ANTIINFLAMMATORY ACTIVITY OF THE FATTY SEED EXTRACT OF Vitellaria paradoxa (SAPOTACEAE)
BY
MUOGHALU, G. U (PG/M.PHARM/08/48526)
DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA NSUKKA
MARCH 2016
1 ANTIINFLAMMATORY ACTIVITY OF THE FATTY SEED EXTRACT OF
Vitellaria paradoxa (SAPOTACEAE)
BY
MUOGHALU, G. U (PG/M.PHARM/08/48526)
A PROJECT REPORT PRESENTED TO THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHARMACY DEGREE.
SUPERVISORS: PROF. PETER A. AKAH & PROF. CHARLES O. OKOLI DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA
MARCH, 2016.
CERTIFICATION
Muoghalu, G. U, a postgraduate student of the Department of Pharmacology & Toxicology with registration number PG/M.Pharm/08/48526 has satisfactorily completed the requirement for the award of Master of Pharmacy (M.Pharm) of the Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka.
The work embodied in this project is original and has not been submitted in part or full for any other diploma or degree in this or other institution.
______Prof. P.A Akah Prof. C.O. Okoli (Supervisor) (Supervisor)
Dr. A.C. Ezike (Head of Department)
DEDICATION
I dedicate this work to the Sacred Heart of Jesus for I fear all things out of my own weakness but I hope for all things from His Goodness.
ACKNOWLEDGEMENT
First of all, I acknowledge God almighty for His goodness beyond measure and what can I say? All I have to say is thank you Lord.
I owe the success of this work to my supervisors Professor C. O. Okoli and Professor P.A Akah. Working with Professor Charles, I learnt not to meet people with a preformed opinion about them. He helped immensely in design of this work and made out time to see it through. I thank him for the force he added to my heel when I gave up. It is easy for me to be grateful to him silently in my heart but it is obvious that silent appreciation profits no one. So am using this opportunity to say thank sir! I appreciate your kindness.
I sincerely appreciate my family members; Dr. Chijioke Muoghalu was the man behind the mask. He is my husband, I call him my best friend and brother. I thank him for the freedom, listening ear and financial muscle he gives and of course for all his ‘troubles’ which makes the world go round. May God bless you my dear. My mother, Mrs. Margaret Okafor-Obi (Evidence) is the reliable giant on whose shoulder I stand on to see far. She represents her husband very well that even the death of Anthony Okafor-obi seems a myth. Without her help, I would not have been able to scale through in this programme. She was there all the way and I pray that God grant you long life and excellent health. I am highly indebted to my children – Chidera, Chisom, Chinonso, Chimamanda and Chijioke. They were at the center of the whole process. I appreciate my siblings- Eucharia, Chiedu, Ifeyinwa, Nwakaego, Chioma, Oluchukwu, Emeka and Izuchukwu for their immense help both financially and otherwise. I thank them for those “provoking” calls asking of my progress.
The initial driving force was from my amiable Dr. Mrs. Ezike. The push and pieces of advice she offered are what I see today embodied in this project work. I thank her so much for her motherly love.
At my work place I owe gratitude to Pharm. (Mrs.) Chinwe Onyeka and Pharm. Ola Okpi. They did all that was necessary for me to run a successful programme.
At home I sincerely thank Mrs Eneje Uzouwa, Obianuju Udaka, Ngozi Ibemesi, Agozie Oyitabu, Ifeoma Okafor-obi and Tochukwu Okoye for backing me up in the house at various points in time during this my work.
To my friends, Collins, Florence and Ifeoma, I owe respect for having the courage to continue in research. Dr. Ubaka Chukwuemeka is the backbone of the analytical work. I pray that God reward him abundantly for his understanding and help. Ubaka is a friend indeed.
The family of Mr. and Mrs. Maurice Onwuka and their children Kosisochukwu and Kaodilinyechukwu has been my shelter and comfort throughout. I owe them love and prayers. Mr. and Mrs. Ibemesi are and will continue to be my own people. May God bless and keep them.
I appreciate workers at the Pharmacology Department especially Mrs. Ugwu Florence for her prompt supply of rats and mice. I am also indebted to workers at the Department of Veterinary Medicine especially Dr. Sunny Udegbunam for having time for me. He handled my case like a brother.
I will not forget Olisa Raphael and his friend Victor for their immense help without which I will still not be able to finish.
I cannot end this acknowledgement without thanking my favoured friend and sister Dr. (Mrs.) Augustina Charles-Okoli. Strength is not measured by how muscular a person looks but in the mind. She is the ideal woman when it comes to strength and wisdom, a woman with diplomacy to be reckoned with. She set me on the fire that made me to jump up. I call her favoured, she knows why. Believing that God’s favour is for a life time, I pray that she lives in abundance of the favour of God.
I sincerely acknowledge all the authors that I used their publications.
TABLE OF CONTENT
Title page……………………………………………………………………………………….. i Certification ………………………………………………………………………………...... ii Dedication ……………………………………………………………………………………... iii Acknowledgement …………………………………………………………………………….. iv Table of Content……………………………………………………………………………….. vi List of Abbreviations………………………………………………………………………….. ix List of Figures …………………………………………………………………………………. xi List of Tables…………………………………………………………………………………... xii Abstract ……………………………………………………………………………………….. xiii
CHAPTER ONE: INTRODUCTION
1.1 Inflammation……………………………………………………………………... 1 1.2 Causes of Inflammation…………………………………………………………... 2 1.3 Types of Inflammation…………………………………………………………… 2 1.3.1 Acute Inflammation……………………………………………………………… 3 1.3.2 Chronic Inflammation…………………………………………………………….. 4 1.4 The Inflammatory Response……………………………………………………… 5 1.5 Mediators of Inflammation……………………………………………………….. 7 1.5.1 Plasma Derived Mediators………………………………………………………… 7 1.5.2 Cell Derived Mediators……………………………………………………………. 8 1.5.2.1 Lipid Derived Mediators………………………………………………………… 10 1.6 Biomarkers of Inflammation……………………………………………………… 12 1.7 Disorders of Acute Inflammation…………………………………………………. 13 1.8 Disorders of Chronic Inflammation………………………………………………. 13 1.8.1 Arthritis …………………………………………………………………………… 14 1.8.1.1 Types of Arthritis………………………………………………………………….. 14 1.8.1.1.1 Non-inflammatory Arthritis………………………………………………………. 14 1.8.1.1.2 Inflammatory Arthritis……………………………………………………………. 15 1.9 Agents Used in Management of Inflammation/ Arthritis……………………….. 16 1.9.1 Non-steroidal Antiinflammatory Drugs………………………………………….. 17 1.9.1.1 Non selective COX Inhibitors………………………………………...... 18 1.9.1.2 Selective COX -2 Inhibitors……………………………………………………. 20 1.9.2 Corticosteroids…………………………………………………………………… 21 1.9.3 Disease Modifying Anti-Rheumatic Drugs………………………………………. 21 1.9.4 Biological Disease Modifying anti-rheumatic drugs (bDMARDs)………………. 23 1.9.5 Medicinal Plants………………………………………………………………….. 24 1.10 Botanical Profile of Vitellaria paradoxa……………………………………….. 26 1.10.1 Plant Taxonomy…………………………………………………………………. 26
1.10.2 Plant Description………………………………………………………………… 27 1.10.3 Geographical Distribution…………………………………………………………. 28 1.10.4 Ethnomedicinal Uses………………………………………………………………. 28 1.10.5 Literature Review……………………………………………………...... 29 1.11 Aim and Scope of Study……………………………………………………… 30
CHAPTER TWO: MATERIALS AND METHODS
2.0 Materials and methods…………………………………………………………….. 31 2.1 Materials…………………………………………………………………………… 31 2.1.1 Animals……………………………………………………………….…………... 31 2.1.2 Chemical and Solvents…………………………………………………………….. 31 2.1.3 Drugs………………………………………………………………………………. 31 2.1.4 Equipment…………………………………………………………………………. 31 2.1.5 Patients…………………………………………………………………………….. 32 2.1.6 Shea butter…………………………………………………………………………. 32 2.2 Methods……………………………………………………………………………. 32 2.2.1 Data collection…………………………………………………………………….. 32 2.2.2 Pharmacological tests…………………………………………………………… 33 2.2.2.1 Systemic acute inflammation of the rat paw…………………………………….. 33 2.2.2.2 Topical acute edema of the mouse ear…………………………………………. 33 2.2.2.3 Formaldehyde induced arthritis in rats………………………………………….. 34 2.2.2.4 Cotton pellet induced granuloma in rats……………………………...... 35 2.2.3 Statistical analysis………………………………………………………………. 36
CHAPTER THREE: RESULTS
3.0 Results……………………………………………………………………………… 37 3.1 Demographic characteristics of respondents……………………………………... 37 3.2 Respondents’ knowledge of their disease Condition………………………………. 37 3.3 Respondents’ knowledge and use of shea butter………………………………….. 37 3.4 Effects of shea butter on systemic acute inflammation………………………… 48 3.5 Effects of shea butter on topical acute inflammation………………...... 48 3.6 Effects of shea butter on experimental arthritis………………………………… 48 3.2.4 Effects of shea butter granuloma formation………………………...... 48
CHAPTER FOUR: DISCUSSION AND CONCLUSION
4.0 Discussion and conclusion………………………………………………………………. 53
4.1 Discussion……………………………………………………………………………….. 53 4.2 Conclusion……………………………………………………………………………….. 56 References………………………………………………………………………………………. 57 Appendix 1……………………………………………………………………………………… 67 Appendix 2……………………………………………………………………………………… 69
LIST OF ABBREVIATIONS
AA Arachidonic acid
ACPA Anti citrullinated protein antibody
ANOVA Analysis of variance
AUC Area under curve
COX Cyclooxygenase enzyme
CRP C-reactive protein
CTLA Cytotoxic T-lymphocyte antigen
DMARD Disease modifying anti rheumatic drugs
EDHF Endothelium derived hyperpolarizing factor
ESR Erythrocyte sedimentation rate
FDA Food and drug agency
G-CSF Granulocyte colony stimulating factor
GM-CSF Granulocyte macrophage colony stimulating factor
ICAM Intercellular cell adhesion molecules
IFN Interferon
IgG Immunoglobulin G.
IL Interleukins
IL-1-RA Interleukin 1 receptor-antagonist
LOX lipoxygenase enzyme
LSD Least significant difference
M-CFS Macrophage colony stimulating factor
MTX Methotrexate
NI No inhibition
NO Nitric oxide
NSAID Non-steroidal anti-inflammatory drugs
OA Ostearthritis
PAF Platelet activating factor
PLA2 Phospholipase A2 RA Rheumatoid arthritis
SEM Standard error of mean
TNF Tumour necrotic factor
TXA Thromboxane
UV Ultra violet
VCAM Vascular cell adhesion molecules
LIST OF FIGURES
Figure 1 Gender of arthritic patient respondents 38
Figure 2 Occupation of respondents 39
Figure 3 Respondents knowledge of their disease condition 40
Figure 4 Part of body affected by arthritis 41
Figure 5 Factors that worsen arthritic pain 42
Figure 6 Respondents knowledge of shea butter 43
Figure 7 Respondents mode of use of shea butter 44
Figure 8 Respondents frequency of use of shea butter 45
Figure 9 Respondents that get relief from use of shea butter only 46
Figure 10 Duration of relief of pain after shea butter use 47
LIST OF TABLES
Table 1 Effect of shea butter on systemic acute edema of the rat paw 49
Table 2 Effect of shea butter on acute ear edema in mice 50
Table 3 Effect of shea butter on global edematous response to formaldehyde 51 arthritis
Table 4 Effect of shea butter on granuloma formation 52
ABSTRACT
Fatty seed extracts of Vitellaria paradoxa (Sapotaceae), an indigenous plant of West Africa is a popular remedy for arthritis and swellings. This study investigated the knowledge, prevalence and pattern of use of the fatty extract also known as shea butter in clinically diagnosed arthritic patients as well as its effects on acute (topical and systemic) and chronic inflammation in rodents. The knowledge, prevalence and pattern of use were determined using pretested questionnaire in clinically diagnosed patients. The antiinflammatory activity was studied using xylene-induced edema of the mouse ear, carragennan-induced edema of the rat paw, formaldehyde-induced arthritis and cotton pellet granuloma test in rats. The result showed that of the 164 respondents, 94.1% know about shea butter and 59.6% have used it mainly as a massage ointment once or twice daily. However, 73.7% of the users combine this remedy with analgesics to achieve relief. The pharmacological tests showed that topical application of shea butter inhibited the acute edema of the mouse ear. Oral administration also inhibited the development of systemic acute edema of the rat paw in a non-dose related manner. The inhibitory effect was significant (p<0.05) within 1 h post administration of irritant. Twice daily topical application of shea butter inhibited the global edematous response to formaldehyde arthritis whereas once daily administration was not effective. Shea butter also caused a significant (P<0.05) non-dose related inhibition of granuloma tissue growth on implanted cotton pellets. These findings showed that shea butter possesses antinflammatory action for both acute and chronic inflammations and thus provide a scientific rationale for its use in treatment of disorders of inflammation in traditional medicine.
CHAPTER ONE
INTRODUCTION
1.1 Inflammation
Inflammation is a defensive response that begins after cellular injury which may be caused by microbes, physical agents (burns, radiation, and trauma), chemicals (toxins, caustic substances), necrotic tissues and/or immunological reaction (Villarreal et al., 2001). It is also defined as a reactive state of hyperemia and exudation from blood vessels with consequent redness, heat, swelling and pain which a tissue manifests in response to physical or chemical injury or bacterial invasion (Macdonald, 1988). It is a condition involving localized increase in the number of leucocytes and variety of complex mediator molecules (Santosh et al.,
2010).
The history of inflammation is as old as man’s existence in this planet. Today, it is recognized that inflammation is far more complex than might first appear from the simple definitions given above. It is a major response of the immune system to tissue damage and infection, although not all infection gives rise to inflammation (Punchard et al., 2004).
