EVALUATION OF WOUND HEALING ACTIVITY OF METHANOLIC LEAVES EXTRACT OF Piliostigma thoninngii IN ALBINO RATS
EVALUATION OF WOUND HEALING ACTIVITY OF METHANOLIC LEAVES EXTRACT OF Piliostigma thoninngii
IN ALBINO RATS
BY
AKAH, SUNDAY OKWURU
1410203022
A Research Project
Submitted to the Department of Biochemistry, Faculty of Science and Science Education
KEBBI STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY, ALIERO, NIGERIA
In Partial Fulfilment of the Requirements for the Award of the Degree of
BACHELOR OF SCIENCE (B.Sc. HONS.) IN BIOCHEMISTRY
October, 2018.
DEDICATION
This work is dedicated to Almighty God, the creator of heaven and earth and the fountain of all knowledge and source of all wisdom and to my lovely parents Mr. Akah Emmanuel and Mrs. Akah Lydia.
CERTIFICATION
This project report titled “Evaluation of Wound Healing Effect of Methanolic Leaves Extract of Piliostigma Thoninngii in Albino Rats” by Akah Sunday O (1410203022) has met one of the requirements for the award of Bachelor of Science (B.Sc. Hons.) Degree in Biochemistry of the Department of Biochemistry, Kebbi State University of Science and Technology, Aliero and is hereby approved for its contribution to knowledge.
…………………………………… ……………………........………………..
Dr. (Mrs.) Angela N. U. Kwaja Date
(Project Supervisor)
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Dr. Ibrahim Abubakar Babangida Date
(Head of Department)
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Prof. M. S Sule Date
(External Supervisor)
ACKNOWLEDGEMENTS
My gratitude goes to the Almighty God for giving me the necessary strength to undertake and conclude this work. This work would not have been successfully completed without the input of many people. However space does not allow me to recognize all of them individually. I am particularly indebted to my supervisor Dr. (Mrs.) Angela N. Ukwuani-Kwaja for tirelessly providing guidance, suggestions, and comments which resulted in the successful completion of this work. I equally appreciate the entire lecturers of Biochemistry Department, Chief Technologist and other laboratory staff for their effort in impacting knowledge and their encouragement.
My deepest gratitude goes to my sweet, precious and lovely parents Mr. Akah Emmanuel and Mrs. Akah Lydia for supporting me morally and financially. And my special appreciation goes to my siblings; Simon, Janet, Maria, David, Silas, James, Jimaimah and also to my relatives and classmates who have in one way or the other contributed to the success of this work, I say thank you all and may Almighty God bless you for been there for me when I needed you the most. My profound gratitude goes to my friends; Daniel Dauda, Abuzaki, Williams, Saifullahi (Finnest), Abubakar, Ojo Damilola (Skrtel), Analyst, HK, MMK, MB Suru and Safiya for their help in bringing this work to reality. I also want to acknowledge the effort of Mr. Lesley and Dalhatu Rufai for guiding me through my practical work. May God almighty richly bless and reward you all, Amen.
Finally, I wish to appreciate and acknowledge all the numerous authorities, whose work were cited in the course of carrying out this research study. Thank you all, may God bless each and every one of us, Amen.
