urokinase, which does not display the same high degree of selectivity for fibrin as tPA. Urokinase is secreted by epithelial cells lining excretory ducts (e.g. renal tubules), is probably involved in lysing any fibrin that may be deposited (Murray and Harenist, 1990).

2.5 The immune systems inflammatory response during blood coagulation.

After an infectious insult, endothelial damage occurs. Subsequently, there is activation of neutrophils and increased vascular permeability with resulting tissue oedema. Tissue factor is expressed by monocytes and the damaged vascular endothelium. Inflammatory cytokines, such as tumour necrosing factor-α, interleukin1, and interleukin-6 are then secreted and coagulation is activated with the final release of thrombin and the formation of a fibrin clot. There is accumulating laboratory and clinical data showing that in sepsis there is a deficiency of some coagulation inhibitors resulting in widespread coagulopathy (Bernard, Vincent, and Laterre, 2001).

There is a complex interaction between coagulation and inflammation. Not only does inflammation promote coagulation, but coagulation may also induce inflammation by the release of inflammatory cytokines. Coagulation factor Xa was found to produce a pro-inflammatory response in endothelial cells (Senden, Jeunhomme, and Heemskerk,

22 1998). Besides promoting fibrinolysis, active Protein C also inhibits the production of inflammatory cytokines, decreases rolling of neutrophils and inhibits the expression of tissue factor on endothelial cells. Further supporting this concept, antithrombin was shown to inhibit nuclear factor B activation in human monocytes and vascular endothelial cells. Nuclear factor B is a transcription factor involved in immediate early gene activation during inflammation (Oelschlager, Romisch, and Staubitz, 2002).

2.6 Coagulopathies

The following conditions affect the coagulation of blood (Berkow et al., 1992):

— Purpura Simplex (Easy bruising)

— Hereditary Hemorrhagic Telangiectasia (Rendu — Osler — Weber Disease)

— Ehlers — Danlos Syndrome and other hereditary connective tissue disorders.

— Allergic Purpura (Henoch — Schonlein or Anaphylactoid Purpura)

— Vascular Purpuras due to Dysproteinemias

— Scurvy (Vitamin C deficiency)

— Platelet Disorders

o Thrombocytopenia . Immunologic Idiopathic Thrombocytopenia Purpura (ITP). . HIV related Thrombocytopenia. . Lymphoproliferative disease . Drug related Thrombocytopenia (including Heparin — Induced Thrombocytopenia) . Hyperspienism

23 . Adult Respiratory Distress Syndrome (ARDS) . Thrombotic Thrombocytopenic Purpura (TTP)

o Metastatic Tumour emboli o Haemolytic — Ureamic Syndrome (HUS) o Abnormalities of platelet function. . von Willebrands’ Disease (VWD) . Bernard —Soulier Syndrome

— Hereditary coagulation disorders

o Haemophilias o Hereditary a2-antiplasmin deficiency

— Vitamin K deficiency

— Disseminated Intravascular Coagulation (DIC)

— Systemic Lupus Erythematosus (SLE)

— Liver disorders

— Protein C and Protein S deficiency

— Antithrombin deficiency

— Drugs with anticoagulative properties:

o Aspirin o Non-steroidal anti-inflammatory drugs (NSAID) o Warfarin o Heparin o Urokinase o Streptokinase

24 2.7 Homoeopathy

In simple terms homoeopathy can be described as “when less is best”. Homeopathic medicines or remedies belong to a pharmaceutical system that prescribes extremely small doses of substances. Its sources are mainly derived from the plant, the animal, and the mineral kingdoms Today in Southern Africa the word homoeopathy has become synonymous with natural, non-toxic, and safe treatments (Sollars, 2001).

2.7.1 The principles of homoeopathy

2.7.1.1 The vital force

According to homeopathic philosophy the human body consists of the sum of its components. This includes the physical, mental, and emotional aspects of who we are as individuals with our dynamic interaction in our environment. Governing this body is an unknown force often referred to as vitalism or vital force (Vithoulkas, 1981).

An organism’s quality of life depends on the presence of a strong and active vital force (Sollars, 2001). Any deviation from this state is thus seen as a pathological insult to the organism, which may develop into a diseased state (Vithoulkas, 1981).

This vital force plays a key role in the maintenance of homoeostasis. Homoeostasis can be chronically affected by many factors. These factors include age, general state of health, and the compensatory mechanisms available to the organism (Hahnemann, 1996).

25 2.7.1.2 The law of similars

The word homoeopathy is derived from the Greek words homos (similar), and pathos (suffering). This “similar suffering” is the basis for homoeopathic prescription where the remedy prescribed is closely matched to the presenting symptom picture of the patient (Vithoulkas, 1981). For accurate remedy-symptom matching, the totality of the patient’s symptoms should be taken into consideration (Hahnemann, 1996).