Therefore, inflammation is also the innate immune system response to attack on the body and a major and complex reaction of the body against infection upon tissue injury (Khan et al.,
2010).
Inflammation is diverse, ranging from the acute inflammation associated with Staphylococcus aureus infection of the skin (the humble boil), through to chronic inflammatory processes resulting in remodeling of the artery wall in atherosclerosis; the bronchial wall in asthma and chronic bronchitis, and the debilitating destruction of the joints associated with rheumatoid arthritis (Punchard et al., 2004).
However, inflammation is a protective attempt by the body to remove the injurious agent/stimuli and initiate a healing process although the complex events and mediators
involved in the inflammatory reaction can induce, maintain or aggravate many diseases (Sosa et al., 2002). Hence, the control of inflammation is of great relevance in the treatment of such pathologies.
Inflammation serves no useful purpose in certain disorders like rheumatoid arthritis and becomes actually a component of the disease rather than part of the healing process.
1.2 Causes of inflammation
As derived from the first definition, the causes of inflammation include;
1. Physical agents: These include trauma, ultraviolet radiation, ionizing radiation, burn
and excessive cooling.
2. Irritants and corrosive chemicals: Acids, alkali and oxidizing agents.
3. Microbial infections: This is a common cause of inflammation. Some organisms cause
immunologically mediated inflammation through hypersensitivity reactions as is
obtained with parasitic infections and tuberculosis inflammation. While viruses cause
death of individual cells by intracellular multiplication, bacteria release exotoxins and
endotoxins which result in inflammatory reactions.
4. Tissue necrosis: Tissue necrosis could be the result of lack of oxygen or nutrient
resulting to inadequate blood flow to tissues. This leads to tissue death causing a
potent inflammatory stimulus.
5. Hypersensitivity reactions: hypersensitivity occurs when an altered state of
immunological response causes an inappropriate or excessive immune reaction with
damages to tissues. These reactions have cellular or chemical mediators similar to
those involved in inflammation (Hiley and Barber, 2000).
1.3 Types of inflammation
Inflammation can be variously classified;
Based on causative agent: Inflammation can said to be aseptic (sterile) or septic.
Based on histological features: It can be specific or non-specific (Heymer, 1985).
Based on exudates: It can be granulomatous, ulcerative, hermorrhagic, necrotizing, pseudomembranous, fibrinous, catarrhal, serous or suppurative (Porth, 2007).
Based on onset/duration: It is classified as per acute, acute, sub-acute or chronic.
Clinically, however, depending on the defense capacity of the host and duration of response, there are two major types of inflammation
(a) Acute inflammation
(b) Chronic inflammation (Mohan,2006).
This also largely depends on extent of injury, type of injury and vascularity of tissue involved
(Khan et al., 2010).
1.3.1 Acute inflammation
Acute inflammation occurs at the time scale of hours to days and represents an initial effort to eliminate the injury. It is characterized by the cardinal signs of inflammation which are pain, redness, swelling, heat and loss of function. The first four (classical signs) were described by
Celsius (30 BC-38 AD), while loss of function was added later by Galen even though this attribute is disputed and the fifth sign has also been ascribed to Thomas Sydenham and
Virchow (Cotran et al., 1998; Chandrasoma and Taylor, 2005). The more the cardinal signs present, the more acute an inflammation is said to be.
The process of acute inflammation is initiated by cells already present in all tissues, mainly resident macrophages, dendritic cells, histocytes, Kupffer cells and mastocytes. At the onset of an infection, burn or other injuries, these cells undergo activation and release inflammatory mediators responsible for the clinical signs of inflammation. Vasodilation and its resulting increased blood flow cause the redness (rubor) and increased heat (calor). Increased permeability of the blood vessels results in exudation (leakage) of plasma proteins and fluids in the tissue (edema), which manifests itself as swelling (tumor). Some of the released
mediators such as bradykinin increase sensitivity to pain (hyperalgesia, dolor). The mediator molecules also alter the blood vessels to permit the migration of leukocytes, mainly neutrophils, outside of the blood vessels (extravasation) into the tissue. The neutrophils migrate along a chemotactic gradient created by the local cells to reach the site of injury. The loss of function (function laesa) is probably the fault of a neurological reflex in the response to pain.
In addition to cell-derived mediators, several acellular biochemical cascade systems consisting of preformed plasma protein act in parallel to initiate and propagate the inflammatory response. These include the complement system activated by bacteria, and the coagulation and fibrinolysis systems activated by necrosis, e.g. a burn or a trauma.
Finally, downregulation of the inflammatory response concludes acute inflammation.
Removal of the injurious stimuli halts the response of the inflammatory mechanism, which requires constant stimulation to propagate the process. Additionally, many inflammatory mediators have short half-lives and are quickly degraded in the tissue. Hence, inflammation ceases once the stimulus has been removed (Cotran et al., 1998).
1.3.2 Chronic inflammation
Chronic inflammation is a prolonged and persistent acute inflammation usually due to the presence of non-degradable pathogens, persistent foreign bodies and autoimmune reactions.
The prolongation leads to, a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue for the inflammation process. It is usually delayed in onset and may last up to several weeks, many months or years and primarily mediated by interferon γ (IFN-γ)and other cytokines, growth factors IL-1, IL-6, TNF-α, reactive oxygen species and hydrolytic enzymes (Ferrero-
Miliani et al., 2007). Chronic inflammation may arise following acute inflammation (e.g. pneumonia) and also without acute inflammation (e.g. tuberculosis, viral infection and
rheumatoid arthritis). It is generally irreversible (Howarth et al., 1991). Chronic inflammation may result in either tissues destruction (fibrosis) or a host of diseases such as hay fever, atherosclerosis, and rheumatoid arthritis. The mononuclear cells: monocytes, macrophages, lymphocytes, plasma cells and fibroblasts are usually involved in chronic inflammation. It is characterized by a dominating presence of macrophages in the injured tissue (Chandrosoma and Taylor, 2005).
1.4 The Inflammatory Response
Generally, the process of inflammation involves a series of events and each type of stimuli provokes a characteristic pattern of response. Three distinct phases mediated by different mechanisms are recognized in inflammatory response;
1. An acute transient phase characterized by local vasodilation and increased
capillary permeability;
2. A delayed subacute phase, most prominently characterized by infiltration of
leukocytic and phagocytic cells and;
3. A chronic proliferative phase in which tissue degeneration and fibrosis occur
(Robert and Morrow, 2001).
The mechanisms of host defense involve mainly the antibodies and leukocytes which are found in bloodstream. This explains why vascular phenomenon plays a key role in inflammation. During inflammation, blood vessels undergo series of changes that are designed to maximize the movement of plasma proteins and circulating cells out of the circulation and into the site of injury or infection.
Vasodilation is one of the earliest manifestations of acute phase of inflammation; sometimes, it follows a transient constriction of arterioles, lasting a few seconds. Vasodilation is quickly followed by increased permeability of the microvasculature, with the outpouring of protein- rich fluid into the extravascular tissues.
The hallmark of acute phase of inflammation is increased vascular permeability which leads to the escape of a protein-rich fluid (exudate) into the extravascular tissue. This loss of fluid results in increased viscosity of blood and stasis.
Subsequently, the hemodynamic condition changes, and more white cells assume a peripheral position along the endothelial surface. This process of leukocyte accumulation is called margination. As a result, individual and then rows of leukocytes tumble slowly along the endothelium and adhere transiently (a process called rolling), finally coming to rest at some point where they adhere firmly. In time, the endothelium can be virtually lined by white cells, an appearance called pavementing. After firm adhesion, leucocytes insert pseudopods into the junctions between the endothelial cells, squeeze through inter endothelial junctions and assume a position between the endothelial cell and the basement membrane. Eventually, they traverse the basement membrane and escape into the extravascular space. Neutrophils, monocytes, lymphocytes, eosinophils, and basophils all use the same pathway to migrate from the blood into tissues. The process of margination, rolling, adhesion and transmigration of leukocytes serve to deliver the leukocytes to the site of injury or infection (Khan and
Khan, 2010).
The following adhesion molecules are involved in the recruitment of circulating blood cells and consequent transmigration:
1. The integrins,
2. Immunoglobulin-like proteins known as intercellular adhesion molecule (ICAM) 1
and 2, and vascular cell adhesion molecule (VCAM),
3. The selectins (L-, P- and E-selectin) and
4. The mucin-like selectin ligands (Villareal et al., 2001).
The ultimate function of cellular events in inflammation is to deliver leukocytes to the site of injury or infection. Because resident tissue macrophages, mast cells, and endothelial cells
respond to injurious agents by secreting the cytokines TNF, IL-1, and chemokines
(chemoattractant cytokines), leukocytes migrate to tissues toward the site of injury by the process of chemotaxis, defined simply as locomotion oriented along a chemical gradient.
Leukocytes ingest offending agents, kill bacteria and other microbes, and get rid of necrotic tissue and foreign substances (Nairn, 2004).
1.5 Mediators of inflammation
Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. A variety of substances are released upon damage to cells while others are synthesized during the events that follow tissue injury
(Dray, 1995).
1.5.1 Plasma derived mediators
Mediators derived from plasma include complements, complement-derived peptides and kinins.
(a). The complement and complement derived peptides
The complement system is made up of serum and membrane bound proteins named due to their ability to augment the effect of other components of the immune system. Their functions include; i. Lysis of cell ii. Production of mediators that participate in inflammation and attract phagocytes, iii. Opsonisation of organisms and immune complexes for clearance by phagocytosis and iv. Enhancement of antibody-mediated immune responses (Nairn, 2004).
Released through the classic or alternative pathways of the complement cascade, complement-derived peptides (C3a, C3b, and C5a) increase vascular permeability, cause smooth muscle contraction, activate leukocytes, and induce mast-cell degranulation. C5a is
a potent chemotactic factor for neutrophils and mononuclear phagocytes (Edward,2014).
Mast cell degranulation triggered by complements is independent of IgE (Delves, 2014).
(b). The kinin system
The kinins are important inflammatory mediators. The most important kinin is bradykinin, a vasoactive protein which increases vascular permeability and vasodilation and, importantly, activates phospholipase A2 (PLA2) to liberate arachidonic acid (AA). The vasodilatory effect of bradykinin is largely mediated through stimulated release of endothelium derived nitric oxide, prostacyclin and EDHF (endothelium derived hyperpolarizing factor)
(Mombouli et al., 1992). Bradykinin causes smooth muscle contraction and is a major mediator involved in the pain response (Dray and Perkins, 1993).
1.5.2 Cell derived mediators
These mediators are derived from injured tissue cells or leukocytes recruited to the site of inflammation. Mast cells, platelets, and basophils produce the vasoactive amines- serotonin and histamine while macrophages and lymphocytes produce the cytokines.
(a) Histamine
Histamine causes arteriolar dilation, increased capillary permeability (Meager, 1999), contraction of nonvascular smooth muscle, and eosinophil chemotaxis. It can stimulate nociceptors responsible for the pain response. Its release is stimulated by the complement components C3a and C5a and by lysosomal proteins released from neutrophils. Histamine activity is mediated through the activation of one of four specific histamine receptors, designated H1, H2, H3, or H4, in target cells. Most histamine-induced vascular effects are mediated by H1 receptors. H2 receptors mediate some vascular effects but are more important for their role in histamine-induced gastric secretion. Less is understood about the role of H3 receptors, which may be localized to the central nervous system. H4 receptors are located on
cells of hematopoietic origin, and H4 antagonists are promising drug candidates to treat inflammatory conditions involving mast cells and eosinophils (allergic conditions) (Edward,
2014).
(b) Serotonin
Serotonin (5-hydroxytryptamine) is a vasoactive mediator similar to histamine found in mast cells and platelets in the gastrointestinal tract and central nervous system. Serotonin also increases vascular permeability, dilates capillaries, and causes contraction of nonvascular smooth muscles (Edward, 2014).
(c) Cytokines
Cytokines, including interleukins 1–10, tumor necrosis factor α (TNF-α), and interferon γ
(IFN-γ) are produced predominantly by macrophages and lymphocytes but can be synthesized by other cell types as well (Burke et al., 2006). Their role in inflammation is complex. These polypeptides modulate the activity and function of other cells to coordinate and control the inflammatory response. Two of the more important cytokines, interleukin-1
(IL-1) and TNF-α, mobilize and activate leukocytes, enhance proliferation of B and T cells and natural killer cell cytotoxicity, and are involved in the biologic response to endotoxins.
IL-1, IL-6, and TNF-α mediate the acute phase response and pyrexia that may accompany infection and can induce systemic clinical signs, including sleep and anorexia. In the acute phase response, interleukins stimulate the liver to synthesize acute-phase proteins, including complement components, coagulation factors, protease inhibitors, and metal-binding proteins.
By increasing intracellular Ca2+ concentrations in leukocytes, cytokines are also important in the induction of phospholipase A2 (PLA2). Colony-stimulating factors (GM-CSF, G-CSF, and
M-CSF) are cytokines that promote expansion of neutrophil, eosinophil, and macrophage colonies in bone marrow. In chronic inflammation, cytokines IL-1, IL-6, and TNF-α contribute to the activation of fibroblasts and osteoblasts and to the release of enzymes such
as collagenase and stromelysin that can cause cartilage and bone resorption. Experimental evidence also suggests that cytokines stimulate synovial cells and chondrocytes to release pain-inducing mediators (Edward, 2014). Chemokines are chemoattrractant cytokines
(Meager, 1999).
1.5.2.1 Lipid derived mediators
Lipid-derived autacoids play important roles in the inflammatory response and are a major focus of research into new antiinflammatory drugs. These compounds include the eicosanoids such as prostaglandins, prostacyclin, leukotrienes, and thromboxane A and the modified phospholipids such as platelet activating factor (PAF).
(a). Eicosanoids
Eicosanoids are synthesized from 20-carbon polyunsaturated fatty acids by many cells, including activated leukocytes, mast cells, and platelets and are therefore widely distributed.