TABLE OF CONTENTS
LIST OF FIGURE
Figure 1.1 Wound healing process………………………………………………..6
Figure 1.2: Hemostatic process…………………………………………………….7
Figure 1.3: Inflammatory process………………………………………………….8
Figure 1.4 Proliferatory process…………………………………………………...9
Figure 1.5 Remodeling process………………………………………………….10
Figure 1.6 Structure of penicillin.………………………………………………..13
Figure 3.1 Effect of methanolic leaf extract of P. thonningii on wound contraction……………………………………………………………30
LIST OF TABLES
Table 1.1 List of plants used in treatment of wound in Kebbi State, Nigeria……..16
Table 2.1 List of apparatus and equipment used………………………………….21
Table 2.2 List of chemicals or reagents used……………………………...………22
Table 3.1 Phytochemical constituents of methanolic leaf extract of P. thonningii……………………………………………………...………27
Table 3.2 Effect of methanolic leaf extract of P. thonningii on wound contraction…………………………………………………………….29
Table 3.3 effect of methanolic leaf extract of P. thonningii on epitheliazation period………………………………………………………………….31
ABSTRACT
Leaves of P. thonningii are used in many parts of West Africa including Kebbi State, Nigeria for management of wounds, chronic ulcer, gastric heart pain and headache. The present study was carried out to evaluate the wound healing effect of methanolic leaves extract of P. thonningii. The wound healing effect was evaluated using excision wound model, 10 and 50% w/w of the extract were used. Significant wound contraction and decrease in epitheliazation period were observed in animals treated with P. thonningii ointment extract when compared with control group. These findings may justify the medicinal uses of P. thonningii for management of wound.
CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 Introduction
Traditional medicine also known as herbal medicine is defined as the sum of the knowledge, skills, and practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health, as well as in the prevention, diagnosis, improvement, treatment of physical and mental illness (WHO, 2000). According to WHO estimate in 1983, a majority of the population in developing countries depend on traditional and herbal medicines as their primary source of health care (Bannerman et al., 1983). Over the past decade, there has been an increased global interest in traditional medical approaches and herbal medicinal products. However, many evidenced showed that the popularity of Complementary and Alternative Medicine (CAM) has drawn significant attention (Dev and Sukh, 2001). There is an increased and concerted efforts to the adoption and integration of traditional medicine and complementary or/and alternative medicine in both developing and developed countries in their health system (WHO, 2005).
Nearly 80% of people living in the developing countries especially in Africa depend on herbal medicine for their health needs (Agyare et al., 2009). The choice of herbal products for the treatment of diseases in Nigeria varies between regions and cultures (Sofowora, 1993). In most of the developing world like Nigeria, plants or herbal products play an important role in the treatment of diseases (Phillipson, 2001; Mensah et al., 2006). Plants have the immense potential for the management and treatment of wounds. A large number of plants are used by tribal and folklore in many countries for the treatment of wounds and burns. These natural agents induce healing and regeneration of the lost tissue by multiple mechanisms. These phytomedicine are not only cheap, effective and affordable but are also safe. The presence of various life-sustaining constituents in plants has urged scientist to examine these plants with a view to determine potential wound healing properties. Medicinal plants are coming into prominence because of the over-use of conventional medicines such as antibiotics which has resulted in the development of resistance in many infectious organisms. Thus, herbal preparations can be more effective than conventional medicines and their non-toxic nature means that they can be administered over long periods (Angolo et al., 2008).
Though several conventional drugs are known to increase healing in different type of wounds, these drugs are complicated, expensive and their availability is still limited (Adedapo et al., 2008; Abraham et al., 2012). Current estimates indicate nearly 6 million people suffer from chronic wounds worldwide (Rhoads, 2012). Unhealed wounds constantly produce inflammatory mediators that produce pain and swelling at the wound site. Chronic wounds may even lead to multiple organ failure of death of the patients (Rhoads, 2012).
The aim of wound care is to promote wound healing in the shortest time possible with minimal pain, discomfort, and scarring to the patient and must occur in a physiological environment, conducive to tissue repair and regeneration (Nayak, 2007). Wound care and maintenance involve a number of measures including dressing and administration of painkillers, use of anti-inflammatory agents, topical and systemic antimicrobial agents and healing drugs (Nayak, 2007). A large number of plants/plant extracts/decoctions or pastes are equally used by tribal’s and folklore traditions in for treatment of cuts, wounds, and burns. Plants or chemical entities derived from plants need to be identified and formulated for the treatment and management of wounds. Many plant drugs have been used in management and treatment of wounds over the years. Plants and their extracts have immense potential in the management and treatment of wounds. (Kumara et al., 2007) Therefore, the present research was designed to evaluate the wound healing potential of Piliostigma thonningii leaves.