Samuel Hahnemann, the founder of homoeopathy, developed his theories through observation of the law of similars. This law of similars is derived from the Latin phrase “similia similibus curantur”, meaning “like cures like” (Hahnemann, 1996).

Homoeopathic prescriptions are based on a remedy picture that is produced during a homoeopathic proving. The remedies used in these provings are prescribed to healthy individuals. As the remedy takes effect, the signs and symptoms are noted and recorded. These recorded symptoms are matched to the presenting symptoms of the ill, and the same remedy is then prescribed to effect cure (Hahnemann, 1996).

2.7.1.3 Minimum dose

For homoeopathic prescriptions, patients receive one remedy at a time. The rational behind this is that a minuscule dose of a single substance should be sufficient to stimulate the vital force and begin the healing process. Thus, when taking the homoeopathic remedy the medication strengthens the body’s natural healing abilities when matching the remedy and the symptoms (Vithoulkas, 1981).

26 Due to this minimum dose approach, homoeopathic medicines are compared to allopathic vaccination by the layperson. This claim is untrue as no active biological or chemical materials enter the body during homoeopathic treatment (Sollars, 2001). Vaccinations rely on inactivated infectious agents to stimulate the specific immune system. These agents are bound to active protein carriers to aid absorption within the vaccinated organism (Berkow et al., 1992).

Samuel Hahnemann developed the minimum dose theory. He believed that a physician should cure rapidly, gently and permanently. This cure is dependant on the organism’s response to the homoeopathic remedy stimulation. As a strong external stimulus to an organism can kill it, a weak stimulus stimulates the same organism to can help it. This occurs generally in a positive manner. By utilising high potency, low dose prescriptions, one gives the organism sufficient stimulus to respond homeostatically to the treatment (Hahnemann, 1996).

Homoeopathic treatment is thus based on a natural cure. The prescribed homoeopathic remedy merely mediates the curative process and does not directly force physiological change to occur. This results in a gentle effective cure of disease (Hahnemann, 1996).

2.7.1.4 Homoeopathic potencies

Through serial dilution and sucussion homoeopathic potencies are made. A toxic substance can be taken and made safe through this process, harnessing its powers to cure (Hahnemann, 1996).

Two scales of homoeopathic potencies exist, the low decimal scale potencies (DH) and the higher centesimal potencies (CH). The decimal scale is used in lower potencies

27 where one part active ingredient is added to nine parts of carrier solution. These DH or X potencies are usually prescribed where little homoeopathic stimulation is required. The Centesimal Hahnemanian (CH) scale of serial dilution is used to produce remedies in the hundredth scale. For example a 1CH will consist of one part active ingredient and ninety-nine parts of carrier solution. This carrier solution is usually thirty percent ethanol, but for this study 0.9% sterile saline was used to prevent any action that may have influenced the samples. This process is repeated by removing one part of the 1CH potency and added to another ninety-nine parts of carrier solution to produce a 2CH. This process is continued till a 200CH potency of medicine is produced. In between each of these phases the homoeopathic remedy is vigorously shaken during a process referred to as sucussion. The amount of percussions that are required to transfer the energy of the original substance to the potency varies according to the pharmacopoeia used (Vithoulkas, 1981).

2.7.2 The science behind homoeopathy

In homoeopathy it is believed that a natural dynamic state exists where the human organism attempts to veer towards homeostasis. By administering homoeopathic remedies to the sick we aid them to help themselves and cure using the organism’s natural available resources in-vivo by manipulating the normal homoeostatic mechanisms involved in cure (Vithoulkas, 1981).

Samuel Hahnemann, the farther of homoeopathy based his medicine on observable or empirical studies of his patients, rather than a theoretical model. Despite clinical results that were obtained in the treatment of individuals during the development of the science, homoeopathy came under heavy criticism as the basic sciences of biology, chemistry

28 and physiology, which were still in their infancy. Only recently has the scientific community developed potential explanations for the pathophysiological mechanisms involved in homoeopathy (Sollars, 2001).

2.7.2.1 The cluster theory

One of the challenging aspects of homoeopathic theory has been the continual dilution and the addition of kinetic energy through the shaking of homoeopathic medicines termed potentisation. Dr. Shui-Yin Lo, a senior research scientist with American Technologies group of Los Angeles conducted breakthrough research in 1997 (Sollars, 2001).