Hormones and other inflammatory mediators (TNF-α, bradykinin) stimulate eicosanoid
2+ production either by direct activation of PLA2, or indirectly by increasing intracellular Ca concentrations, which in turn activate the enzyme. Cell membrane damage can also cause an
2+ increase in intracellular Ca . Activated PLA2 directly hydrolyzes AA, which is rapidly metabolized via one of two enzyme pathways—the cyclooxygenase (COX) pathway leading to the formation of prostaglandin and thromboxanes, or the 5-lipoxygenase (5-LOX) pathway that produces the leukotrienes (Samuelsson, 1983)
(i). Prostaglandins
Cyclooxygenase catalyzes the oxygenation of AA to form the cyclic endoperoxide prostaglandin G2 (PGG2), which is converted to the closely related PGH2. Both PGG2 and
PGH2 are inherently unstable and rapidly converted to various prostaglandins, thromboxane
A2 (TXA2), and prostacyclin (PGI1). In the vascular beds of most animals, PGE1, PGE2, and
PGI1 are potent arteriolar dilators and enhance the effects of other mediators by increasing small vein permeability. Other prostaglandins, including PGF2α and thromboxane, cause smooth muscle contraction and vasoconstriction. Prostaglandins sensitize nociceptors to pain- provoking mediators such as bradykinin and histamine and, in high concentrations, can directly stimulate sensory nerve endings. TXA2 is a potent platelet-aggregating agent involved in thrombus formation (Edward, 2014).
(ii). Leukotrienes
Found predominately in platelets, leukocytes, and the lungs, 5-lipoxygenase (5-LOX) catalyzes the formation of unstable hydroxyperoxides from AA. These hydroxyperoxides are subsequently converted to the peptide leukotrienes. Leukotriene B4 (LTB4) and 5- hydroxyeicosatetranoate (5-HETE) are strong chemoattractants stimulating polymorphonuclear leukocyte movement (Pelletier, 2003). LTB4 also stimulates production of cytokines in neutrophils, monocytes, and eosinophils and enhances the expression of C3b receptors. Other leukotrienes facilitate the release of histamine and other autacoids from mast cells and stimulate bronchiolar constriction and mucous secretion. In some species, leukotrienes C4 and D4 are more potent than histamine in contracting bronchial smooth muscle (Edward, 2014).
(iii). Platelet activating factor (PAF)
Platelet activating factor (PAF) is also derived from cell membrane phospholipids by the action of PLA2. PAF, synthesized by mast cells, platelets, neutrophils, and eosinophils, induces platelet aggregation and stimulates platelets to release vasoactive amines and synthesize thromboxanes. PAF also increases vascular permeability and causes neutrophils to aggregate and degranulate (Vane et al., 1998).
(b). Nitric oxide
The role of the free radical gas nitric oxide (NO) in inflammation is well established. NO is an important cell-signaling messenger in a wide range of physiologic and pathophysiologic processes. Small amounts of NO play a role in maintaining resting vascular tone, vasodilation, and anti-aggregation of platelets. In response to certain cytokines (TNF-α, IL-1) and other inflammatory mediators, the production of relatively large quantities of NO is stimulated. In larger quantities, NO is a potent vasodilator, facilitates macrophage-induced cytotoxicity, and may contribute to joint destruction in some types of arthritis (Willoughby and Flower, 1993; Edward, 2014).
(c). Substance P
This is a neuropeptide that works hand in hand with cytokines to mediate vascular permeability and stimulate immune cell secretion. It also transmits pain and regulates blood pressure (Burke et al., 2006).
1.6 Biomarkers of inflammation
Biomarker is anything that can be used as an indicator of a particular disease state or some other physiological state of an organism. Inflammation is known to induce high levels of acute phase proteins which include C-reactive protein, serum amyloid A, serum amyloid P, fibrinogen and vasopressin. These proteins mediate a range of systemic effects including fever, malaise and somnolence (Cotran and Kumar, 1998).Apart from acute phase reactant proteins, other biomarkers include increases in white cell count, ESR, albumin (Hiley and
Barber, 2000) and increases in cytokines especially interleukin1-6 and TNFα. Also the adhesion molecules are increased especially E-selectin, P-selectin, intercellular and vascular cell adhesion molecules.
Plasma levels of circulating biomarkers of inflammation are believed to reflect the severity of inflammation and extent of underlying condition. C-reactive protein (CRP) – the acute phase protein synthesized primarily by the liver that is stable and readily measured – is currently the most widely used biomarker of inflammation. Albumin, the most abundant protein in the body is also employed (Khan et al., 2010).
1.7 Disorders of Acute Inflammation
Disorders associated with acute inflammation comprise of large, unrelated abnormalities that underlie a vast variety of human diseases. Acute inflammation has been said to underlie skin inflammation/blisters, myocardial infarction, appendicitis, prostatitis, bronchopneumonia and gastric ulcer (Beck, 2014).
1.8 Disorders of Chronic Inflammation
Chronic inflammatory diseases are defined by long-term inflammatory processes directed at a particular endogenous or exogenous antigen. However, some non-immune diseases have their etiological origin in inflammatory processes e.g. cancer, atherosclerosis and ischaemic heart disease (Cotran et al., 1998). Classical chronic inflammatory disorders include; arthritis, inflammatory bowel disease, idiopathic pulmonary fibrosis. Within them however, there is considerable overlap with autoimmune conditions such as Multiple Sclerosis and Type 1 diabetes (Heap, 2009).
More contemporary revelations show chronic inflammation to be a major factor in the development of degenerative diseases and loss of youthful functions. Human aging is characterized by a chronic, low-grade inflammation, and this phenomenon has been termed
“inflammaging.” Inflammaging is a highly significant risk factor for both morbidity and mortality in elderly people, as most if not all age-related diseases share an inflammatory pathogenesis (Franceschi and Campisi, 2014).
1.8.1 Arthritis
Arthritis (which literally means inflammation of the joint) is defined as inflammation of the intra-articular tissue of one or more joints and characterized by an increased volume of intra- articular fluid with specific features that vary depending on the cause. Features that may have diagnostic significance include color, turbidity, hemorrhage, or exudate. In addition, arthritis may be classified according to the cause, duration (acute or chronic), or the components of the exudates (serous, fibrinous, purulent, macrophagic or lymphoplasmacytic) (Weisbrode and Doige, 2001).It often results in stiffness, soreness, and in many cases, swelling. It is a common disease with peak incidence in third to fourth decades of life and 3 to 5 times higher in females (Mohan, 2000).
1.8.1.1 Types of arthritis
Inflammatory and non-inflammatory arthritis are the two most common forms of arthritic condition. However, there are dozens of different arthritis types (Godman, 2013).
1.8.1.1.1 Non-inflammatory arthritis
(a). Osteoarthritis
Non-inflammatory arthritis refers mainly to osteoarthritis (OA). Though it still results in inflammation of the joints, this inflammation is the result of wear and tear. In particular, OA results from the breakdown of cartilage which allows the bones to rub together. This can be painful. It usually occurs later in life. Injuring the joint can accelerate the progression of OA, but even everyday activities can contribute to OA later in life. Being overweight and putting extra strain on the joints can also cause OA. Non-inflammatory arthritis is most commonly found in the knees, hips, spine, and hands (Godman, 2013). It can also cause problem in shoulder or any other joint.
(b). Gout (metabolic arthritis)
This occurs when uric acid crystals form in and around joints, causing sudden and intense pain, redness and swelling.
1.8.1.1.2 Inflammatory arthritis
Inflammatory arthritis is a term used to describe a group of conditions which affects the immune system.
(a). Septic arthritis
This is as a result of infection in a joint that causes pain, redness, heat and swelling of the affected joint. The patient may also have a fever and feel unwell. The most commonly affected joint is the knee, but it can also occur in the ankle, hip or wrist. This is a serious condition that needs treatment with antibiotics and an operation in which the joint is washed out.
(b). Rheumatoid arthritis (RA)
Rheumatoid arthritis is an autoimmune disease in which there is joint inflammation, synovial proliferation and destruction of articular cartilage (Tripathi, 2003). It is a typical example of inflammatory arthritis. It is a chronic disorder that affects about 1% of the population globally with females 3 times more prone to attack than males (Shikha, 2010). The condition is associated with progressive disability, systemic complications, early death and socioeconomic costs (Firestein, 2003).This type of arthritis is less common but more severe.
It occurs when the immune system attacks the tissue lining the joints, and can lead to pain, swelling, stiffness and joint deformity. The joints most affected are the hands, wrists, shoulders, knees or feet. The cause is not known.
Various leucocyte populations, orchestrated by several cytokines, chemokines, growth factors and hormones, infiltrate rheumatoid tissues and increase injury (Montecucco and Mach,
2009). The lipid mediator PGE2 is produced during inflammatory responses and is thought to
be a major PG species working in RA pathogenesis, since a high level of PGE2 is detected in the synovial fluid and tissues of RA patients (Sano, 2011) and PGE2 exhibits pleiotropic biological actions. For example, PGE2 mediates pain and inflammatory responses (Walsh et al., 2005).
Rheumatoid arthritis is said to be characterized by synovial inflammation and hyperplasia
(“swelling”), autoantibody production (rheumatoid factor and anti–citrullinated protein antibody [ACPA]), cartilage and bone destruction (“deformity”), and systemic features, including cardiovascular, pulmonary, psychological, and skeletal disorders (Mclnnes and
Schett, 2011).
It is diagnosed by rheumatoid factor, which are abnormal antibodies (IgG) which are present in blood. These are reacted with antigen and form antigen-antibody complex that leads to pain and inflammation of synovial membrane (Kaur et al., 2012). The American College of
Rheumatology requires at least four of the following seven criteria to confirm the diagnosis
(Rindflisch and Muller, 2005).
i. Morning stiffness around the joint that lasts at least 1 hour
ii. Arthritis of three or more joints for at least 6 weeks
iii. Arthritis of hand joints for at least 6 weeks
iv. Arthritis on both sides of the body for at least 6 weeks
v. Rheumatoid nodules under the skin
vi. Rheumatoid factor present in blood testing vii. Evidence of rheumatoid arthritis on X-rays
1.9 Agents used in management of inflammation/ arthritis
Ideally, antiinflammatory drugs should have;
1) Effect on the prime causative factor,
2) Inhibitory or blocking effect on initial reaction set in a biological model by the prime
cause and thereby inhibit the established inflammation and
3) Effect on end result of established inflammation which probably modifies non-
specifically the underlying symptoms of inflammation or enhances the repair process.
(Naik and Shett, 1976).
The goals of treatment of rheumatoid arthritis are to alleviate pain, control inflammation, preserve and improve activities of daily living, and prevent progressive joint destruction.
Treatment of rheumatoid arthritis is characterized by a steady evolution of new agents and new approaches. For over 50 years, medical treatment has been based on the use of non- steroidal anti-inflammatory drugs (NSAIDs), corticosteroids and synthetic disease-modifying antirheumatic drugs (sDMARDs) (Favalli et al., 2014). Equally important in the management of RA is nonmedical treatment, including patient education, physical therapy, occupational therapy, orthotics, and rarely, surgery (Jisna et al., 2014).
1.9.1 Non- sterodial anti-inflammatory drugs
The non-steroidal antiinflammatory drugs (NSAIDs) are chemically heterogeneous groups of compounds, which though chemically unrelated, share certain therapeutic actions and adverse effects (Burke et al., 2006). They alleviate pain by counteracting the cycloxygenase (COX) enzyme which synthesizes prostaglandins, creating inflammation. Most of these drugs have in addition to their antiinflammatory property, analgesic and antipyretic activities. However, these drugs cause adverse gastric reactions, inhibit renal function, reduce the efficacy of diuretics and retard the angiotensin converting enzyme inhibitors (Dobrilla et al., 1997; Gaddi et al., 2004). Some long term use of NSAIDs can cause gastric erosions leading to stomach ulcers and in extreme cases, severe hemorrhage resulting in death (Hayliyar et al., 1992;
Ament and Childers, 1997). Other adverse effects of NSAIDs are exacerbation of asthma and
kidney damage (Rang et al., 2007).NSAIDs are among the most frequently prescribed drugs in modern medicine. They are very effective in the alleviation of pain, fever and inflammation, and millions of patients worldwide have found relief in their use since the discovery of the soothing properties of willow bark more than 3,500 years ago (Meek et al.,
2010). In 1963, indomethacin was introduced to treat rheumatoid arthritis, and this was followed by the development of many other antiinflammatory agents.
NSAIDs can be broadly grouped into the non-selective COX inhibitors and selective COX-2 inhibitors. Some drugs, notably pyrazolones and acetaminophen, were previously not classified into this group because they did not inhibit COX enzymes. In recent years, new
COX isoenzymes have been described, such as COX-2b and COX-3 that can be selectively antagonized by these drugs, and therefore would fit into the NSAID category
(Chandrasekharan et al., 2002).
1.9.1.1Non-selective COX inhibitors
The non-selective COX inhibitors affect both COX-1 and COX-2 enzymes. They include the salicylic acid derivatives, indole and indene acetic acids, arylpropionic acids, anthranillic acids, heteroaryl acetic acids, enolic acids and the alkanones (Burke et al., 2006).
(a) Salicylic acid derivatives
These comprise esters of salicylic acids and salicylate esters of organic acids and salt of salicylic acid (Burke et al., 2006). They have antipyretic, analgesic and anti-inflammatory properties. A typical one is aspirin which is widely consumed and is the standard for comparison and evaluation of the others (Amann and Peskar, 2002).
Apart from their effect on biosynthesis of prostaglandins, it has been observed that salicylates have the capacity to suppress a variety of antigen-antibody reactions e.g. inhibition of anti- body production, for antigen-antibody aggregation and of antigen-induced release of
histamine (Guyton and Hall, 2007). At high dose, salicylates also inhibit the activation of NF-
κB in vitro (Yin et al., 1998).
(b). Indole and indene acetic acids
Indomethacin is a methylated indole derivative with prominent antiinflammatory property.
Others include sulindac, a prodrug whose antiinflammatory activity resides in its sulfide metabolite (Haanen, 2001).