1.2 Justification
Wounds are inescapable events of life, which arise due to physical, chemical or microbial injury. Management of wounds is complicated and expensive and research on drugs that increase wound healing activity is a developing area in modern biomedical sciences (Adedapo et al., 2008). Several conventional drugs known to increase healing in different type of wounds are complicated, expensive and their availability in rural areas is still limited (Adedapo et al., 2008; Abraham et al., 2012). Traditional medicine remains popular in rural areas due to its availability, affordability and accessibility as well. In spite of recorded traditional uses of the Piliostigma thonningii leaves, there is no scientific literature to the best of my knowledge that confirms the wound healing effect Piliostigma thonningii leaves. Thus, this research work was undertaken to investigate wound healing effects of Piliostigma thonningii leaves.
1.3 Aims and Objectives
1.3.1 Aim
This research aimed at evaluating the wound healing activity of methanolic leaves extract of Piliostigma thonningii in albino rats.
1.3.2 Objectives
⦁ To prepare methanolic leaves extract of Piliostigma thonningii
⦁ To determine the phytochemical constituents of Piliostigma thonningii leaves.
⦁ To create exicision wound model and evaluate the wound healing activity of methanolic leaves extract of Piliostigma thonningii in albino rats.
1.4 Literature Review
1.4.1 Wound
Wounds are physical, chemical or thermal injuries that result in an opening or breaking in the integrity of the skin or may also be defined as the disruption of anatomical and functional integrity of living tissue. According to the Wound Healing Society, wounds are physical injuries that result in an opening or break of the skin that causes disturbance in the normal skin anatomy and function. They result in the loss of continuity of epithelium with or without the loss of underlying connective tissue (Ramzi et al., 1994; Strodtbeck, 2001).
Wounds can be classified as open or closed wound depending on the underlying cause of wound creation and acute or chronic wound on the basis of physiology of wound healing.
⦁ Open Wounds
In open wounds, the skin is cracked open, leaving the underlying tissue exposed to the outside environment, which makes it more vulnerable to bleeding and infection. Incised wound, laceration or tear wound, abrasions or superficial wounds, puncture wounds, penetration wounds and gunshot wounds (Schultz, 1999).
⦁ Closed Wounds
In closed wounds blood escapes the circulatory system but remains in the body. It includes Contusion or bruises, heamatomas or blood tumor, Crush injury etc (Schultz, 1999).
⦁ Acute Wound
Acute wound is a tissue injury that normally precedes through an orderly and timely reparative process that result in sustained restoration of anatomic and functional integrity. Acute wounds are usually caused by cuts or surgical incisions and complete the wound healing process within the expected time frame (Lazarus et al., 1998).
⦁ Chronic wounds
Chronic wounds on the other hand are wounds that have failed to progress through the normal stages of healing and therefore enter a state of pathologic inflammation. Chronic wounds either require a prolonged time to heal or recur frequently or in most cases require extensive treatment to heal. Local infection, hypoxia, trauma, foreign bodies and systemic problems such as diabetes mellitus, malnutrition, immunodeficiency or medications are the most frequent causes of chronic wounds (Menke et al., 2007; Krishnan, 2006).
1.5 Wound Healing process
Wound healing is complex and dynamic process involving the reconstruction and regeneration of devitalized and damaged cellular structures and tissue layers as closely as possible to the original state (Nguyen et al., 2009). The process comprises of several critical biochemical events that can be grouped into four sequential, but overlapping and distinct phases of homeostasis, inflammation, proliferation or granulation and tissue remodeling (Nguyen et al., 2009; Guo and Dipietro, 2010). See figure 1.1
Figure 1.1: Wound Healing Process
1.5 1 Hemostasic (blood clotting) Process
Hemostatic (blood clotting) phase occurs in order to stop blood loss by way of a fibrin clot. When tissue is first wounded, blood comes in contact with collagen, triggering blood platelets to secrete inflammatory factors (Rosenberg and de la Torre, 2006), and also express sticky glycoproteins on their cell membranes that allow them to aggregate, forming a mass (Midwood et al., 2004). Fibrin and fibronectin cross-link together and form a plug that traps proteins and particles and prevents further blood loss (Sandeman et al., 2000).