Dr. Lo’s research found that when a solution is diluted, placed in water, and shaken as in homoeopathic remedy preparation, the solution begins to form water clusters or even ice at room temperature. In the research, the formation of TE clusters (I for ice, and E for electromagnetic forces) was witnessed, which was not influenced by the temperature of the solution. These clusters were quantitatively measured by quantum electrodynamic calculations .The research further revealed that the clusters were not formed purely by simple dilution or shaking. Only the combination of these two actions, which are similar processes to the method used in the preparation of homoeopathic medicines, yielded these unique cluster formations. The research also showed that if the solution is further diluted and vigorously shaken, the concentration of the solid water clusters increases (Sollars, 2001).

For the last two centuries homoeopaths have argued that each remedy has a unique action and effect on the human body. According to the above-mentioned research the

29 original substance that was placed into chemical solution determines the unique shape of the water clusters. This would explain the individual characteristics of each remedy (Sollars, 2001).

2.7.2.2 Homoeopathic human studies: The proving

Homoeopathic medicines were first tested on humans during vigorous studies referred to as provings. In the past few years, interest has been shown in homoeopathic research due to the medicines non-toxic and cost-effective potential. Ullman’s research (1992) points out that in one hundred and seven controlled clinical trails, eighty-one of these trials showed successful results from homoeopathic medicines.

The homoeopathic proving is the cornerstone of homoeopathy. During this process a person ingests a homoeopathic medication and records in detail all his or her thoughts and feelings. This would include; physical, emotional, and mental symptoms. These symptoms are then filtered to exclude symptoms that a healthy person would normally experience. These symptoms are then combined with other individuals’ experiences that have been ingesting the same substance during the same proving. The gathered information is collected and forms a huge database used by practitioners to match the patient’s symptoms experienced during illness. The more detailed and accurate the proving notes are, the greater the possibility practitioners have of matching the symptoms of the prover and the patient together (Vithoulkas, 1981). This concise compilation of the gathered information is termed the homoeopathic Materia Medica (Eizayaga, 1991).

30 2.7.2.3 The coherent frequencies in living systems and homoeopathic medication

The study of coherent frequencies in living systems and homoeopathic medication L concluded that through analysis of the coherent frequencies of water in homoeopathic remedies, it was found that there appeared to be a stored imprint of these frequencies L during potentisation. This would imply that a water memory of the original substance is maintained during the process of potentisation (Smith, 1998).

2.7.2.4 The chaos theory and homoeopathy

In recent years the chaos theory has been widely used to describe various aspects of science that is as yet unexplained. This theory basically states that no matter how unorganised an event may seem, in totality it is organised to some extent. Some basic aspects of this theory have relevance to homoeopathy (Gamer and Hock, 1991).

Serial dilution follows a dynamic process that mimics the chaos theory. A study conducted by Gamer and Hock (1991), indicated that in high homoeopathic potencies a given structure (i.e. the mother tincture) could reappear, in a similar shape in later potencies. The corresponding structure is thus not lost by serial dilution as would be expected. This would include dilutions far exceeding Avogadro’s number. Instead the number and quality of the proving pictures are increased with higher potencies. Also, clarity of the remedy increases with the increased number of dilutions. The study claims that it is not foreseeable which symptoms a homoeopathic remedy can cause in the proving symptoms, or in the cure of the patient, therefore the necessity of a drug provings become evident.

31 It could be possible that a living organism has the capacity to “calculate” forwards and backwards, by taking the appropriate homoeopathic remedy. This would explain the development of proving symptoms during therapy and also the reappearance of old symptoms of the patient during treatment (Garner and Hock, 1991).

2.8 Arnica montana

2.8.1 Classification

Arnica montana’s taxonomy is classified as follows within the plant kingdom: Kingdom: Plantae Subkingdom: Tracheobionta Division: Magnoliophyta Class: Magnoliopsida Subclass: Asteridae Order: Asterales Family: Asteraceae Genus: Arnica Species: Arnica montana (The plants database, 1996).

Arnica montana belongs to the greater botanical family from the natural order Compositae. Calendula officinalis and Bellis perenis are also included in this family (Gordon Ross, 1977).

Figure 2.1 is an artist’s impression of the herb Arnica montana.

32 Figure 2.1: Arnica montana

Also known as Leopard’s Bane, Wolf’s Bane and Mountain tobacco, Panacea Laprosum, Fall-Kraut (fall-herb), and Mountain Daisy (Gordon Ross, 1977).The herb is most commonly referred to by its Latin description as Arnica montana (Vithoulkas, 1995). The German name “Fall-Kraut” or “Fall-herb” originated from the German mountainous regions. Here it was observed that after a sheep sustained a fall down the mountainside, the animal would instinctively nibble at the Arnica montana herb (Gordon Ross, 1977).