(c). Oxicams
Oxicams inhibit COX-I and II and possess good antiinflammatory properties. Oxicams include piroxicam, meloxicam and tenoxicam (Nilsen, 1994; Burke et al., 2006).Meloxicam, has a lower frequency for gastrointestinal side effects than piroxicam and several other
NSAIDs (Karen and Knox, 2007).
(d). Alkanones
Nabumetone was approved in 1991 and has substantial efficacy in treatment of rheumatoid arthritis and osteoarthritis (Davies, 1997). It is principally 6-methoxy-2-naphythyl acetic acid, potent non-selective inhibitor of COX enzyme (Patrignani et al., 2003).
(e). Heteroaryl acetic acids
These include tolmetin and ketorolac which are structurally related but with different pharmacological features. Tolmetin was introduced in 1976 in the USA possessing typical
NSAID properties with strong antiinflammatory effects (Morely et al., 1982). Ketorolac has moderately effective antiinflammatory effect with more potent analgesic property (Buckley and Brodgan, 1990). Diclofenac on the other hand is a phenylacetic acid derivative specifically developed as an antiinflammatory agent which also reduces intracellular concentration of free arachidonate in leucocytes (Ebadi, 1997).
(f).Arylpropionic acids
These are approved for use in symptomatic treatment of rheumatoid arthritis, ankylosing spondylitis, acute gouty arthritis and osteoarthritis. Ibuprofen is the most commonly used and first member of the class to come into general use. Others include naproxen, ketoprofen, flurbiprofen, fenoprofen and oxaprozin (Burke et al., 2006).
(g).Anthranillic acids
The phenylanthranillic acid was discovered in the 1950s as derivatives of N-phenylacetic acids. They comprise the fenamates such as mefenamic acid, meclofenamic acid and flufenamic acid (Burke et al., 2006; Guyton and Hall, 2007). They show no clear superiority in antiinflammatory activity and may produce more adverse effects than other NSAIDs
(Karen and Knox, 2007).
1.9.1.2Selective COX-2 inhibitors
These are agents that selectively inhibit COX-2 while sparing COX-1 enzymes. They include the diaryl substituted pyrazole (celecoxib) analogous to diclofenac, the diaryl substituted furanone (rofecoxib), the indole acetic acid (etodolac), the sulphonanilide (Nimesulide)
(Robert and Morrow, 2001) and the new agents valdecoxib, peracoxib, lumiracoxib and etoricoxib. Valdecoxib and rofecoxib have been withdrawn from the market in view of their adverse event profile. The relative degree of selectivity for COX-2 inhibition by these agents is shown below with the first approved member having the lowest degree of COX-2 selectivity. Lumiracoxib=Etoricoxib>Valdecoxib=Rofecoxib>>Celecoxib (Brune and Hinz,
2004). Actually, COX-2 inhibitors are effective for decreasing pain in RA with less gastrointestinal side effects (Sano, 2011), although some concerns of risk of cardiovascular events have been expressed (Mukherjee and Topol, 2003).
1.9.2 Steroidal antiinflammatory drugs (corticosteroids)
Glucocorticoids reduce inflammation or swelling by binding to cortisol receptors. These drugs are often referred to as corticosteroids. They suppress inflammation rather than address its underlying causes by inhibiting phospholipase A2 and suppressing expression of COX II and the prostaglandin production it mediates. Corticosteroids also inhibit the transcription of many genes coding for pro-inflammatory proteins like the cytokines (Newton et al., 1998).
The glucocorticoids are the most potent antiinflammatory drugs available (Ukwe, 2004).
Examples are prednisolone and dexamethasone.
1.9.3 Disease modifying anti-rheumatoid drugs (DMARDs)
The disease-modifying anti-rheumatic drugs are a category of unrelated drugs defined by their use in rheumatoid arthritis to slow down disease progression. The term is often used in contrast to non-steroidal antiinflammatory which refers to agents that treat the inflammation but not the underlying cause. Although their use was first propagated in rheumatoid arthritis
(hence their name), the term has come to pertain to many other diseases, such as Crohn’s disease, systemic lupus erythematosus (SLE), idiopathic thrombocytopenic purpurea (ITP), myasthenia gravis and various others. Many of these are autoimmune disorders (Laurence et al., 2006). The DMARDs include hydroxychloroquine, tumor necrotic factor (TNF) inhibitors such as adalimumab, etanercept and golimumab; gold salts like sodium aurothiomalate and auranofin; methotrexate, azathioprine, cyclophosphamide, biologics (infliximab), leflunomide and D-penicillamine.
Some DMARDs are mild chemotherapeutic agents but use immunosuppression, a side-effect of chemotherapy as its main therapeutic benefit. The term was originally introduced to indicate drugs that reduce evidence of processes thought to underlie inflammatory diseases, such as a raised erythrocyte sedimentation rate, reduced haemoglobin level, raised
rheumatoid factor level and more recently, raised C-reactive protein level. More recently, the term has been used to indicate a drug that reduces the rate of damage to bone and cartilage.
DMARDs can be further subdivided into traditional small molecular mass drugs synthesized chemically and newer ‘biological’ agents produced through genetic engineering (Nandi et al.,
2008).
DMARDs work by curbing the underlying processes that cause certain forms of inflammatory arthritis including rheumatoid arthritis (RA), ankylosing spondylitis and psoriatic arthritis. These drugs not only treat arthritis symptoms, but they can also slow down progressive joint destruction. Some of these medications have been used to treat other conditions, such as cancer or inflammatory bowel disease, or to reduce the risk of rejection of a transplanted organ. Although these agents operate by different mechanism, many of them can have similar impact on the course of a condition (Nandi et al., 2008).
Combinations of DMARDs are often used together, because each drug in the combination can be used in smaller dosage than if it were given alone, thus reducing the risk of side effects (Capell et al., 2007). DMARDs help control arthritis but do not cure the disease or cause a “rebound flare” with no assurance that disease control will be reestablished upon resumption of the medication (Nandi et al., 2008). They have varied mechanisms of action.
For instance, sulfasalazine suppresses IL-1 and TNF-alpha, induces apoptosis of inflammatory cells and increases chemotactic factors. The gold salt inhibits macrophage activation; cyclosporine inhibits calcineurin while methotrexate and azathiopurine have antifolate and purine synthesis inhibitory activities (Capell et al., 2007) respectively.
Methotrexate (MTX) has long been considered the ‘gold standard’ disease modifying anti- rheumatic drug (DMARD) for RA (McGeough et al., 2011). Methotrexate was first introduced in rheumatology in 1962 for treatment of psoriatic arthritis (Black et al., 1964) based on the wrong assumption of a possible interference with proliferation of connective
tissue. Its mode of action was not clear but the increase in adenosine levels and reduction in proinflammatory cytokines seems to play a more predominant role than inhibition of cell proliferation (Cutollo et al., 2002). Methotrexate has been recognized as the DMARD with the most long term effectiveness and safety for RA before the introduction of biologic agents
(Sokka and Pincus, 2002).
With the identification of TNF as a key player in inflammatory and destructive pathway of the disease, interest was shifted away from agents with poorly understood mechanism of action towards therapies targeted to key molecules and cells involved in RA pathogenesis
(Feldmann et al., 1996). The first biological agents that were registered were tumour necrosis factor alpha (TNFα) inhibitors: etanercept (FDA approved 1998) and infliximab (1999), followed by adalimumab (2002). Infliximab is a chimeric monoclonal antibody, administered intravenously. Etanercept is a fusion protein consisting of two identical chains of the recombinant human TNF-receptor p75 monomer fused with the Fc domain of human IgG1, and adalimumab is a human monoclonal antibody against TNFα. The latter 2 drugs are both administered subcutaneously. Advances in understanding the role of T cells, B cells and cytokines such as IL-6 has paved way to development of additional biologic drugs beyond
TNF inhibitors.
1.9.4 Biologic disease modifying anti-rheumatic drugs (bDMARDs)
These biologic agents(bDMARDs) include: Anakinra, a recombinant form of the IL1 receptor antagonist (IL1-RA, administered subcutaneously); Rituximab, a β-cell depleting agent;
Abatacept, a recombinant dimerized form of cytotoxic T-lymphocyte antigen 4 (CTLA4) that blocks T-cell co-stimulation, administered intravenously; Tocilizumab, a human monoclonal antibody against the IL-6 receptor administered intravenously; and two recent TNFα blockers, which are both administered subcutaneously: certulizumab pegol, a pegylated Fab
fragment from a humanized monoclonal antibody; and golimumab, a human monoclonal antibody (2009)) (Bugatti et al., 2007; Fonseca et al., 2009; Hetland, 2011 ). They have shown good efficacy and safety in patients with RA and are now widely used in clinical practice (Favalli et al., 2009; Atzeni et al., 2013).
1.9.5 Medicinal plants with antiinflammatory activity
Since time immemorial, indigenous plants have been a major source of medicine because the different constituents present in them have immense therapeutic value (Meher et al., 2011).
About 80%of people in developing countries still rely on traditional medicines of both plant and animal origin. Side effects remain one of the problems of the long term use of medicines as required in inflammatory rheumatoid arthritis and other inflammatory conditions. Thus there is need for anti-arthritic drug with less severe side effects. Interest in alternative treatment of arthritis (Gaby, 1999; Jacobs et al., 2001) has promoted use of alternative medicine in western world but scientific evidence of anti-arthritic efficacy is lacking in some cases. Also, there is growing realization that apart from being safer, economical and easily available, herbs, phytochemicals and herbal products can influence the course of inflammatory diseases and may provide an amalgamation of nutritional substances, which help in restoring and maintaining wear and tear of tissues (Santosh et al., 2010). The use of herbal medicine is becoming popular due to toxicity and side effect of allopathic medicine and potential benefit of herbs.
Plants are important source of new therapeutic agents. Some medicinal plants that have been studied for antiinflammatory activity include Aspila africana (Okoli et al., 2006b),
Schewenckia americana. L. (Solancaceae) (Nwabunike et al., 2014), Securidaca longipedunculata Fres. (Polygalaceae) (Okoli et al., 2006a), Culcasia scadens P. Beauv
(Okoli and Akah, 2000), Acanthus monthanus (Okoli et al., 2008), Polygonum cuspidatum
(Bralley et al., 2008) and Sphaeranthus indius (Asteraceae) (Meher et al., 2011).A comprehensive review on antiinflamatory activity of plants has shown medicinal plants as reservoir for development of potent and safer drugs (Okoli et al., 2003). On the other hand, plants used in rheumatoid arthritis include Piper nigrum L. (Piperaceae), Calotropis procera
L. (Asclepiadaceae) and Mangifera indica L. (Anacardiaceae) (Kaur et al., 2012).
Plant constituents that can modulate the expression of pro-inflammatory signals have potential against arthritis. They include flavonoids, terpenoids, quinines, catechins, alkaloid, anthocyanins and anthoxanthins.
Flavonoids are known to have anti-inflammatory activity (Bellik and Laid, 2013). It has been elucidated that flavonoids are major anti-inflammatory agents. Some of them act as phospholipase inhibitors while others act as TNF-α inhibitors in different inflammatory conditions. Biochemical investigations have also shown that flavonoids can inhibit both cyclooxygenase and lipoxygenase pathways of arachidonic metabolism depending upon their chemical structures (Jang et al., 2002).Terpenoids significantly inhibit the development of chronic joint swelling. Terpenoids may affect different mechanisms relevant to inflammation arising in response to varied etiological factors (Changa et al., 2008). Antiinflammatory and antinociceptive activity of terpenoids has been reported (Santos and Rao, 2000).It has also been reported that terpenoids are natural inhibitors of NF-kB signaling with both antiinflammatory and anticancer potentials (Salminen et al., 2008). The topical antiinflammatory action of sesquiterpene is caused by inhibition of arachidonic acid metabolism (Kumar et al., 2013). Phenolic compounds are reported to have antiinflammatory and antioxidant activities (Frautschy et al., 2001).Alkaloids based on pyridine ring system have been reported to have striking anti-inflammatory activity, e.g Berberine from Berberis is a traditional remedy against rheumatism (Kupeli et al., 2002).
Plants commonly used to treat arthritis include Allium sativum, Allium cepa (Liliaceae), Aloe vera (Liliaceae), Zingiber officinale (Zingiberaceae), Ginkgo biloba (Ginkgobaceae) and
Ananas comosus (Bromeliaceae) amongst others (Vikrant et al., 2011).
Moreover, scientific studies on anti-arthriticactivity have been carried out with significant positive result in a lot of the medicinal plants; e.g.Costus speciousus koen (Costaceae) (Shruti et. al., 2012), Strychnos potatorum (Longiniaceae) (Ekambaram et. al., 2010) and bark extract of Alangium salvifolium Wang (Alangiaceae) (Jubie et al, 2008). Carpolobia lutea was reported to possess both antiinflammatory and anti-arthritic properties (Iwu and Anyanwu,
1982) and has also been studied for analgesic action (Jackson et al., 2011). However, many herbal medicines for inflammation and rheumatism have not undergone thorough scientific investigations.
1.10 Botanical profile of Vitellaria paradoxa
1.10.1 Plant Taxonomy
Kingdom - Plantae
Division - Tracheophyta
Class - Angiospermae
Sub-class - Eudicots
Orders - Ericales
Family - Sapotaceae
Genus - Vitellaria (C.F Gaertn)
Specie - Vitellaria paradoxa
Subspecie - Vitellaria paradoxa nolitica
Synonyms - Butyrospemum parkii (Kotschy) and Butyrospemum paradoxum
(Henry and Nair, 1983).
Vernacular names - “Shea tree” (English),
“Dan ka’raye”, “K’awara” and “Ka’danya” (Hausa),
“Aku makpa” or “Emi-emi” (Yoruba),
“Okwuma” (Igbo) (Hall et al., 1996).
1.10.2 Plant description
The shea tree, which resembles an oak (Quercus species) in its general size and form, grows up to 20 m (65 ft) tall and 1 m (3 ft) in diameter, with a dense, many-branched crown. It is deciduous, but appears evergreen, because new leaves emerge as the old ones fall.