Figure 1.2: Hemostasic Process
1.5.2 Inflammatory Process
Immediately after a blood vessel is breached, ruptured cell membranes release inflammatory factors like thromboxanes and prostaglandins that cause the vessel to spasm and prevent blood loss and to collect inflammatory cells and factors in the wound area. The inflammatory phase is characterized by the five classical signs of inflammation (i.e. redness, pain, heat, swelling and loss of function; also known as rubor, dolor, calor, tumor and functiolaesa, respectively). The inflammatory response causes the blood vessels to become leaky, releasing plasma and neutrophils into the surrounding tissue (Wahl etal., 1992). The neutrophils phagocytose debris and microorganisms and provide the first line of defence against infection. As they digest bacteria and debris, neutrophils die and release intracellular enzymes into the surrounding matrix, which further digest tissue (See figure 1.3).
Figure 1.3: Inflammatory process: By the first day following injury, neutrophils attach to endothelial cells in the vessel walls surrounding the wound (margination), then change shape to move through the cell junctions (diapedesis) and migrate to the wound site (chemotaxis). This is the beginning of the inflammatory phase.
Circulating monocytes differentiate into macrophages after they exit the blood vessels and come in contact with the extracellular matrix. Macrophages are able to phagocytose bacteria and provide a second line of defence by secreting extracellular enzymes to degrade necrotic tissue at the wound site (Sussman and Bates-Jensen, 2007).
1.5.3 Proliferatory Process
The proliferation phase starts approximately 4 days after wounding and usually lasts until day 21 in acute wounds, depending on the size of the wound and the health of the patient. It is characterized by angiogenesis, collagen deposition, granulation tissue formation, wound contraction and epithelialization (Lorenz and Longaker, 2003). Clinically, proliferation is observed by the presence of pebbled red tissue or collagen in the wound base and involves replacement of dermal tissues and sometimes subdermal tissues in deeper wounds, as well as contraction of the wound. In the final stage of epithelialization, contracture occurs as the keratinocytes differentiate to form the protective outer layer (Greenhalgh, 1998).
Figure 1.4: Proliferatory Process
1.5.4 Remodeling Process
When the levels of collagen production and degradation equalize, the maturation phase of tissue repair is said to have begun (Greenhalgh, 1998). During maturation, type III collagen, which is prevalent during proliferation, is replaced by type I collagen (Dealey, 1999). Originally disorganized collagen fibers are rearranged, cross-linked, and aligned along tension lines (Lorenz and Longaker, 2003). As the phase progresses, the tensile strength of the wound increases (Mercadetti and Cohen, 2005). Since activity at the wound site is reduced, the scar loses its red appearance as blood vessels that are no longer needed are removed by apoptosis. (Greenhalgh, 1998)
Figure 1.5: Remodeling Process
1.6 Factors affecting wound healing
Wound healing is normal biological process in the human body. Many factors can adversely affect this process and lead to improper and impaired wound healing. A thought understanding of these factors and their influence on wound healing is essential for better therapeutic option for wound treatment (Kerstein, 2007).
1.6.1 Improper diet
Wound healing is anabolic process that requires both energy and nutritrive substrates. It is reported that serum albumin level of 3.5gm dL-1 or more is necessary for proper wound healind site (Hanna, and Giacopelli, 1997). Protein is essential collagen synthesis on wound site. A state malnutrition may provide an inadequateb amount of protein and this can decreased the rate of collagen synthesis, wound tensile strength or increased chance of infection (Albritton, 1991; Rosen, and Cleary, 1991).
1.6.1 Infection at the wound site
Wound infection is probably the most common reason of impaired wound healing (Lazarus et al., 1994). Streptococcus aurous, Streptococcus pyrogenes, Escherichia coli and Pseudomonas aeraginosa (Kumar et al., 2006).