2.8.2 Description

Arnica montana is a perennial herb. It has hairy leaves, and large, deep yellow flower heads (van Wyk and Wink, 2004). The herb grows horizontally, with long elliptically shaped leaves. These leaves tend to be dark at the top and lighter underneath (Gordon Ross, 1977). Up to three flower heads in bright chrome yellow to orange colourations are found per plant (Mills and Bone, 2000).

33 2.8.3 Origin of the herb

The plant is found in high altitude, mountainous areas. These areas include Europe, Russia, Siberia and in the northwest region of the United States of America (Encyclopaedia of Homoeopathic Pharmacopoeia, 2002).

2.8.4 Parts of the herb used

Herbal preparations use mainly the flower heads (Arnicae flos). Tinctures and volatile oils are procured from the herb. Rarely the roots or even the whole plant is used for herbal preparation (van Wyk and Wink, 2004). Figure 2.3 is a close-up photograph taken from the flower head of the herb Arnica montana.

Figure 2.3: Arnicae flos

2.8.5 Herbal therapeutic category

In herbal preparation Arnica montana contains anti-inflammatory, counter irritant and wound healing properties. These properties make it ideal for the treatment of traumatic wounds, preventing inflammation and speeding up healing of the affected part (van Wyk and Wink, 2004).

34 2.8.6 Uses and properties

Arnica montana in herbal preparation is traditionally used to treat bruises, haematomas, sprains, strains, bums (including sunburn), nappy rashes, and acts as a counter irritant to treat rheumatism. In herbal preparation it should only be applied externally or used as a mouthwash (when treating inflammation of the oral mucosa). Ingestion of the herb is no longer recommended as toxicity may develop quickly, and sensitivity may follow (van Wyk and Wink, 2004).

2.8.7 Active ingredients

The main active ingredients (0.2-0.5%) are helenalin and related sesquinterpene lactones. These agents give the herb a peculiar odour, not unlike apples, and an astringent taste (Gordon Ross, 1977). Other ingredients include flavones, flavonols (volatile oils with thymol, thymolmethylether and azulene), triterpenoids, phenolic acids and polysaccharides (van Wyk and Wink, 2004).

2.8.8 Pharmacological effects

Sesquinterpene lactones, such as helenalin, can form covalent bonds with proteins and thus alter their properties. These and the other Arnica compounds have mutagenic, antimicrobial and anti-inflammatory properties. If topically applied these agents show analgesic, antiseptic, hyperaemic, and wound healing effects (van Wyk and Wink, 2004).

35 2.9 Homeopathic Arnica montana

The effects of homoeopathic Arnica montana have been well documented in in-vivo studies, also known as provings. In these, the entire organism has to be considered for prescription, including the physical, mental, and emotional aspects. This makes it exceptionally difficult to pinpoint the direct physiological action of a particular remedy (Harisch and Dittmann, 1997).

For homoeopathic use of Arnica montana, the plant has to be prepared according to the homeopathic pharmacopoeia and potentised into a homoeopathically active form (Vermeulen, 1997). The mother tincture or plant tincture is prepared from the herbs’ roots, flowers and leaves (Gordon Ross, 1977). According to the German Homoeopathic Pharmacopoeia, the homoeopathic remedies used in this study are made from the dried underground parts of Arnica montana L., containing no less than 15m1 of essential oil per kg of herbal drug (German Homoeopathic Pharmacopoeia, 2003).

2.9.1 The prescription of Arnica montana

Homoeopathic Arnica montana is prescribed for pathological conditions that have a sudden onset, are traumatic in nature, and result from the complications of the initial trauma (Vithoulkas, 1995). It has been prescribed for various thrombotic disorders. It is known that it speeds up healing and revascularisation of the surrounding tissue (Savage and Roe, 1977). It is a short acting homoeopathic medication, as it needs to be repeated often, but it is quick in its actions (Gordon Ross, 1977).

36 2.9.2 Indications for homoeopathic Arnica montana

2.9.2.1 Angina

The homoeopathic symptom picture for the prescription of Arnica montana in angina is as follows:

• Angina waking the patient from sleep, resulting in great fear. • Angina with a bruised sore sensation across the chest. • Pain that causes the patient to grasp at their heart. • The chest sensitive to touch; patient fears to be touched. • Pain worse on exertion. “Strained” heart from continuous over-exertion or lifting anything heavy. • Pain relieved by gentle pressure. • Pain radiating to the left elbow. • Stitching pains on both sides of the chest, which inhibit adequate breathing (Vithoulkas, 1995).

2.9.2.2 Bruises and blunt trauma

Arnica montana is prescribed as the single most important remedy for bruises, blunt trauma and crush injuries. The indications for the prescription of the remedy include:

• Marked soreness tender to touch or pressure, bed feels too hard, feels as if beaten. • Worse for jarring, touch, pressure, or cold.

