Inhibitory Activity of Triterpenoids, Eugenol and Eugenol Acetate in Thrombin, Adenosine Diphosphate and Epinephrine-Induced Platelet Aggregation.
ABSTRACT
Platelet hyper-aggregation is the underlying cause of cardiovascular diseases. The clinical limitation and adverse side effect associated with currently used anti-platelet aggregation agents have fuelled the search for more effective agents of natural origin.
Oleanolic acid, maslinic acid and eugenol from Syzigium aromaticum and betulinic acid from Melaleuca bracteata were isolated. The isolated compounds were modified to their acetates to mimic the functionality of aspirin.
The isolated and modified compounds were characterized using nuclear magnetic resonance (NMR) spectroscopic techniques to ascertain the structure of the compounds. The isolated and modified compounds were evaluated for platelet aggregation inhibition induced by Thrombin, Adenosine diphosphate (ADP) or epinephrine.
The result of the platelet aggregation inhibition using thrombin, ADP and epinephrine as agonist on the test compounds showed that the highest inhibition exhibited by betulinic acetate on the thrombin-induced platelet aggregation (54.5±0.01 at 1mg/ml, 63.5±0.17.
At 3mg/ml and 73.5±0.15 at 10mg/ml, IC50 0.81mg/ml) which was similar (P<0.05) to the values of Aspirin (65.4±0.07 at 1mg/ml, 72.1±0.03 at 3mg/ml and 76.5±1.22 at 10mg/ml, IC50 0.33mg/ml) and Heparin (65.9±0.59 at 1mg/ml, 66.9±0.03 at 3mg/ml and 70.6±0.16 at 10mg/ml, IC50 0.26) used as standards.
Antioxidant activity was investigated using 1,1′-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS+ )) assay to suggest a mechanism of action of the test compounds.
The anti-oxidant activities of the compounds showed that eugenol possessed significant (P<0.05) free radical scavenging activity with highest activity in the ABTS+ assay (57.7±0.002, at 0.0625mg/100ml, 75.3±0.003, at 0.125mg/100ml, 89±0.002.
At 0.25mg/100ml, 89.7±0.001, at 0.5mg/100ml, 90.7±0.001, at 1mg/100ml and an IC50 0.05mg/100ml) which is more than those of the standards Ascobic acid (9.7±0.001, at 0.0625mg/100ml, 19.5±0.003, at 0.125mg/100ml, 26.0±0.001, at 0.25mg/100ml, 47.7±0.001.
At 0.5mg/100ml, 60.9±0.002, at 1mg/100ml and an IC50 0.66mg/100ml) and Butylated hydroxyanisole (9.7±0.005, at 0.0625mg/100ml, 15.4±0.001, at 0.125mg/100ml, 21.9±0.002, at 0.25mg/100ml, 52.0±0.001, at 0.5mg/100ml, 65.8±0.001, at 1mg/100ml and an IC50 0.48mg/100ml)) used.
The study suggests that the isolated and modified compounds be used as antiplatelet aggregation agents in the management of blood-clotting related diseases.
TABLE OF CONTENTS
Title page – – – – – – – – – i
Declaration – – – – – – – – – ii
Certification – – – – – – – – – iii
Acknowledgement – – – – – – – – iv
Abstract – – – – – – – – – v
Table of content – – – – – – – – vi
List of Figures – – – – – – – – – xi
List of Plates – – – – – – – – – xii
List of Tables – – – – – – – – – xiii
List of Appendices – – – – – – – – xiv
List of Abbreviations – – – – – – – – xviii
CHAPTER ONE
1.0 Introduction – – – – – – – – 1
1.1 Statement of Research Problem – – – – – 4
1.2 Justification – – – – – – – – 4
1.3 Aims – – – – – – – – – 4
1.4 Research Objectives – – – – – – – 5
1.5 Hypothesis – – – – – – – – 5
CHAPTER TWO
2.0 Literature Review- – – – – – – – 6
2.1 Blood-Clotting- – – – – – – – 6
2.1.1 Platelet Activation and Aggregation – – – – – 7
2.1.2 Mechanism of Blood Clotting (Coagulation cascade) – – 9
2.2 Platelet Agonist – – – – – – – 13
2.2.1 Thrombin – – – – – – – – 14
2.2.2 Adenosine Diphosphate – – – – – – 14
2.2.3 Epinephrine – – – – – – – – 15
2.2.4 Free Radicals – – – – – – – – 16
2.3 Anti-Platelet Therapy, Limitation and Future Prospects – – 18
2.3.1 Mechanism of Action of Aspirin – – – – – 19
2.4 Syzigium Aromaticum – – – – – – – 21
2.5 Melalueca Bracteata – – – – – – – 23
2.6 Triterpenes – – – – – – – – 24
2.6.1 Pentacyclic Triterpenes from Nature – – – – – 27
2.6.2 Betulinic Acid – – – – – – – 29
2.6.3 Oleanolic Acid – – – – – – – 29
2.6.4 Maslinic Acid – – – – – – – – 29
2.6.5 Eugenol – – – – – – – – 29
CHAPTER THREE
3.0 Materials and Methods – – – – – – 30
3.1 Materials – – – – – – – – 30
3.1.1 Equipment – – – – – – – – 30
3.1.2 Chemicals – – – – – – – – 30
3.1.3 Plant Materials – – – – – – – 31
3.1.3.1 Syzigium aromaticum and Melaleuca bracteata – – – 31
3.1.4 Animals – – – – – – – – 32
3.2 Methods – – – – – – – – 32
3.2.1 Extraction and Isolation of Oleanolic acid and Maslinic acid
from S. aromaticum – – – – – – – 32
3.2.2 Formation of Oleanolic and Maslinic Acetate – – – 33
3.2.3 Isolation of Eugenol and formation of Eugeno Acetate – – 33
3.2.4 Extraction and Isolation of Betulinic Acid and preparation