1.6.2 Insufficient oxygen supply and tissue perfusion to the wound area
Adequate blood supply and tissue perfusion is extremely important for wound healing. Excessive pain, cold and anxiety can cause local vasoconstriction and increased healing time (Cuzzell and Stotts, 1990). Smooking and use of tobaccodecreased tissue perfusion and oxygen tension in wound ( LaVan and Hunt, 1990)
1.6.3 Drugs
Many drugs are known to impair wound healing. Chemotherapeutic drugs used in cancer are the largest group well known to delay wound repair. Systemic glucocorticoids interferes normal healing process by reducing collagen synthesis and fibroblast proliferation (Franz et al., 2007).
1.6.4 Elderly age
Elderly age is found to associated with delay wound healing. It is reported that the fibroblast growth and activity diminishes and collagen production, wound contraction is slow in older individuals (Sherman, 1997).
1.6.5 Diabetes and other diseases conditions
Diabetic patients are more susceptible to wound healing. In study wound infection rate was found 11% higher in diabetic patients than in general patient’s population. Acute and chronic liver diseases also associated with delay wound healing. Patients with altered immune faction have an increased susceptibility to wound infection (Greenhalgh, 2003).
1.7 Treatment of Wounds
Some wounds may be treated at home and others may require a medical approach. Minor wounds can be treated at home by washing and disinfecting the wound to remove all dirt and debris and the use of direct pressure and elevation to control bleeding and swelling. Pains typically accompany a wound. Acetaminophen (Tylenol) can be taken as directed on the package and products with aspirin should be avoided since they can cause or prolong bleeding.
Another treatment includes pain medication. Antimicrobials including iodine based preparations and silver releasing agents are used to treat infected wounds. Antimicrobial agents target bacteria at several level (cell membrane, cytoplasmic organelle, and nucleic acid), thus minimising bacterial resistance. They can be used either on their own or in conjunction with systemic antibiotics. The many silver releasing agents, in dressing form, aim to deliver sustained doses of silver to the wound. In addition to the microbicidal effect of silver on common wound contaminants, silver may also be effective against methicillin resistant Staphylococcus aureus (MRSA). Penicillin and other antibiotics may also be prescribed if there’s an infection or high risk for developing an infection.
1.7.1 Penicillin
In 1928, Alexander Fleming isolated penicillin from a sample of the mold Penicillinium notatum in his laboratory at St. Mary’s Hospital in London. This antibacterial substance was named penicillin by Fleming. It was not introduced into clinical practice until 1941, when Florey, Chain, and their colleagues had been successful in extracting enough penicillin (Fleming, 1946; Florey, 1949). Penicillins are one of most important antibiotics groups. They are extremely effective and still widely used. Moreover, they are the drugs of choice for a large number of infectious diseases. Penicillins belong to the β-lactam group, which also include cephalosporins, monobactams, and carbapenems. Β-lactam antibiotics have four-membered ring structure (Chain, 1954; Abraham, 1949).
1.7.2 Chemistry
The basic structure of penicillin includes a nucleus (6-aminopenicillanic acid, 6-APA) and side chain (R). The penicillin nucleus consists of thiazolidine ring linked to a β-lactam ring and it is a requirement for biologic activity of these molecules. The side chain determines many of the antibacterial and pharmacologic characteristics of penicillins (Davies, 1994).
Figure 1.6: Structure of Penicillin
1.7.3 Pharmacokinetics
Absorption: Most of the penicillins are incompletely absorbed after oral administration, and they reach the intestine in sufficient amounts to affect the composition of the intestinal flora. However, amoxicillin is almost completely absorbed. Consequently, it is not appropriate therapy for the treatment of Shigella- or Salmonella-derived enteritis, because therapeutically effective levels do not reach the organisms in the intestinal crypts. Absorption of all the penicillinase-resistant penicillins is decreased by food in the stomach, because gastric emptying time is lengthened, and the drugs are destroyed in the acidic environment. Therefore, they must be administered 30 to 60 minutes before meals or 2 to 3 hours postprandial. Other penicillins are less affected by food.