37 • Black eyes from blows. • Post surgical bruising. • Epistaxis post blow to the nose. • Dental procedures, extrications with extreme soreness. • Surgical trauma with marked extravasation (e.g. orthopaedic surgery where the incision is less but the trauma is greater). • Labour and trauma of delivery. • Traumatic arthritis. • Sore muscles after unaccustomed exertion or exercise (Morrison, 1998).

According to Hahnemann Arnica montana is very beneficial not only in injuries caused by severe contusions and lacerations but also in the most severe wounds caused by other penetrating trauma, dental extraction, surgical operations, dislocations of joints, and setting of fractures of bones (Gordon Ross, 1977).

Other indications include the immediate control of haemorrhage and the absorption of extravasated blood in cerebro-vascular accidents (Moiloa, 2000). It is also prescribed in the treatment of angina pectoris resulting from myocardial infarction, thrombosis and the treatment of ecchymoses post trauma (Vermeulen, 1997).

In conclusion, homoeopathic Arnica montana is prescribed for the treatment of both thrombus formation and haemorrhage. Its indications require that its prescription be preceded by a traumatic incident or injury, resulting in the coagulopathy (Vermeulen, 1997).

38

39 CHAPTER 3

METHODOLOGY

3.1 Sample group

The research sample group consisted of fifteen male participants, between eighteen and thirty years of age. Only male participants were selected to prevent any gender variables that may influence the study. These participants were recruited from the Gauteng area, via advertisement. These individuals were required to complete a medical examination (Appendix B) and to sign a consent form (Appendix A) on entrance to the study.

3.1.1 Inclusion criteria

• The research was limited to individuals living in the Gauteng area. • All participants were subjected to a thorough medical examination (Appendix B). • All participants were male. • All participants were between the ages of eighteen and thirty years.

3.1.2 Exclusion criteria

• No participants should suffer from any diseases affecting blood coagulation.

3.2 Research procedure

Four blood samples each consisting of five millilitres was taken from each participant. Current and efficient phlebotomy techniques were used and the samples were placed in

39 non-treated plastic phlebotomy containers to allow speedy clot formation. Each container was numerically labelled for reference purposes. These samples were left to clot for a further period of fifteen minutes, at room temperature.

After the fifteen-minute clotting time passed, two samples were randomly selected for each subject. One sample was treated with one drop of 30CH Arnica montana and the other with one drop of 200CH Arnica montana. These homoeopathic remedies were prepared in 0.9% sterile saline, and were donated by W. Last Homoeopathics cc. for the purpose of the study.

The third sample for each subject was used as a control where one drop of 0.9% sterile saline was added. Thus, consistency concerning diluting effects was maintained. All three samples were left to stand for a period of one hour to allow time for any reaction. D-dimer levels were measured using the D-DI Test from Diagnostica Stago, which is a rapid latex agglutination slide test. A semi-quantitative testing mode will be used to gather the relevant research information.

The D-DI test was designed to have a positive cut-off at 0.5 micrograms per millilitre of fibrinogen equivalent units (FEU). Undiluted plasma that gives a positive D-DI test result should contain at least 0.5 micrograms per millilitre FEU.

To achieve a semi-quantitative D-dimer level, a buffer was used to serially dilute the samples. The highest dilution that still produces positive agglutination will contain at least 0.5micrograms per millilitre FEU. A backward calculation provides semi- quantisation of the sample. For example if the 1:8 dilution is the highest that produces

40 positive agglutination, then the sample D-dimer level is equal to or greater than 4 micrograms per millilitre FEU (8 x 0.5 micrograms per millilitre).

All the testing was done at Ampath Laboratories at Garden City Clinic, Mayfair, Johannesburg.

In the study, the data was collected in several different ways:

• The participant’s past medical, surgical, and physical history was recorded in detail (Appendix B). • Each blood sample was numbered to avoid laboratory errors. • The following times were noted (Appendix C):

o The time each sample was taken was noted. o The time of visible clotting was noted. o The time of addition of the homoeopathic remedies was noted. o The time of addition of the buffered 0.9% saline for the control group was noted.

o The time allowed for reaction will be noted for each sample. o The spin down time was noted. o The times D-dimer testing was commenced and completed was noted. o The D-dimer results of each sample were recorded, as well as the D- dimer score.

41 3.3 Analysis of Data

After testing of the blood samples, the values obtained from the D-DI Test cards were statistically interpreted to asses whether or not the homoeopathic medicines Arnica montana 30CH and 200CH, had an influence on in-vitro thrombolysis. By using the prescribed methodology the results could be assessed semi- quantitatively. These values obtained from this research were statistically analysed using the quantitative frequency distribution of the one-way analysis of variants (ANOVA) method.