of Betulinic Acetate – – – – – – – 35
3.2.5 Spectroscopy – – – – – – – – 37
3.2.6 Acute Toxicity – – – – – – – 37
3.2.7 In vitro Anti-platelet Aggregation Study – – – – 38
3.2.7.1 Preparation of Blood Platelets – – – – – 38
3.2.7.2 Anti-platelet Aggregation Activity – – – – – 39
3.2.7.3 Calculation of Percentage Inhibitory Effect of the Compounds
on Platelet Aggregation – – – – – – 39
3.2.8 In vitro Antioxidant Activity – – – – – – 39
3.2.8.1 Thin Layer Chromatography Bioautography using DPPH
and Bete-carotene Method – – – – – – 39
3.2.8.2 DPPH Radical Assay – – – – – – – 40
3.2.8.3 ABTS Radical Assay – – – – – – – 40
3.2.8.4 Calculation of Percentage Radical Scavenging Activity
of the Compounds – – – – – – – 41
CHAPTER FOUR
4.0 Results – – – – – – – – 42
4.1 Isolation of Bioactive Compounds – – – – – 42
4.2 Modification of Isolated Compounds – – – – 42
4.3 Characterization of Isolated and Modified Compounds – – 43
4.3.1 Characterization of Oleanolic Acid – – – – – 43
4.3.2 Characterization of Oleanolic Acetate- – – – – 45
4.3.3 Characterization of Betulinic Acid – – – – – 45
4.3.4 Characterization of Betulinic Acetate – – – – – 46
4.3.5 Characterization of Maslinic Acetate – – – – – 46
4.3.6 Characterization of Eugenol – – – – – – 47
4.3.7 Characterization of Eugenol Acetate – – – – – 47
4.4 Anti-Aggregation Activity of Isolated and Modified Compounds – 47
4.4.1 Anti-aggregation Activity of Isolated and Modified Compounds in
Thrombin-induced Platelet Aggregation – – – – 47
4.4.2 Anti-aggregation Activity of Isolated and Modified Compounds in
ADP-induced Platelet Aggregation – – – – – 50
4.4.3 Anti-aggregation Activity of Isolated and Modified Compounds in
Epinephrine-induced Platelet Aggregation – – – – 52
4.5 Antioxidant Activity of Isolated and Modified Compounds – – 54
4.5.1 Thin Layer Chromatography Bioautography of Isolated and
Modified Compounds – – – – – – 54
4.5.2 Antioxidant Activity of Isolated and Modified Compounds
Using DPPH Assay – – – – – – – 56
4.5.3 Antioxidant Activity of Isolated and Modified Compounds
Using ABTS Assay – – – – – – – 58
CHAPTER FIVE
5.0 Discussion – – – – – – – – 60
CHAPTER SIX
6.0 Conclusion and Recommendation – – – – – 64
6.1 Conclusion – – – – – – – – 64
6.2 Recommendation – – – – – – – 65
References – – – – – – – – 66
Appendices – – – – – – – – 77
INTRODUCTION
Platelets are disk-shaped, non-nucleated blood cells that help to make blood clot. They are also called thrombocytes with a very fragile membrane. They play a major role in blood clotting process and tend to adhere to uneven or damaged surfaces.
They average about 250,000 per cubic millimeter of blood and are formed in the red bone marrow by fragmentation of megakaryocytes, the largest of the bone marrow cells. Platelet production is controlled by a hormone thrombopoietin, and regulatory lymphocytes acting at the stem cell level.
At any given time about one-third of the total blood platelets can be found in the spleen; the remaining two-thirds are in the circulating blood (Holt and Chandra, 2002).
It is well known that platelets play an important role in the physiology of primary hemostasis and pathophysiological processes such as thrombosis (Shattil et al., 1998; Stouffer and Smyth, 2003).
Blood clots can become a problem especially if they are in the heart, brain or other arteries in the body (Ruggeri, 2002).
Blood clotting forms an important part of haemostasis; it is a complex process that needs to be carefully controlled.
It involves platelets, enzymes and clotting factors in a cascade of reactions that lead to the catalytic conversion of a soluble fibrinogen by thrombin to insoluble fibrin polymers.
The fibrin polymers form a meshwork around platelet to form a fibrin clot (Murray et al., 2003).
REFERENCES
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Adedapo, A.A. Jimoh, F.O. Afolayan, A.J. and Masika, P.J. (2008). Antioxidant activities and phenolic contents of the methanol extracts of the stems of Acokanthera oppositifolia and Adenia gummifera. BMC Complementary and Alternative Medicine, 8(54): 1472-6682.
Akular, U.S. and Odhav, B. (2008). In vitro 5-lipoxygenase Inhibition of Phenolic Antioxidants from Undomesticated Plants of South Africa. Journal of Medicinal Plants research, 2(9): 207-212.
Alqareer, A. Alyahya, A and Andersson, L. (2006). The Effect of Clove and Benzocaine Versus Placebo as Topical Anesthetics. Journal of Dentistry, 43(10): 747-750.
Ambrosio, G. Tritto, I. and Golino, P. (1997). Reactive Oxygen Metabolites and arterial thrombosis (Review). Cardiovascular Research, 34: 445-452.
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