3. Distribution: The β-lactam antibiotics distribute well throughout the body. All the penicillins cross the placental barrier, but none has been shown to be teratogenic. However, penetration into certain sites, such as bone or cerebrospinal fluid (CSF), is insufficient for therapy unless these sites are inflamed. Penicillin levels in the prostate are insufficient to be effective against infections.
4. Metabolism: Host metabolism of the β-lactam antibiotics is usually insignificant, but some metabolism of penicillin G has been shown to occur in patients with impaired renal function.
5. Excretion: The primary route of excretion is through the organic acid (tubular) secretory system of the kidney as well as by glomerular filtration. Patients with impaired renal function must have dosage regimens adjusted. Thus, the half-life of penicillin G can increase in the presence of renal dysfunction. Probenecid inhibits the secretion of penicillins by competing for active tubular secretion via the organic acid transporter and, thus, can increase blood levels. Nafcillin, dicloxacillin and oxacillin are exceptions to the rule and are not eliminated by the kidneys. The penicillins are also excreted into breast milk.
1.7.4 Mechanisms of Action
All β-lactam antibiotics share general mechanisms of antibacterial action. These mechanisms involve: attachment to specific penicillin-binding proteins (Waxman and Strominger, 1983; Yocum et al., 1980), inhibition of the bacterial cell wall peptidoglycan synthesis (Tipper, 1987), and inactivation of an inhibitor of the autolytic enzymes in the cell wall, which initiate bacterial cell lysis and death (Tomasz, 1979). The relationship between inhibition of PBP’s activity and activation of autolysins is unclear. Some organisms have defective autolytic enzymes and are inhibited but not lysed they are referred to as tolerant (Tomasz and Holtje, 1977). The PBPs vary in their affinities for different β-lactam antibiotics.
1.8 Medicinal plants used for treatment of wound in Kebbi State, Nigeria.
Some medicinal plant used to treat wounds in Kebbi State are shown in the table below (table 1.1)
Table 1.1: List of plant used in the treatment of wounds in Kebbi State, Nigeria.
S/N Botanical name Local Name Part used LGA
1 Vitellaria paradoxa Kade Seeds and barks Matseri (Fakai)
2 Parkia biglobosa Doruwa Barks and roots Buk (Fakai)
3 Euphorbia balsamifera Aguwa Leaves and roots Marafa (fakai)
4 Acacia nilotica Bagaruwa Seeds Yauri
5 Terminalia avicenmoidu Baushe Roots Kele (D/wasagu)
6 Balanites aegyptiaca Aduwa Seeds Yauri
7 Calotropis procera Tumfafiya Milk content Mahuta
8 Euphorbia hirta Nonon kurciya Latex D/wasagu
9 Mormodica balsamifera Garafuni Leaves and bark Tungan yawo and Augie
10 Annona senegalensis Gwandan daji Barks Yauri, Zuru and aleiro
11 Anogeissu leiocarpus Marke Seeds Matseri (Fakai))
12 Daniela olivera Maje Barks Zuru and Yauri
13 Acacia albida Gawo Barks Pisabu (Yauri)
14 Diospyros mespliformis Kanya Barks Uchiri (Fakai)
15 Guiera senegalensis Sabara Roots and leaves Yauri, Argungu and Zuru
16 Ficus gnaphalocapa Cediya Roots Bagida (Fakai)
17 Piliostigma thonningii Kalgo leaves Kalgo, Koko and Aleiro
18 Pennisetum pedicellatum Kasuwa Leaves and roots B/K, Aleiro and Zuru
1.9 PILIOSTIGMA THONNINGII
1.9.1 Taxonomy
Kingdom: Plantae
Phylum: Angiosperms
Class: Eudicots
(Unranked): Rosids
Order: Fabales
Family: Fabaceae
Subfamily: Cercidoideae
Tribe: Bauhinieae
Genus: Piliostigma
Species: P. thonningii
Binomial name: Piliostigma thonningii (Schum.)