42 CHAPTER 4

RESULTS

The efficacy of the homoeopathic remedies, Arnica montana 30CH and 200CH was evaluated. This was assessed, by establishing if a significant difference existed in the D- dimer results obtained from the blood samples.

The research sample group consisted of fifteen healthy male participants, between the ages of eighteen and thirty years of age. All of the participants were recruited from the Gauteng region. All the participants under went a full medical examination, and thorough assessment, ensuring that they were not suffering from any diseases of blood coagulation. The entire sample group successfully met the inclusion criteria for the study. None of the participants were excluded from the study by the set exclusion criteria.

The methodology of the study was closely followed as was set by the parameters of the research procedure. Current efficient phlebotomy techniques were used in obtaining the blood samples from the study participants.

All the samples were tested using the D-DI Test from Diagnostica Stago post in- vitro coagulation. The first sample drawn was discarded to avoid cross contamination.

43 4.1 Control samples

The control samples were medicated with one drop of sterile 0.9% saline solution. These samples did not show any agglutination reaction when they were combined with the D- dimer latex agglutination tests. If agglutination had been observed in this sample, it would have indicated a defect in the methodology or a problem with the D-dimer test kit.

The results are reflected in Table 4.1 below. As can be clearly seen, no significant results were obtained within the control samples.

Graph 4.1: Control samples

2 1.8 1.6 1.4 1.2 1 mg/ml FEU 0.8 0.6 0.4 0.2 0 1 3 5 7 9 11 13 15

44 4.2 Arnica montana 30CH samples

These samples were medicated each with one drop of Arnica montana 30CH. These samples also had no positive agglutination reactions when they were combined with the D-dimer latex agglutination tests, indicating a D-dimer level well below 0.5 micrograms per millilitre of FEU.

The results are reflected in Table 4.2 below. As can be clearly seen, no significant resuks were obtained within the Arnica montana 30CH samples.

Graph 4.2: Arnica montana 30CH samples

2 1.8 1.6 1.4 1.2 1 mg/ml FEU 0.8 0.6 0.4 0.2 0 1 3 5 7 9 11 13 15

45 4.3 Arnica montana 200CH samples

These samples were medicated each with one drop of Arnica montana 200CH. These samples also had no positive agglutination reactions when they were combined with the D-dimer latex agglutination tests, indicating a D-dimer level well below 0.5 micrograms per millilitre of FEU.

The results are reflected in Table 4.3 below. As can be clearly seen, no significant results were obtained within the Arnica montana 200CH samples.

Graph 4.3: Arnica montana 200CH samples

2 1.8 1.6 1.4 1.2 1 mg/ml FEU 0.8 0.6 0.4 0.2 0 1 3 5 7 9 11 13 15

With regard to the methodology chosen for this study, the results if any were below detectable values. All the samples tested did not show any form of agglutination on the test cards when combined with the D-DI Test from Diagnostica Stago. There are several possibilities why there were no recordable results; these are discussed in Chapter 5.

46 CHAPTER 5

DISCUSSION

Homoeopathic Arnica montana is often prescribed for pathological conditions that have a sudden onset, are traumatic in nature, and result from the complications of the initial insult to the organism (Vermeulen, 1997). Although extensive research has been conducted on Arnica montana, little is known of the direct pathophysiological pathways involved in its role in thrombolysis. The aim of the study was to test the efficacy of Arnica montana 30CH and 200CH to thrombolise a blood clot in an in-vitro sample. This was preformed using the D-DI Test from Diagnostica Stago, measuring the D- dimer levels of the samples through latex agglutination. For the purpose of the research the study was conducted in an in-vitro, eliminating the effects of an in-vivo environment. The reasoning behind this was to establish if Arnica montana 30CH and 200CH acted locally to cause thrombolysis.

5.1 Control samples

The control samples did not show any agglutination reaction when they were combined with the D-dimer latex agglutination tests. If agglutination had been observed in this sample, it would have indicated a defect in the methodology or a problem with the D- dimer test kit.

5.2 Arnica montana 30CH samples

All the Arnica montana 3OCH samples when tested did not show any D-dimer results. It was thought that homoeopathic Arnica montana 3OCH may replace tPA in the

47 activation of plasmin. As a certain amount of plasminogen is available in an in-vitro sample, Arnica montana 30CH would have to act directly on the plasminogen to convert it to plasmin. As there was no increase in D-dimer levels after the administration of the Arnica montana 30CH potency, it can be assumed, using the study methodology, that the selected homoeopathic medicine had no thrombolytic effect.