The bark is thick and corky, deeply fissured both horizontally and longitudinally, and fire- resistant. It is slash pale pink, secreting white latex when cut as do the broken twigs or petioles (Orwa et al., 2009)
The leaves are tough, leathery and elliptical to oblong, with entire margins, and are clustered at the branch tips or twig (alternate to whorled). Juvenile leaves rust-red and pubescent, later coriaceous, glabrous and shiny dark green. The leave is 12-25 cm long and 4-7 cm wide.
The flowers are cream to brown, in dense terminal clusters, and are insect-pollinated. The flowers develop in the axils of scale leaves, at the extremities of dormant twigs, from buds formed 2 years previously. Each inflorescence usually contains 30-40 flowers, though 80-100 have been recorded. Individual flowers are subtended by scarious, brown, ovate or lanceolate bracteoles, which are abscised before flower opening (Orwa et al., 2009).
The fruits are round to elliptical drupes, 3 to 6 cm (1.1-2.25 in) long, borne on peduncles
(fruit stalks) 1 to 3 cm (0.5 to 1.25 in) long. The fruits are drupes, with fleshy greenish yellow pulp surrounding a hard- but thin-shelled seed, an egg-shaped kernel that weighs around 3g
(about 1/10 of an ounce) (Kar and Mital, 1981).The shea nut is surrounded by a fragile shining shell with a large, round, rough hilum on a broad base (Orwa et al., 2009).
1.10.3 Geographical distribution
The shea tree is an indigenous plant to West Africa occupying Mali, Cameroon, Cote d’voire,
Ghana, Guinea, Togo, Nigeria, Sudan, Senegal and Ethiopia (Okullo et al., 2004) Burkina
Faso and Uganda (Lovett and Haq, 2000). However, the genus Vitellaria is considered by botanical authorities as monospecific. The subspecie V. Paradoxa is restricted to Western
Africa while V. nilotica to Eastern Africa.
1.10.4 Ethnomedicinal uses
The fruit pulp of V. paradoxa is edible and is said to have a laxative effect (Soladoye, et al.,
1989). Its roots and barks are ground to paste and administered orally to cure jaundice
(Ampofo, 1983). Over the years, the fatty extract of the seeds of V. paradoxa (shea butter) has been used in cosmetology and confectionary. The shea bark has been employed to treat minor cuts and scratches. The leaves can be eaten raw while extracts of the leaves are used to relieve headache and as an eyebath. The nut shell has in-built mosquito repellant and the butter is used as moisturizing creams and lotions and for soap and chocolate manufacturing
(Abidemi et al., 2009; George et al., 2011). It has been claimed to have antiinflammatory, emollient and humectant properties (Akihisia et al., 2010).
The main use of shea butter in the Western world is in chocolate manufacturing where the similarity in composition and crystallisation properties between shea butter and cocoa butter is utilized. Shea butter has served as an antiinflammation balm and used to heal bruises, dermatitis and all forms of massage therapy. Shea butter is used as a base for medicinal and cosmetic ointment, as pomade, as a hair cream, for soap production and as an illuminant
(Abbiw, 1990).
Unrefined shea butter is claimed to be good for dry skin, skin rashes, skin peeling after tanning, sunburn, blemishes, cracked heels and skin, itchy skin, frost bite, stretch marks,
scars, chapped lips, eczema, small wounds or scrapes, diaper rash, hair moisturizer, burns, athlete's foot, insect bites and stings, arthritis, muscle fatigue, pets' (dogs and horses) dry skin, sunburn, scrapes, and as a natural mechanics lubricant (Wan and Wakilyn, 1997).
In southeast Nigeria, shea butter is a common remedy for swellings. It is used as massage ointment in arthritis, as cough remedy and nasal decongestant in children. It is also used to enhance hair growth in women.
1.10.5 Literature review
Studies on V. paradoxa have ranged from domestication of shea tree (Yidana, 2003), increase in yield efficiency and purification of shea butter to morphological character variation studies of shea tree (Djekota et al., 2014). Because of its role in combating food insecurity and sustaining rural livelihoods, an assessment of the nutritional composition of shea fruit pulp was carried out in Uganda. It was found to be rich in vitamin C, total carbohydrates and crude fiber (Okullo et al., 2010). Phytochemical screening of the plant parts revealed the presence of carbohydrates (free reducing sugars, ketoses, pentoses and starch), saponins, steroids, tannins and alkaloids (Ndukwe et al., 2007).A unique healing property has been ascribed to shea tree which justifies the name ‘tree of life’ though there is limited scientific backing to the healing claims. Its fatty extract (shea butter) has been shown to be efficacious in nasal congestion as decongestant (Tella, 1979). The bioactive substance in shea butter resides in the unsaponifiable fraction and includes vitamin E, catechins and triterpenes (cinnamic acid esters, alpha- and beta-amyrin, parkeol, buytospermol, and lupeol) (Badifu, 1989).Shea butter is composed of five principal fatty acids: palmitic, stearic, oleic, linoleic, and arachidic. The fatty acid composition is dominated by stearic and oleic acids, which together account for 85-
90% of the fatty acids. The relative proportions of these two fatty acids produces differences in shea butter consistency (Maranz et al., 2004).
1.11Aim and scope of study
This study was to investigate the antiinflammatory activity of the fatty extract of V. paradoxa
(shea butter), to ascertain the basis for its use in inflammatory disorders. The study involved the determination of the prevalence of use of shea butter amongst clinically diagnosed arthritic patients using pre-tested questionnaire and investigation of the antiinflammatory activity using rodent models of acute and chronic inflammation.
CHAPTER TWO
MATERIALS AND METHODS
2.0 Materials and Methods
2.1 Materials
2.1.1 Animals
Adult Swiss albino rats (80 – 230 g) and mice (15– 23 g) of either sex were used. The animals were obtained from the laboratory animal facility of the Department of
Pharmacology and Toxicology, University of Nigeria, Nsukka and housed in steel cages under standard conditions with natural lighting within the facility. The animals were allowed free access to clean drinking water and standard feed pellets. Prior to commencement of the experiment, the animals were allowed a 2 week acclamitization period. Animal experiments and handling were in accordance with the National Institute of Health Guide for care and use of Laboratory Animal (pub No. 85-23, revised 1985).
2.1.2 Chemicals and solvents
Carrageenan, Ketamine, xylene, chloroform, formalin (2%v/v), Normal saline, Olive oil
2.1.3 Drugs
Indomethacin (Indolab®, Embassy Nig. Ltd. Nigeria) and methyl salicylate cream
(Neurogesic®, Drugfield Nig. Ltd. Nigeria).
2.1.4 Equipment
Animal weighing balance, cotton wool (Sterilized), Oven, Surgical blade, Sterile silk suture, test tubes, measuring cylinder, electronic weighing balance, mortar and pestle.
2.1.5 Patients
Patients that were clinically diagnosed of arthritis (as evidenced from the case notes) and visited National Orthopaedic Hospital, Enugu between July and November 2012 were recruited. Patients at all ages and of any gender were included in the study. Patients who have arthritis but did not or had not presented the complaint to the doctor (new patients and patients with other complaints other than arthritis) were excluded from the study. Accident victims were also excluded.
2.1.6 Shea butter
The shea butter used in this study was purchased from dealer in new market Enugu in January
2013. The shea butter was produced by the dealers using local extraction technique. The technique involved cracking the shea seed to remove the outer shell, drying the exposed nut under sun before crushing, frying/roasting the crushed nut before grinding it to smooth paste.
The paste was kneaded by hand in large basins and water is gradually added to help separate out the butter oils. As they float to the top, the butter oils, which are in a curd state, are removed and excess water squeezed out. The butter oil curds are then melted in large open pots over slow fires. A period of slow boiling will evaporate any remaining water. The shea butter, which is creamy or golden yellow at this point, is ladled from the top of the pots and put in cool places to harden. It was put in airtight container and stored at room temperature until used.
2.2 Methods
2.2.1 Data collection on prevalence of use
Pre- tested questionnaire was issued to patients with arthritis at the Outpatient Department of
National Orthopaedic Hospital, Enugu after due explanation and consent sought orally. The
questionnaires were returned after completion on daily basis (Monday-Thursday). A total of
164 arthritis patients responded. Illiterate patients were however interviewed based on the content of the questionnaire (Appendix 1). The questionnaire comprised of three sections ;
(a). Demographic characteristic of the patient (b). Knowledge of disease condition and (c).
Knowledge and shea butter use pattern amongst the arthritis patients.
2.2.2 Pharmacological activity tests
2.2.2.1 Systemic acute inflammation of the rat paw
The method of Winter et al. (1962) was used. Adult albino rats (80-230 g)of both sexes were divided into 5 groups of 5 animals each, Groups 1, 2 and 3 were the treatment group and received oral administration of 100,200 and 400 mg/kg of shea butter (suspended in olive oil) respectively. The shea butter was suspended in olive oil. Group 4 received indomethacin (10 mg/kg) while group 5 was given an equivalent volume of olive oil. One hour after shea butter administration, the rats were injected with carrageenan (0.1 ml, 1% solution in normal saline) into the sub plantar region of the right hind paw. The paw size was measured by volume of distilled water displaced before (0 h) and at 0.5, 1, 2, 3,4 and 5 h after carrageenan injection.
Increase in paw volume was calculated as the difference in paw volume at zero time and its volume at different times after carrageenan injection. The level of inhibition (%) of edema was calculated for each group using the relation:
Inhibition (%) = 100 [1-(Vt/Vc)]
Where
Vt: Average paw volume of the treated group
Vc: Average paw of the control group
2.2.2.2 Topical acute ear edema in mice
The effect of shea butter on topical acute inflammation was studied using xylene induced ear edema. Adult albino mice (15-23 g) were divided into 3 groups of 5 animals each. Group 1
received topical application of shea butter (5 mg/ear). Group 2 received indomethacin (10 mg/ear) on the anterior surface of the right ear while group 3 (control) received an equivalent volume of olive oil. Xylene (0.05 ml) was instantly applied on the posterior surface of same right ear for all animals. The left ear was left untreated and served as control. The animals were sacrificed 3h after xylene application and a circular disk of uniform diameter punched out with a cork borer (6 mm diameter) and weighed. The difference in weight of discs from treated and untreated ears was calculated and used as a measure of inflammation (Attah and
Alkohafi, 1998). The level of inhibition (%) of edema was calculated using the relation:
Inhibition (% ) = 100 [1-(Et/Ec)]
Where
Et: Average edema of the treated group
Ec: Average edema of the control group
2.2.2.3 Formaldehyde induced arthritis in rats
The formaldehyde arthritis model of Selye (1949) was used with modification. Adult albino rats of both sexes (80-230 g) were divided into 6 groups (n=5). The basal right hind paw volume was measured by water displacement. On day 1 of the experiment, groups 1, 2 and 3 were treated by liberal topical application of shea butter, olive oil and methylsalicylate cream respectively. Inflammation was induced after 30 min. by sub-plantar injection of 0.1ml of
(2%v/v) formaldehyde solution. The paw volume was measured again after 4h. The animals were then treated once daily (24 hourly) for 10 days with a re-challenge with formaldehyde injection on day 3. Measurement by water displacement was also taken daily for 10 days.
Animals in groups 4, 5 and 6 received similar treatment but measurement and treatment were taken twice daily (12 hourly) for 10 days. The increase in paw volume was used as a measure of inflammation. The global edematous response to formaldehyde arthritis was quantified as
the area under the curve (AUC) of the time-course of the arthritic event. The AUC was calculated using the trapezoidal rule. The level of inhibition (%) of edema was calculated using the relation:
Inhibition (%) = 100 [1-(AUCt/AUCc)]
Where;
AUCt: AUC of the treated group
AUCc: AUC of the control group
2.2.2.4 Cotton pellet-induced granuloma in rats
Adult albino rats (80-230 g) of both sexes were used in this study. The method described by
Swingle and Shideman (1972) was used. On day 0, animals were divided into 5 groups (n=5).
Groups 1, 2 and 3 received oral administration of 100, 200 and 400mg/kg of shea butter respectively. Group 4 received indomethacin 10 mg/kg, while group 5 (control) received 2.5 ml/kg of olive oil. The animals were immediately anesthetized with xylazine (10 mg/kg)/ketamine (50 mg/kg). The axilla region was quickly shaved with razor blade and swabbed with methylated spirit. Autoclaved cotton pellets (20 mg) were aseptically inserted in the axilla region of the rats through small subcutaneous incisions of about 1 cm length on each side of the axilla. The incisions were sutured with sterile nylon. Treatment was continued once daily for 7 consecutive days. On day 8, the animals were sacrificed by overdose of anesthesia and the cotton pellets removed. The pellets were freed from extraneous tissues and dried in an oven at 60°C for 48 h and weighed. The net dry weight was determined. The average weight of the pellet of the control group as well as of test groups was calculated.
The level of inhibition (%) of edema was calculated using the relation:
Inhibition (%) = 100 [1-(Et/Ec)]
Where;
Et: Average granuloma weight of the treated group
Ec: Average granuloma weight of the control group
2.2.3 Statistical Analysis
Data obtained from the questionnaire were analyzed using descriptive statistics. Data obtained from pharmacological tests were analyzed using One Way ANOVA and subjected to LSD post-hoc test (SPSS version 20). Differences between means were accepted significant at P< 0.05. Results were presented as Mean± SEM.
CHAPTER THREE
RESULTS
3.1 Demographic characteristics of respondents
The demographic characteristics showed that the mean age of the respondents in years was
50.95. A greater proportion of the respondents were female (65.9%) (Fig.1). Most of the patients seen were educated and were either employed (50.6%) or traders (19.6%). However some were unemployed (29.7%) (Fig.2).One hundred and forty five (145) of the 164 respondents were married (89.5%) while the single patients were 16 (9.9%).
3.2 Respondents’ knowledge of their disease condition
About 94% of the patients had heard of arthritis (Fig.3) and 85.1% admitted that they had it.