Common name: Camel’s foot, Monkey bread
Local names: abefe (Yoruba), kalgo (Hausa) Okpoatu (Igbo), ejei-jei (Igala), omepa (Igede) and nyihar (Tiv).
1.9.2 Description of Piliostigma thoningii
Piliostigma thoningii is a deciduous, single-stem leguminous tree belonging to the family Caesalpiniaceae. It is a perennial in habit with large, simple, two-lobed, leathery leaves which resemble a camel’s foot and account for the common name ‘Camel foot’. The name ‘Piliostigma’ means cap-shaped stigma, while specific name, thonningii was given after the Danish Botanist, Peter Thonning. It was formerly called Bauhinia thonningii, but later differentiated from Bauhinia by its unisexual flowers and indehiscent pods.
Figure 1.7: Piliostigma thonningii Fresh Leaves
The flowers have five white to pinkish pendulous petals with male and female organs on separate trees produced during November and April (Jimoh and Oladeji, 2005). The fruit is a hairy, hard, flattish pod which turns rusty brown at ripening and split; it is usually persistent on the tree and produced between June and September (Lock and Simpson, 1999). Piliostigma thonningii is also known across Africa and other sub-Saharan countries as Mukolokote (Venda); Mokgoropo (North Sotho). In Nigeria, the plant bears local names such as abefe (Yoruba), kalgo (Hausa) Okpoatu (Igbo), ejei-jei (Igala), omepa (Igede) and nyihar (Tiv) (Dasofunjo, 2000). It grows abundantly in the wild in some parts of Nigeria such as Zaria, Bauchi, Ilorin, Plateau, Lagos, Abeokuta and Lagos (Schultes and Hofmann, 1973; Djuma, 2003) and some parts of Kogi, Benue, Kebbi, Sokoto and Nasarawa States. (Jimoh and Oladiji, 2005)
1.9.3 Medicinal Uses of Piliostigma thonningii
In Africa, Piliostigma thonningii the root and twig have been used for the treatment of dysentery, fever, respiratory ailments, snake bites, hookworm and skin diseases (Jimoh and Oladiji, 2005). It is also used in the treatment of malaria fever, wounds, ulcers, gastric heart pain, arthritis, headache, hemorrhoids backache and gingivitis (Egharevba and Kunle, 2010). The leaves are used for the treatments of wounds, chronic ulcers, diarrhea, toothache and gingivitis, cough, and bronchitis. The leaf, stem bark or root extracts are taken as cough medicine, whereas the leaves as menorrhagia medicine (Ighodaro et al., 2012). The stem bark, in addition to dysentery, toothache and snakebite, is also used as an anthelmintic (Lewis and Elvin-lewis, 1979). Traditional healers in “Doila” refer to this plant as “child remedy” as it is mainly used as a remedy for children (Egharevba and Kunle, 2010). The bark of Piliostigma thonningii is used as a remedy for cough, usually as an infusion or by chewing. A common use in Uganda is to stop diarrhoea, dysentery and intestinal upsets (Ajali, 2002; Burkill, 1995). Also, the infusion is also used in the treatment of malaria and leprosy. Other uses of the bark include analgesic, remedy for sore throat, toothache, stomachache and earache (Cowan, 1999). D-3-0 methylchiroinositol, an anthelmintic compound isolated from the stem bark of Piliostigma thonningii is used to treat helminthiasis in African traditional medicine (Cowan, 1999).
1.9.4 Other Uses of Piliostigma thonningii
The bark is commonly used for tying hut, fence and bridge building. The bark provides tanning material and yields a red-brown dye used for cloth and wooden tools. The root yields a red-brown or black dye, and the fruit and seeds a black or blue one. In Ghana, Nigeria and DR Congo women chew the root-bark to redden their lips; the bark is also rubbed on the lips to colour these red. Hausa in Nigeria stain their teeth red by chewing (Neuwinger, 2000).