This indicates that it is more likely that homoeopathic Arnica montana 30CH may act on the vascular endothelium once thrombus formation has occurred. As Arnica montana 30CH is mostly indicated in trauma, this hypothesis could be more accurate in assessing the thrombolytic properties of the remedy.

5.3 Arnica montana 200CH samples

The Arnica montana 200CH samples also showed no D-dimer results when tested. As for the Arnica montana 30CH samples, no conversion of plasminogen occurred to result in thrombolysis.

The reasons for the results obtained could be due to the follow explanations:

• The quantity of medication that should be used is controversial. Since homoeopathy as a science is based on the use of a single and a minimum dose, there is no established methodology to determine the relevant dose required to medicate a particular sample. This area of homoeopathy should be researched further as no data is available on the calculation of the actual dose of a specific homoeopathic medication. • According to homoeopathic philosophy, it can be argued that as the specimens were tested in-vitro, the vital force of the organism was weakened as the blood

48 samples were removed from an in-vivo state. That would imply that this weakened vital force was not strong enough to be stimulated by the homoeopathic Arnica montana 30CH and 200CH.

5.4 The importance of this study

Initially the study was designed to establish if Arnica montana’s thrombolytic effects could be explained in an in-vitro model. Since this model was designed and tested for the first time, several errors have occurred in the methodology, as no other similar models are available.

Although the study has failed to demonstrate a positive outcome regarding the hypothesis, it holds scientific relevance to the possible explanation of the thrombolytic effects of Arnica montana 30CH and 200CH.

It has been established that as separate entities homoeopathic Arnica montana 30CH and 200CH have little or no direct effect on a clotted in-vitro sample. This indicates that if the medicine has thrombolytic properties, it has to utilise or activate other endogenous factors in-vivo. As these factors require the presence of vascular endothelium, it makes it potentially difficult to conduct studies due to ethical reasons.

During the coarse of the study several variables were identified that could influence the outcome. These variables can be manipulated in future studies of homoeopathic medication in the field of thrombolysis.

49 CHAPTER 6

CONCLUSION AND RECOMMENDATIONS

6.1 Conclusion

Arnica montana is a commonly used homoeopathic medicine prescribed for various conditions. These conditions include acute conditions with sudden onset, circulatory conditions and trauma or injury.

Although research is available on both the herbal and the homoeopathic forms of Arnica montana, little research has explained the medicines effectiveness in the treatment of thrombosis and other circulatory disorders. As most of the medicines characteristics were gathered through homoeopathic provings and toxicology, very little is known about the actual pathophysiological mechanisms involved.

This study aimed to test the efficacy of Arnica montana 30CH and 200CH to thrombolise a blood clot in an in-vitro sample. This study failed to prove that homoeopathic Arnica montana 30CH and 200CH had an effect on blood clot thrombolysis in-vitro. This does not however prove that the medicine has no effect on thrombolysis in-vivo, but merely indicates a lack of results in relation to the methodology chosen for this particular study.

No previous study designed according to this format has ever been published, locally or internationally. Due to inexperience in using this methodology, many unknown variables were encountered during the testing procedure. In future use, this study may serve as a

50 guide to others seeking to establish the relationship between Arnica montana and thrombolysis.

6.2 Recommendations

The methodology used in this study may be used as a guideline for future studies in haematology and homoeopathy. The problematic variables concerning the methodology used should be carefully considered.

The following are recommendations regarding further research in this field:

• Other potencies of Arnica montana should be tested to asses their effects on thrombolysis. Very high potencies such as Arnica montana M and 1 OM potencies should be considered. • An in-vivo study to assess the fibrinolytic effects of homoeopathic Arnica montana should be devised. This study should be able to isolate the actual pathophysiological pathways involved in thrombolysis, explaining scientifically, on a cellular and chemical level which factors are involved. Ethics should be carefully taken into consideration upon designing such a study. • A more accurate method of assessing D-dimer levels should be used. The Enzyme-linked immunosorbent assay (ELISA) method is more accurate but could not be used in this study due to budget constraints. • A greater volume of the homoeopathic medication should be administered to the samples. No accurate dose requirements have been established homoeopathically. Developing an accurate dose regiment will help improve homoeopathic prescriptions and therapeutic effects.

51 • More than one dose of the homoeopathic medication should be administered to the samples. As homoeopathic medication acts as an energetic model, continuous stimulation may be required to elicit a thrombolytic effect. • Other homoeopathic remedies such as Lachesis mutus and Bellis perenis should be tested for their effects on thrombolysis.

Taking the abovementioned recommendations into consideration, further scientific research may explain the pathophysiological role of Arnica montana in thrombolysis.