Up to 38.3% have had the ailment for over 3 years. Arthritis of the knee and hips had the highest frequency (73 and 53% respectively) (Fig.4). The respondents acknowledged that arthritic pain was worse at night (49.2%), in the morning (33.3%) and after a long distance walk (31.1%) (Fig.5). All the patients made one form of effort or another such as use of orthodox drugs (78%), herbs (12.1%) and massage therapies to manage the pain and swelling associated with their condition.
3.1.3 Respondents’ knowledge and use of Shea butter
On the knowledge and use of shea butter, 94.1% of the patients had heard of Shea butter while only 59.6% of them had used it (Fig.6). Majority of the users (63.7%) used it as a massage ointment (Fig.7) once (35.1%) or twice (48.9%) daily (Fig.8). Also, majority of the users (76.9%) used it for 6 months or more. Up to 80% of the users admitted getting relief
(Fig.9) in form of reduction in pain (71%) mainly for periods of less than one hour (78.3%)
(Fig.10). Some of the users (73.7%) combine shea butter use with other massage therapies or
n=164
Figure1: Gender of arthritic patient respondents
n=164
Figure 2: Occupation of respondents
39
n=164
Figure 3: Respondents’ knowledge of their disease condition
40
n=164
Figure 4: Part of body affected by arthritis
41
n=164
Figure 5: Factors that worsen arthritic pain
42
n=164
Figure 6: Respondents knowledge of Shea butter.
n=164
Figure 7: Respondents mode of use of Shea butter.
n=164
Figure 8: Respondents frequency of use of Shea butter.
n=164
Figure 9: Respondents that get relief from use of Shea butter only.
n=164
Figure 10: Duration of relief of pain after Shea butter use.
drugs especially analgesics(68.2%). Most of the users (91.3%) obtain relief when they use shea butter with other remedies.
3.4 Effect of shea butter on systemic acute inflammation
Single oral administration of shea butter inhibited the development of systemic acute edema of the rat paw. The inhibitory effect was non-dose dependent but significant (p<0.05) within
1 h post administration of irritant (Table 1).
3.5 Effect of shea butter on topical acute inflammation
Topical administration of shea butter inhibited the acute edema of the mouse ear induced with xylene. However, the inhibitory effect was not significant (p<0.05) but comparable to that of indomethacin (Table 2).
3.6 Effect of shea butter on experimental arthritis
In the global edematous response to formaldehyde arthritis, once daily chronic application of shea butter was not effective in inhibiting the development of arthritis. However, twice daily application caused inhibitory effects greater than that of methylsalicylate (Table 3).
3.7 Effect of shea butter on granuloma formation
Shea butter caused a significant (P<0.05) inhibition of granuloma tissue growth on the implanted cotton pellets. The effect was non-dose-dependent and was less than the inhibition caused by indomethacin (Table 4).
Table 1:Effect of shea butter on systemic acute edema of the rat paw
Treatment Dose Edema (ml)
(mg/kg) 0.5 h 1 h 2 h 3 h 4 h 5 h
Shea butter 100 0.26±0.09* 0.44±0.15* 0.50±0.17 0.52±0.11 0.38±0.08 0.56±0.15
(59.38) (29.03) (3.85) NI (17.4) NI
200 0.16±0.05* 0.36±0.18 0.42±0.17 0.40±0.21 0.40±0.16 0.36±0.25
(75.00) (41.94) (19.24) (23.08) (13.04) (14.29)
400 0.26±0.11* 0.48±0.13 0.42±0.08 0.50±0.19 0.36±0.11 0.32±0.22
(59.38) (22.58) (19.24) (3.85) (21.74) (23.81)
Indomethacin 10 0.14±0.09* 0.18±0.08* 0.26±0.11* 0.14±0.11* 0.10±0.1* 0.18±0.11*
(78.13) (70.97) (50.00) (73.08) (78.26) (57.14)
Control 2.5 0.64±0.05 0.62±0.11 0.52±0.13 0.52±0.13 0.46±0.15 0.42±0.11
n=5;*P < 0.05 compared to control (One way ANOVA; LSD post hoc test); Values shown
are Mean±SEM; Values in parenthesis are Inhibition (%) calculated relative to the Control.
NI= No Inhibition
Table 2: Effect of shea butter on acute ear edema in mice
Treatment Dose Edema weight Inhibition
(mg/ear) (mg) (%)
Shea butter 5 8.2±1.36 21.15
Indomethacin 5 8.4±1.03 19.23
Control - 10.4±2.23 - n=5; Values shown are Mean± SEM; Percent inhibition was calculated relative to the control.
50
Table 3: Effect of shea butter on global edematous response to formaldehyde arthritis
Treatment AUC(ml/d)
Once Daily Twice daily
Morning Evening
Shea butter 4.18 ±0.36 2.51 ±0.52 2.12 ±0.44*
(NI) (22.53) (26.39)
Methylsalicylate 3.78 ±0.41 3.78 ±0.37 3.40 ±0.44
(NI) (NI) (NI)
Control 3.62 ±0.42 3.24 ±0.43 2.88 ±0.32
n=5;*P < 0.05 compared to control (One Way ANOVA; LSD post hoc test); Values shown are Mean±SEM; Values in parenthesis are Inhibition (%) calculated relative to the
Control;AUC= Area Under Curve. NI= No Inhibition
51
Table 4: Effectof shea butter on granuloma formation
Treatment Dose Granuloma weight Inhibition
(mg/kg) (mg) (%)
Shea butter 100 51.90±3.62* 28.9
200 56.20±4.60* 23.01
400 56.30±2.88* 22.88
Indomethacin 10 38.20±0.85* 47.67
Control - 73.00±8.29 - n=5; *P < 0.05 compared to Control (One Way ANOVA; .LSD post hoc test); Values shown are Mean± SEM; Percent inhibition was calculated relative to the Control.
52
CHAPTER FOUR
DISCUSSION AND CONCLUSION
4.1 Discussion
A greater majority of the respondents in this study were females. Arthritis is a common disease with peak incidence in third to fourth decades of life and 3 to 5 times higher in females (Mohan, 2000; Sikha, 2010). The result showed that in more than 60 percent of the time, women have more incidence of arthritis than men. The mean age (in years) observed in the field study was 50.95. The respondents had good knowledge of their arthritis condition and a good percentage admitted having the condition. They also affirmed the pain worsened on taking a long distance walk which is consistent with osteoarthritis (Godman, 2013).
Patients with arthritis of the knee and hip were in the majority and points also to non- inflammatory arthritis (Godman, 2013). Rheumatoid arthritis affects only about 1% of the population (Godman, 2013). Thus, the number of these patients with arthritis suggests these may be more of osteoarthritis cases than other forms of arthritis such as rheumatoid.
Obesity is a major modifiable factor in arthritis (Wilkins, 2004). As most of the patients were females and married, child bearing and body weight of the patients could be factors in osteoarthritis.
Shea butter is a common traditional remedy for pains and swellings in southeastern Nigeria.
It is therefore not surprising that most of the patients have heard about it and used it. This further shows that use of shea butter is a common practice among patients with arthritis.
Majority of the patients use it as a massage ointment which is the most common mode of application of this remedy. Many of the patients have used it for 6 months or more. Relief obtained from use of shea butter alone is obviously insufficient and of short duration to adequately relieve pain associated with the chronic condition. This may explain why it is usually combined with other therapies or drugs especially analgesics to achieve better result.
53
In the experimental studies, topical application and systemic administration of shea butter suppressed acute edema. Xylene causes instant irritation of the mouse ear leading to fluid accumulation and edema. Ear edema induced by phlogistic substances is an acute inflammatory response mediated by a variety of agents such as leukocytes and prostanoids
(Tubaro et al., 1985). Shea butter is a fatty extract and its topical anti-inflammatory effect confirms its lipophilic nature which enabled it to cross the membrane barrier and exert action.
Systemic oral administration caused a mild inhibitory effect that was more pronounced very early after induction of edema. The effect started to wane beyond 1 hour post induction of edema and suggests that the fatty extract may be more effective in suppressing the initial phase of acute inflammatory response. The first phase of this acute inflammatory response in the rat paw begins at about 1 hour after challenge and has been known to be mediated by massive release of histamine and serotonin. The second phase starts at about 3 hours after challenge and attributed to bradykinin, prostaglandins (Willoughby and Flower, 1993) and lysozymes. Therefore, the antiinflammatory effect of shea butter may be largely due to antihistaminic action. The brief effect may be attributable to suppression of histamine release.
The rapid onset of antiinflammatory effect may be responsible for the reduction in pain experienced by the users. The relief in pain is usually brief and may be as a result of the brief systemic antiinflammatory activity.
Chronic inflammation usually arises as a consequence of incomplete elimination or containment of the pro-inflammatory stimuli by acute inflammation and is usually marked by neutrophil infiltration, fluid exudation and fibroblast proliferation amongst others. In chronic inflammation of the formaldehyde arthritis, shea butter reduced edema in arthritic rats when it was applied twice daily. Once daily application was not effective. The reason for this is not clear but may not be unrelated to the brief action noticed in systemic acute inflammation. The ineffectiveness of single application may be the reason why patients apply it more than once
54
a day to obtain relief. Formaldehyde is a potent edematous agent and produces inflammation through the release of several inflammatory mediators including prostaglandins (Tjolsen et al., 1992). Inhibition of the global edematous response to formaldehyde indicates that shea butter may contain agents useful in management of chronic inflammation.
The cotton pellet induced granuloma is used to study the transudative and proliferative phase of chronic inflammation (Winter et al., 1957). Chronic inflammation often progresses to development of proliferative cells which can spread or form granuloma. Granuloma tissue formed on an inert foreign body in a dead space comprises an accumulation of modified macrophages and other cells and tissues (Whaley and Burt, 1996). Granulomatous inflammation is a prolonged process often involving the mononuclear cells. Though the exact mechanism of anti-inflammatory activity of shea butter is unknown, its effect in this type of inflammation is an indication of anti-proliferative action.
Earlier studies had documented the phytochemical constituents of shea butter. It has been shown to contain carbohydrates, saponins, steroids, tannins and alkaloids (Ndukwe et al.,
2007) and high levels of UV absorbing triterpene esters such as cinnamic acids, tocopherol, vitamin A and phytosterol (Weisman et al., 2003). The phytosterols make up the unsaponifiable fraction and include campesterol, stigmasterol, β-sitosterol, α-spinosterol, triterpenes (cinnamic acid esters, α- and β-amyrin, parkeol, butyrospermol and lupeol) and a hydrocarbon, karitene (Badifu, 1989). Shea butter is composed of five principal fatty acids: palmitic, oleic, stearic, linoleic and arachidic acid (Maranz et al., 2004). Some of these phytoconstituents have been implicated in the antiinflammatory activity of plants.
Campesterol, stigmasterol, β-sitosterol, α- and β-amyrin (Okoye et al., 2014), lupeol
(Fernández et al., 2001a; Fernández et al., 2001b; Saleem, 2009) and cinnamic acid (Saleem,
2009) are antiinflammatory terpenoids present in many plants. Αlpha amyrin and β- amyrin are triterpenoids found to be responsible for the profound anti-inflammatory activity of the
55 stem bark of Alstonia boonei De Wild (Apocyanaceae) (Okoye et al., 2014). Cinnamic acid was the triterpene found to be responsible for the antiinflammatory activity of Cinnamonium cassia. Lupeol is a dietary triterpene that has been studied extensively for inhibitory effects on inflammation (Saleem, 2009). Terpenoids are reported to be natural inhibitor of NF-κB signaling both antiinflammatory and anticancer activities (Salminen et al., 2004).The antiinflammatory action of sesquiterpenes has been attributed to inhibition of arachidonic acid metabolism (Kumar et al., 2013).
4.2 Conclusion
This study revealed that the fatty extract of Vitellaria paradoxa (shea butter) is commonly used among arthritis patients. The extract possesses antiinflammatory actions in both acute and chronic inflammation. The antiinflammatory activity may be attributed to its terpenoid phytoconstituents. These findings provide a scientific rationale for the use of shea butter in treatment of disorders of inflammation in traditional medicine.
56
REFERENCES
Abbiw, K. D. (1990). The useful plants of Ghana; West African uses of wild and cultivated plants. Intermediate Technology publishing and the Royal Botanic Garden, Kew (Pub) England.pp.5-13.
Abidemi, T.A., Adebayo, O.J., Agbotoba I.O. (2009). Nutrient content and anti-nutritional factors in shea butter (Butryospermum parkii) leaves. Afr. J. Biotechnol. 8 (21):5888- 5890.
Akihisa, T., Kojima, N., Katoh, N., Ichimura, Y., Suzuki, H., Fukatsu, M., Maranz, S., Masters, E.T. (2010). Triterpene alcohol and fatty acid composition of shea nuts from seven African countries. J. Oleo. Sci. 59(7):351-360.
Amann, R., Peskar, R.A. (2002).Antiinflammatory effects of aspirin and sodium salicylate. Eur. J. pharmacol.447:1-9.
Ament, P.W., Childers, R.S. (1997). Prophylaxis and treatment of NSAID-induced gastropathy. Am. Fam. Phys. 4:1323-1326.
Ampofo, O. (1983). “First aid in plant medicine”.Ghana rural reconstruction movement, Annual Report.
Attah, A.H., Alkohafi, A. (1998). Anti-nociceptive and anti-inflammatory effects of some Jordanian medicinal extracts. J. Ethnopharmacol. 60:117-124.
Atzeni, F., Sarzi-Puttini, P., Mutti, A., Bugatti, S., Cavagna, L. Caporali, R. (2013).Long- term safety of abatacept in patients with rheumatoid arthritis.Autoimmun.Rev. 12:1115–1117.
Badifu, G.I.O. (1989). Lipid composition of Nigerian Butyrospermum paradoxum kernel. J. Food Compos. Anal. 2:238-244.
Beck, S. (2014).Acute and chronic inflammation.Available atwww.hopkinsmedicine.org /mcp/education/300,713 lectures. Accessed July 2015.
Bellik, Y., Laid, B. (2013). Molecular mechanism underlying anti-inflammatory and anti- allergic activities of phytochemicals: An update. Molecules 18:321-353
Black, C.M., O'Brien, M., Vanscott, E.J., Auerbach, R., Eisen, A.Z., and Bunim, J.J. (1964) Methotrexate therapy in psoriatic arthritis: double-blind study on 21 patients. JAMA 189:743–747.
Bralley, E. E., Greeopan, P., Hargrove, J. L., Wicker and Hartle (2008). Topical anti- inflammatory activity of polygonum cuspidatum extract in the TPA models of mouse ear inflammation. J. Inflamm. 5: 1-7.
Brune, K., Hinz, B. (2004). Selective cyclooxygenase-2 inhibitors: similarities and differences. Scand. J. Rheumatol. 33:1-6.
Buckley, M.M., and Brogden, R.N. (1990). Ketorolac: a review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential. Drugs 39:86-109.
Bugatti, S., Codullo, V., Caporali, R. and Montecucco, C. (2007).β cells in rheumatoid arthritis. Autoimmun. Rev. 7:137–142.
Burke, A., Smyth, E., FitzGerald, G.A. (2006). Analgesic-Antipyretic and Anti-inflammatory agents: Pharmacotherapy of Gout’. In: Goodman and Gilmans’s The pharmacological Basis of Therapeutics. (Laurence L. Bruton, John S. Lazo and Keith L. Parker eds) 11thedn. McGraw Hill, New York, pp. 671-716.
Capell, H. A., Madhok, R., Porter, D.R. (2007). Combination therapy with sulfasalazine and methotrexate is more effective than either drug alone in patients with rheumatoid arthritis with a suboptimal response to sulfasalazine: results from the double-blind placebo-controlled MASCOT study. Ann. Rheumatic. dis. 66(2):235-241.
Chandrasekharan, N.V., Dai, H., Roos, K.L. (2002). COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure and expression. Proc. Natl. Acad. Sci. U S A. 99:13926-13931.
Chandrosoma, P., Taylor, O. R. (2005). General pathology: The host response to injury; Concise Pathology 3rd edition, McGraw Hill, New York. Available at http://www.accessmedicine.com/content.aspx?aID.Assessed Jan, 2015.
Changa, C., Wena, Z., Wangc, S. and Duha, C. (2008). New anti-inflammatory steroids from the formosan soft coral Clavularia viridis, Steroids. 73:562-567.
Cotran, R.S., Kumar, C. T. (1998). Robbins pathologic Basis of Disease. Philadephia: W. B. Saunders & Company, pp. 50-111.
Cutollo, M., Sulli, A., Craviotto, C., Felli, L., Pizzorni, C., Seriolo, B. (2002). Antiproliferative and anti-inflammatory effects of methotrexate and sex hormones on cultured differentiating myeloid monocytic cells (THP-1). Ann. N. Y. Acad. Sci. 966:232–237.
Davies, N.M. (1997). Clinical pharmacokinetics of nabumetone, the dawn of selective cycloxygenase-2 inhibition. Clin. Pharmacokinet. 33:404-416.
Delves, P.J (2014). Mediators of inflammation. The Merck Manual, MSD professional edition.
Djekota, C., Diouf, D., Sane, S., Mbaye, M. S. and Noba, K. (2014).Morphological Characterization of shea tree (Vitellaria paradoxa) Population in the region of Mandoul in Chad. Int. J. Biodivers. Conserve. 6:184-193.
Dobrilla, G., Benvuenti, S., deGuelmi, A. (1997).The epidemiology of gastroduodenal damage induced by aspirin and other non-steroidal anti-inflammatory drugs. Recent. Prog. Med. 88(5): 202-211.
Dray, A. (1995).Inflammation mediators of pain. Br J. Anaesth, 75: 125-131.
Dray, A., Perkins, M. (1993).Bradykinin and inflammatory pain.Trends Neurosci. 16:99-104. Ebadi, M. (1997).corre concept in pharmacology 1st edn. Lipincott- Raven Publisher. Philadephia, New york pp. 55-75. Edward, H. S. (2014). Chemical mediators of inflammation In: The Merck Veterinary manual.
Ekambaram, S., Perumal, S. S and Subramanian, V. (2010). Evaluation of antharthritic activity of Strychnos potatorium seeds in Freund’s Adjuvant induced arthritis in rat. BMC Complement Altern. Med.,10:56.
Favalli, E.G., Desiati, F., Atzeni, F., Sarzi-Puttini, P., Caporali, R., Pallavicini, F.B. (2009).Serious infections during anti-TNFalpha treatment in rheumatoid arthritis patients. Autoimmun. Rev. 8:266–273.
Feldmann,M., Brennan, F.M., Maini, R.N. (1996) Role of cytokines in rheumatoid arthritis. Annu. Rev. Immunol. 14:397–440.
Ferrero-Milliani, L., Neilsen, O.H., Anderson, P.S., Girardin, S.E. (2007). Chronic Inflammation: Importance of NOD2 and NALP3 in interleukin-1 beta generation. Clin. Exp. Immunol. 147(2): 227-235.
Firestein, G.S. (2003). Evolving concepts of rheumatoid arthritis. Nature. 423:356-361.
Fonseca, J.E., Santos, M.J., Canhao, H., Choy, E. (2009). Interleukin-6 as a key player in systemic inflammation and joint destruction. Autoimmun. Rev. 8:538–542.
Franceschi, C., Campisi, J. (2014). Chronic inflammation (inflammaging)and its potential contribution to age associated disease. J. Gerantol. Biol. Sci. Med. Sci. 69(s1): s4-s9
Frautschy, S.A., Hu, W., Kim, P., Miller, S.A., Chu, T., Harris-White, M.E., Cole, G.M.(2001). Phenolic anti-inflammatory antioxidant reversal of a β-induced cognitive deficits and neuropathology.Neurobiol.Aging.22: 993- 1005.
Gaby, A. R. (1999).Alternative treatments for rheumatoid arthritis.Altern.Med.Rev.4: 392- 402.
Gaddi, A., Cicero, A.F.G., Pedro, E.J. (2004). Clinical perspectives of anti-inflammatory therapy in the elderly: the lipoxygenase (LOX)/cycloxygenase (COX) inhibition concept. Arch. Gerontol. Geriatr. 38:201-212.
Garba, I. D., Nwawe, C. N and Oisakede, I. L., (2011). The potentials of shea nut tree to Nigerian Economy. Intl Journal of Agricultural Economics and Rural development, 4(1):62-73.
George, C., Shams A. N. and Dunkel, F.V. (2011). Lessons Learned in an International Service-Learning Collaborative: Shea Butter Case Study. North American Colleges and Teachers of Agriculture Journal , 71-77.
Godman, H. (2013). Inflammatory vs. non-inflammatory arthritis: what is the difference? www.healthline.com (Assessed Jan. 2015)
Guyton, C.A., Hall, J.E. (2000).Textbook of Medical Physiology.10thedn. W. B. Saunders Company, Philadelphia, pp. 392-401.
Haanen,C., (2001). Sulindac and its derivatives: a novel class of anticancer agents. Curr. Opin. Investing. Drugs. 2: 677-683.
Hall, J., Aebischer, D., Tomlinson, H., Osei-Amaning, E. and Hindle, J. (1996).Vitellaria paradoxa.A monograph of school of Agricultural science publication, University of Wales, Bango, pg.105.
Hayliyar, J., Macpherson, A., Bjarnason, I. (1992). Gastro-protection and nonsteroidal anti- inflammatory drugs (NSAIDS): Rational and Clinical Implications. Drug Safety 7(2):86-105.
Heap, G.A. (2009).The genetic of human inflammatory diseases. Hum. Mol. Genet 18(R1): 101-106
Henry, A. N and Nair, C. V. (1983). Vitellaria paradoxa vs Butyrospemum (Sapotaceae).Taxon 32: 286.
Hetland, M.L. (2011). Modern treatment strategies in rheumatoid arthritis. Dan. Med..Bull. 58(11):B432
Heymer, B. (1985). Causative agents, mediators and histomorphology of inflammation. Pathol. Res. Pract. 180 (2): 143-150.
Hiley, P. and Barber, P.C. (2000).Acute inflammation. Available at http://medweb.am.ac.uk/http/mod/3/1/a/acute.htm/
Howarth, P.H., Djukanovic, R., Walls, A., Wilson, J., Roche, W., Holgate, S.T. (1991). Future developments in anti-inflammatory studies.Eur.Resp.Rev. 4:276-281.
Iwu, M. M., Anyanwu, B. N. (1982). Phytotherapeutic profile of Nigerian herbs: Anti- inflammatory and anti-arthritis agent. J. Ethnopharmacol. 6(3): 263-274.
Jackson, C., Mbagwu, H., Jackson, I., Ekpe, G. and Eitenam, F. (2011). Analgesic activities of ethanolic extracts of the root of Carpolobia lutae. Afr. J. Pharm. Pharmacol. 5(3): 367-370.
Jacobs, J. W. G., Kraaimat, F. W., Bijlsma, J.W. (2001). Why do patients with Rheumatoid arthritis use alternative treatment? Clin. Rheumatol.20: 192-196.
Jang, D., Cuendet,, M., Hawthorne, M., Kardono, L., Kawanishi, K. and Fong H. (2002). Prenylated flavonoids of the leaves of Mucaranga conifer with inhibitory activity against cyclooxygenase 2.Phytomedicine. 61:867-872.
Jisna, R. P., Deepali, S and Prabha, R. (2014). Rheumatoid arthritis In; Handbook of Phamacogenomics and Stratified medicine pp. 653-681.
Jubie, S., Jawahar, N., Koshy, R., Gowramma, B., Murugan, V and Suresh, B. Anti-arthritis activity of bark extracts. Alangium salvifolium Wang.
Kar A., Mital, H. C. (1981). The extraction of shea butter. Qual. plant foods Hum. Nutr.31: 67-69.
Karen, A.W., Knox V.D (2007). Antiinflammatory and Antirheumatic Drugs – Therapeutic aspects of inflammatory and selected other clinical disorders. In: Modern Pharmacology with Clinical Applications (Charles, R. Craig and Robert, E. Stitzel eds.) 6thedn., Lippincott, New York. pp. 423-440.
Kaur, A., Nain, p. and Nain, J. (2012). Herbal plants used in treatment of rheumatoid arthritis: a review. Int. J. Pharm. Pharm. Sci. 4(4): 44-57.
Khan, F.A., and Khan, M. (2010).Inflammation and acute phase response.Int. J. Appl. Biol. Pharm. Tech.1(2).
Kumar, S., Baywa, B.S., Kuldeip, S and Kalia, A. N. (2013). Antinflammatory activity of herbal pbants: A review. Int. J. Adv. Pharm. Biol. Chem. 2 (2): 271-282.
Kupeli, E., Kosar, M., Yesilada, E. and Baser, K.(2002). A comparative study on the anti- inflammatory, antinociceptive and antipyretic effects of isoquinoline alkaloids from roots of Turkish Berberis species, Life Sci. 72:645-652.
Laurence, L. B., John, S.L., Keith, L.P. (2006). Goodman and Gilman’s, The Pharmacological Basis of Therapeutics.11thedn. McGraw Hill, New York. pp. 671- 716.
Lovett, P. N. and Haq, N. (2000).Diversity of the shea nut tree (Vitellaria paradoxa C. F. Gaertn) in Ghana. Genetic research and crop evaluation, 47: 293-304
Macdonald, C. (1988) Butterworth’s Medical Dictionary. 7thed. Butterworth and Co. Ltd: Kent.
Maranz, S, Z., Wiesman, J., Bisgaard and G. Bianchi. 2004. Germplasm resources of Vitellaria paradoxa based on variations in fat composition across the species distribution range. Agroforestry Systems 60:71-76.
McGeough, C.M., Bjourson, A.J. (2012) Diagnostic, Prognostic and Theranostic Genetic Biomarkers for Rheumatoid Arthritis .J. Clin. Cell. Immunol. S6:002. doi:10.4172/2155-9899.S6-002
McInnes, B.L and Schett, G. (2011).The pathogenesis of rheumatoid arthritis. N. Eng. J. Med. 365:2205-2219
Meager, A. (1999).Cytokine regulation of Cellular adhesion molecule expressions in inflammation. Cytokine Growth Factor Rev. 10:27-39.
Meek, L.I., Mart, A.F., Van De Laar and Vonkeman, H.E. (2010). Non-steroidal anti- inflammatory drugs. An overview of cardiovascular risks. Pharm. Res.,3: 2146-2162.
Meher, B.R., Rath, B.G. and Biswal, S. (2011). Evaluation of ethanolic extract of Sphaeranthus indicus. J. Chem. Pharm. Res. 3(3): 831-834.
Mohan, H. (2000). Textbook of Pathology.JP publisher New Dehli.
Mohan, H. (2006). Inflammation and healing In: Textbook of pathology. JP publisher New Delhi.pp.133-179.
Mombouli, J. V., Illiano, S., Nagao, T., Scott-Burden, T., Vanhoutte, P. M. (1992). Potentiation of endothelium dependent relaxation to bradykinin by angiotensin 1 converting enzyme inhibitors in Canine Coronary arteries involves both EDRF and EDHF. Circ. Res. 71: 137-144.
Montecucco,F. and Mach, F. (2009). Common inflammatory mediators orchestrate pathophysiologic process in rheumatoid arthritis and artherosclerosis. Rheumatol.48:11–22.
Morley, J., Hanson, J.M., Rumjanek, U.M. (1989). Lymphokines In: Textbook of Immunopharmacology. 2ndedn. (Dale, M.M and Foreman, J.C. eds.) Blackwell Scientific Publications, Oxford, pp. 168-180.
Morley, P.A., Brogden, R. N., Carmine, A.A. (1982). Zomepirac: A review of its pharmacological properties and analgesic efficacy. Drugs. 23: 250-275.
Mukherjee, D. and Topol, E.J. (2003) Cox-2: where are we in 2003?: cardiovascular risk and Cox-2 inhibitors. Arthritis Res. Ther.. 5: 8-11.