52 REFERENCES

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54 Gordon Ross A.C., (1977) The Amazing Healer Arnica and a dozen other homoeopathic remedies for aches, pains and strains. Thornton Publishers Limited, Northamptonshire, pp. 19-29

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58 APPENDIX A

INFORMATION AND CONSENT FORM

Dear Participant

The purpose of this study is to determine the efficacy of Arnica montana 3OCH and 200CH to thrombolise a blood clot in an in-vitro sample. You will be one of fifteen male participants that will be involved in this study. The study will consist of two days and prior notification of the dates will be arranged with you personally.

For you to participate in the study you will have to meet the selection criteria:

• Participants must be male in gender. • Participants should be between twenty and thirty years of age. • The research will be limited to individuals living in the Gauteng area. • All participants will be subjected to a thorough medical examination. • No participant should suffer from any diseases affecting blood coagulation:

o Purpura Simplex ( Easy bruising) o Hereditary Hemorrhagic Telangiectasia (Rendu — Osler — Weber Disease)

o Ehlers — Danlos Syndrome and other hereditary connective tissue disorders.

o Allergic Purpura (Henoch — Schonlein or Anaphylactoid Purpura) o Vascular Purpuras due to Dysproteinemias

59 o Scurvy (Vitamin C deficiency) o Platelet Disorders . Thrombocytopenia . Immunologic Idiopathic Thrombocytopenia Purpura (ITP). . HIV related Thrombocytopenia. . Lymphoproliferative disease . Drug related Thrombocytopenia (including Heparin — Induced Thrombocytopenia) . Hyperspienism . Adult Respiratory Distress Syndrome (ARDS) . Thrombotic Thrombocytopenic Purpura (TTP) . Metastatic Tumour emboli . Haemolytic — Ureamic Syndrome (HUS) o Abnormalities of platelet function. o Von Willebrands’ Disease (VWD) o Bernard —Soulier Syndrome. o Hereditary coagulation disorders o Haemophilias o Hereditary alpha2 — antiplasmin deficiency o Vitamin K deficiency o Disseminated Intravascular Coagulation (DIC) o Systemic Lupus Erythematosus (SLE) o Liver disorders o Protein C and Protein S deficiency o Antithrombin deficiency

60 • No participant should be on any drugs or medication affecting blood coagulation:

o Drugs with anticoagulative properties: Aspirin, NSAID, Warfarin, Heparin,

o Urokinase, Streptokinase. o Alcohol o Smoking o Recreational drugs o Any Homoeopathic treatment o Drugs that promote coagulation Vitamin K.

Two weeks before and during the study, you will be advised to refrain from an unhealthy diet, alcohol consumption, smoking, and taking recreational drugs. Your participation in the study will consist of two days. On the first day, a full medical examination and history will be conducted. On the second day, samples of venous blood will be taken from you. You will be advised of your appointment time and date forty- eight hours before the event.

Please note that your participation in the research study is voluntary and that you are free to refuse participation or withdraw your consent at any stage. A copy of this consent form will be signed and made available to you. Any information submitted by you will be confidential and only the researcher will have access to it.

I, the participant, fully understand what this research entails and any questions that I have will be directed to the researcher. I understand the study procedure to be followed and agree to abide by them. I agree that any information about my case may be used for

61 discussion by the researcher and colleagues. I am aware that I may refuse participation at any time.

Date: ______Signature: ______

Thank you

I, the researcher, have completely explained the techniques and the purpose of the tests used in this research. Any questions that may arise from the participants will be answered to the best of my ability.

Date: ______Signature: ______

62 APPENDIX B

MEDICAL INFORMATION FORM

Research number: Date Surname First Names DOB Gender Contact details Telephone number Residential Address Postal Address Next of Kin Name and Surname Telephone number Relation Medical History Surgical History Allergies Medication and Supplements Family Medical History Social History Vital Signs Pulse Respiratory rate Blood pressure

63 Temperature Capillary refill Physical examination Head and Neck Skin and Soft tissue

Eyes and ENT

Respiratory System

Cardiovascular System

Abdomen

Extremities

64

65 APPENDIX C

DATA COLLECTION Sample number 1.1 1.2 1.3 2.1 2.2 2.3 Average Time taken Time of visible clotting Time of addition of homeopathic remedy Time of addition of 0.9% buffered saline Time allowed for remedy reaction Spin down time Time D-dimer testing commenced Time D-dimer testing completed

D-dimer results (micrograms I milliliter)

65

66 APPENDIX D

Plasminogen

Pro-urokinase

tPA

Plasmin Urokinase Alpha- antiplasminogen (Negative feedback)

Fibrin Fibrin degradation products

D-dimers

66