Naik, S. R., Sheth, U. K. (1976). Inflammatory process and screening methods for antti- inflammatory agents-A review. J. Postgrad. Med. 22: 5-21
Nairn, R. (2004). Immunology In: Medical Microbiology (Brooks, G.F., Butel, J.S., and Morse , S.A. eds) int. edition McGraw Hill, New York pp 119-145
Nandi, P., Kingsley, G.H., Scott, D.L. (2008). Disease-modifying anti-rheumatic drugs other than methotrexate in rheumatoid arthritis and seronegative arthritis. Curr. Opin. Rheumatol. 20 (3): 251-6.
Ndukwe, I. G., Amupitan, J. O., Isah, Y. and Adegoke, K. S. (2007). Phytochemical and antimicrobial screening of the crude extracts from the root, stem bark and leaves of Vitellaria paradoxa (GAERTN. F).Afr. J. Biotechnol., 6 (16):1905-1909.
Newton, E.L.(1998). “Repression of Cyclooxygenase-2 and Prostaglandin E2 Release by Dexamethasone Occurs by Transcriptional and Post-transcriptional Mechanisms Involving Loss of Polyadenylated mRNA.” J. Biol Chem. 273(48): 32312-32321.
Nilsen,O.G. (1994). Clinical pharmacokinetics of tenoxicam. Clin. Pharmacokinet. 26:16-43.
Nwabunike, I. A., Ezieke, A. C., Nwodo, N. J., Udegbunam, S. V and Okoli, C. O. (2014). Bioactivity Guided Studies of the anti-inflammatory activity of extracts of aerial part of Schewankia americana L (Solanaceae). J. Med. Plant Res., 8(22): 794-801.
Okoli, C. O,. Akah, P.A., (2000). A pilot evaluation of the anti-inflammatory activity of Culcasia scandans, a traditional anti-rheumatic agent. J. Altern. Comp. Med. 6(5): 423-427.
Okoli, C. O., Akah, P. A. and Ezegwuorie, U. (2006a).Anti-inflammatory activity of extracts of root bark of Securidaca longipedunculata fres (Polygalaceae).Afr. J. Trad CAM, 2(3): 54-63.
Okoli, C. O., Akah, P. A., Nwafor, S. V., Anisiobi, A.I., Ibegbulam, I.N., Erojikwe, O. (2006b). Antiinflammatory activity of hexane leaf extract of Aspila africana. J. Ethnopharmacol., 109(2):219-225.
Okoli, C. O., Akah, P. A.and Nwafor, S. V. (2003). Anti-inflammatory activity of plants. J. Nat Remedies. 3(1): 1-30.
Okoli, C. O., Akah, P.A., Onuoha, N.J., Okoye, C.O., Nwoye, A.C., and Nworu, C.S. (2008).Acanthus monthanus: An experimental evaluation of the antimicrobial, anti- inflammatory and immunological properties of a traditional remedy for furuncles. BMC Compel. Altern.Med.,8:2 7061.
Okoye, N. N., Ajaghaku, D. N., Okeke H. N., Ilodigwe, E. E., Nwosu, C. S., Okoye, F. B. (2014). Beta-amyrin and alpha-amyrin acetate isolated from stem bark of Alstonia boonei display profound antiflamatory activity. Pharm. Biol. 52(11): 1478-1486.
Okullo, J. B. L., Hall, J. B and Obua, J. (2004). Leafing, flowering and fruiting of Vitellaria paradoxa subsp nolotica in Savannah parklands of Uganda. Agro-forestry Systems, 60: 77-91.
Okullo, J.B.L., Omujal, F., Agea, J.G., Vuzi, P.C., Namutebi, A., Okello, J.B.A. and Nyanzi, S.A. (2010).Proximate and mineral composition of fruit pulp in Uganda. Afr. J Food, Agric. Nutr. Dev. 10(11): 4430-4443
Orwa, C., Mutua, A., Kindt, R., Jamnadass, R., Anthony, S. (2009) Agroforestry Database: A tree reference and selection guide version 4.0 (http://www.worldagroforestry.org/ sites/treedbs/treedatabases.asp) Accessed January 2015.
Patrignani, P., Capone, M.L., Taconelli, S. (2003). Clinical pharmacology of Etoricoxib: a novel COX 2 inhibitor. Expert. Opin. Pharmacother. 4: 265-284.
Pelletier, J.P., Martel-Pelletier, J., Lajeunesse, D., Reboul, P. (2003).Therapeutic role of dual inhibitors of 5-LOX and COX, selective and non-selective steroidal anitinflammatory drugs.Ann. Rheum. Dis. 62: 501-509.
Perez, G.R.M. (1996). Antiinflammatory activity of Ambrosia artemisiaefolia and Rheo spathaceae. Phytomed. 3:163-167.
Porth, C. (2007). Essentials of Pathology; Concepts of altered health state. Lipincott, William and Wikins.P, 270.
Punchard, N.A., Whelan, C.J. and Adcock, I. (2004) Introduction; Inflammation. J Inflamm. 1:1
Rang, H.P., Dale, M.M., Ritter, J.M., Flower, R.J. (2007).Local Hormones, Inflammation and Immune Reactions. In: Pharmacology. 6th ed. Elsevier, London. pp. 203-247
Rindfleisch, J.A, Muller, D. (2005).Diagnosis and Management of Rheumatoid Arthritis. Am. Fam. Phys. 72(6): 1037-1047.
Robert, L. J, Morrow, J.D. (2001). Analgesic, antipyretic and anti-inflammatory agents and drugs employed in treatment of Gout In: The pharmacological basis of therapeutics 10th ed. McGraw-Hill, pg 687-731.
Salminen A, Lehtonen M, Suuronen T, Kaarniranta K, Huuskonen J. Terpenoids: Natural inhibitors of NF-kappaB signaling with anti-inflammatory and anticancer potential. Cell Mol. Life Sci. 2008; 65: 2979-2999.
Samuelsson, B. (1983). Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation. Science.220:568-575.
Sano, H. (2011). The Role of Lipid mediators in the pathogenesis of rheumatoid arthritis. Inflamm. Regen. 31(2): 151-156
Santos, F.A, Rao, V.S. (2000). Antiinflammatory and antinociceptive effects of 1,8-cineole; a terpenoid oxide present in many plant essential oils. Phytother. Res. 4: 240-244.
Santosh V, Shreesh O, Mohammed R. (2010). Anti-inflammatory activity of Aconitum heterophyllum on cotton pellet-induced granuloma in rats. J. Med. Plant Res. 4(15): 1566-1569.
Selye, H.(1949).Further studies concerning the participation of adrenal cortex in the pathogenesis of arthritis. Brit. Med. J. 2:1129.
Shikha, S. (2010).Experimental animal models of rheumatic arthritis. Biotech. Articles, 8(21): 19.
Shruti, S., Pradeep, S., Keshri, K. J., Garima, M., Sourah, S.Ratan, L. (2012). Evaluation of anti-arthritic potential of methanolic extract of the aerial parts of Costus Speciosus. J. Aryurveda Integr. Med., 3(4): 204-208.
Sokka, T. and Pincus T. (2002). Contemporary disease modifying antirheumatic drugs (DMARD) in patients with recent onset rheumatoid arthritis in a US private practice: methotrexate as the anchor drug in 90% and new DMARD in 30% of patients. J. Rheumatol. 29:2521–2524.
Soladoye, M.O., Orhiere, S.S., Ibimode, B.M. (1989).“Ethnobotanical study of two indigenous multipurpose plants in the Guinea savanna of Kwara state – Vitellaria paradoxa and Parkia biglobosa’’. Biennial Conference of the Ecological Society of Nigeria, 14th August , Forestry research institute, Ibadan, p. 13.
Sosa, S., Balicet, M. J., Arvigo, R., Esposito, R. G., Pizza, C., Altinier, G., Tubaro, A. (2002).Screening of the topical antiinflammatory activity of some central African plants. J. Ethnopharmacol, 8: 211-215. Swingle, K.F., Shideman, F.E. (1972).Phases of the inflammatory response to subcutneous implantation of a cotton pellet and their modification by certain antiinflammatory agents. J Pharmacol Exp Ther 183:226-234.
Tella, A. (1979). Preliminary studies on nasal decongestant activity from the seed of the shea butter tree.Butyrospemum Parkii . Br. J. Clin. Pharmacol.,7(5): 497-499
Tjolsen, A., Berge, O., Hunskaar, S., Rosland, J.H., Hole, K. (1992). The formalin test: An evaluation of the method.Pain 51: 5-14. http://dx.doi.org/10.1016/0304- 3959(92)90003-T
Tripathi, K. D. (2003). Essentials of medical pharmacology.JP, New Dehli.
Tubaro, A., Dri, P., Delbello, G., Zilli, C., Della, L. R. (1985). The croton oil ear test revisited. Agent actions.17:347-349.
Ukwe, C.V.(2004). Rheumatoid arthritis. In: Therapeutics Basis of Clinical Pharmacy in the Tropics. (Aguwa C.N. ed) 2ndedn. Optimal publishers. Enugu, Nigeria. pp. 268-282.
Vane, J.R., Botting, R.M. (1998). Mechanism of action of non-steroidal anti-inflammatory drugs. Am. J. Med. 104:25-85.
Vikrant, A., Vivek, K. G., Ranjeet, K. (2011). A review of plants having anti-arthritis potential. Int. J. Pharm. Sci. Rev. Res. 7(2): 131-136.
Villarreal, G., Zagorski, J., Wahl, S. M. (2001) Inflammation: Acute. Encyclopaedia of life sciences.
Walsh, N., Crotti, T.N., Goldring, S.R., Gravalles, E.M. (2005). Rheumatic diseases: The effects of inflammation on bone. Immunol.Rev. 208: 228-251.
Wan, J. P and Wakelyn, P. J. (1997). AOCS Press Illinois, USA, Gregory Oil Refining pg. 142-146.
Weisbrode, S.E., Doige, D.E., (2001). Bone and joints. In: Macgavin MD, Carlton WW, Zachary JF. Thomson’s Special Veterinary Pathology. 3rd ed. Saint Louis, Missouri: Mosby Inc. pp. 528–529.
Weisman, Z., Maranz, S., Bianchi, G., Biagaard, J (2003). Chemical analysis of fruit of Vitellaria paradoxa In: Improved management of Agroforestry parkland system in Sub-Sahara Africa. Final report ed. School of Agriculture and Forest science University of Wales Banago U.K pp. 131-139.
Whaley, K., Burt, A.D. (1996).Inflammation, healing and repair. In: Muir’s textbook of pathology. (Macsween R.M.N., Whaley, K.C. eds) 13th edn. Arnold, London. Pp.112- 165. Wilkins, K. (2004). Arthritis and excess weight. Statistic Canada Catalogue 82-003: Health report. 15(1): 39-51
Willoughby, D.A., Flower, R.J. (1993). The anti-inflammatory action of the salicylates. In: Aspirin and other salicylates. (John R. Vane and Regina M. Botting eds) Chapman and Hall Medical, New York. pp. 141-159.
Winter, C.A., Risley, E.A. and Nuss G.W.(1962). Carrageenan induced edema in hind paw of rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 111: 544- 547.
Yidana, A. J. (2004). Progress in developing technologies to domesticate the cultivation of shea tree (Vitellaria paradoxa) in Ghana. Agric. Food Sci. J. Ghana, 3: 249-267.
Yin, M. J., Yamamoto, Y., Gaynor, R.B. (1998). The anti-inflammatory agents aspirin and salicylate inhibit the activity of (kappa) B kinase-beta. Nature. 396: 77-80.
APPENDIX 1 Dear Respondent, I am a pharmacist and Postgraduate research student at the University of Nigeria, Nsukka. I am conducting a survey on the prevalence of use of SHEA BUTTER amongst patients with arthritic conditions. Your response would be useful to me and will be kept in strict confidence. SECTION A: DEMOGRAPHIC DATA Age:……..……….…… Gender:……..…………………… Marital Status: Single [ ] Married [ ] Divorced [ ] Education background: Primary [ ] Secondary [ ] Tertiary [ ]
Occupation:……………………………………………………….
SECTION B: KNOWLEDGE OF DISEASE AND ITS TREATMENT
1. Have you heard of arthritis before? Yes [ ] No [ ]
2. Do you have arthritis or any joint pain? Yes [ ] No [ ]
3. If yes, for how long have you had these pains? 0-6 months [ ] 7-12months [ ] 1-3
years [ ] over 3 years [ ]
4. What part of the body is affected? Arm [ ] Knee [ ] Fingers [ ] Toes [ ] Hips
[ ]
5. Is the pain accompanied by swelling? Yes [ ] No [ ]
6. When does it get worse? Morning [ ] Afternoon [ ] Night [ ]
7. What makes the pain get worse?Long distance walk [ ] Cold weather [ ] Sitting at a
place for long [ ] Other……….
8. How do you treat the pains?……………………………………
9. Have you heard of OKWUMA before? Yes [ ] No [ ]
10. Have you used SHEA BUTTER before? Yes [ ] No [ ]
11. How do you use this SHEA BUTTER? By mouth[ ] By rubbing on the skin[ ] By
massaging it on the skin[ ]
12. How often do you use it? Once a day [ ] Twice a day [ ] Three times a day [ ]
Once in two days [ ] Once a week [ ] Others………
13. For how long have you been using SHEA BUTTER? 0-6 months [ ] 6-12 months [
] 1-2 years [ ] Others ………………….
14. Do you use SHEA BUTTER with other medications? Yes [ ] No[ ]
15. Which medications do you use with SHEA BUTTER? ………………………….
16. You get relief when using SHEA BUTTER. Agree [ ] Disagree [ ]
17. What kind of relief do you get? Pain stopped [ ] Pain reduced [ ] Swelling
reduced [ ] Others…………
18. How long does it take for the pain to return? ……………………………..
19. You get relief when using SHEA BUTTER with other drugs. Agree [ ] Disagree [ ]
20. Did you ever stop using SHEA BUTTER? Yes [ ] No [ ]
21. If yes why?......
22. What else do you use SHEA BUTTER for?......