The tree provides poles and timber for local house construction. The wood is also used for kitchen utensils, tool handles, furniture, bedposts, wheel-work and carpentry. The unripe fruits are used as a soap substitute, and the ash of ripe fruits is also used in soap making. Infusions of the bark, leaf or pod are used to coagulate Funtumia latex in making rubber. The bark yields a gum that swells and softens in water and is used for caulking. A resin obtained from the unripe fruit is used as glue for fastening the iron parts of tools and spears to the handles (Burkill, 1995).
Piliostigma thonningii is an agroforestry tree, suitable for intercropping with crops. It provides good shade and serves as shelterbelt in homesteads when in full foliage (World Agroforestry Centre, 2009).
CHAPTER TWO
2.0 MATERIALS AND METHODS
2.1 Materials
2.1.1 Apparatus and Equipments
Table 2.1: List of Apparatus and Equipments Used.
S/N Apparatus/Equipments Model/Type Manufacturer
1 Beaker Glass Pyrex glass England
2 Pipette Glass Pyrex glass England
3 Measuring cylinder Glass Pyrex glass England
4 Conical flask Glass Pyrex glass England
5 Funnel Glass Pyrex glass England
6 Glass rod Glass Pyrex glass England
7 Test tubes Glass Pyrex glass England
8 Spatula Iron Spectrum lab 23A guflex England
9 Motar Wood -
10 Pestle Wood -
11 Muslin cloth Cotton Asaba Texile Mill, Nig.
12 Masking tape Paper -
13 Aluminum foil Aluminium Pacpro Industries ltd Lagos nig.
14 Sample bottles Rubber -
15 Transparent ruler Rubber -
16 Plain container Rubber -
17 Volumetric flask Glass -
18 Syringe and needle Rubber and iron Jiangyin Nanquen macromolecule products co., ltd.
19 Face mask - Anhui kangda medical products co., ltd.
20 Drying oven Paper Whatman
21 Refrigerator Suntex, kontes Matter, London
22 Weighing Balance - -
23 Centrifuge - -
26 Water bath HH-S Stainless -
2.1.2 Chemicals and Reagents
Table 2.2: List of Chemicals Reagents Used
S/N Reagents Manufacturer Conc. Used
1 Chloroform BDH chemical ltd England 99%
2 Distilled water BDH chemical ltd England 50cl
3 Fecl3 - 0.1%
4 Acetic acid - 10%
5 Ammonia BDH chemical ltd England Conc.
6 NaOH - 10%
7 H2SO4 - Conc.
8 HCl - 10%
9 HCl BDH chemical ltd England Dil.
10 Benedict’s reagent - -
11 Wagner’s reagent - -
12 NaOH - Dil.
13 Acetic anhydride - -
14 Methanol - 99.5%
15 Petroleum jelly - -
16 Penicillin - -
17 Ketamine - -
2.2 Methods
2.2.1 Collection and Identification of plant material
Fresh leaves of Piliostigma thonningii were collected within the premises of Kebbi State University of Science and Technology, Aleiro Nigeria, which are then identified and authenticated by Dr. D. Singh of Department of Biological Science, Kebbi State University of Science and Technology, Aleiro. A sample with voucher number 109 has been deposited for future reference at the department’s Herbarium.
2.2.2 Preparation of methanolic extracts of Piliostigma thonningii leaves
The leaves of Piliostigma thonningii were separated from the stem and air dried under room temperature until constant weight was obtained. The dried leaves were then pulverized using a wooden mortar and pestles into powders and was sieved to get uniform powdered. After which, 100g of the pulverized plant material was dissolved in 1000mL of methanol for 72 hours with constant shaking and filtered using muslin cloth. The filtrate was concentrated in an oven at 40°C and stored in a refrigerator at 4°C prior to the commencement of this study.
2.3 Qualitative Phytochemical Screening
The Piliostigma thonningii leaves extracts were analyzed for glycosides, alkaloids, saponins, tannins, flavonoids, steroids, carbonhydrates Antraquinones and quinones using standard procedures.
2.3.1. Test for Saponin
To 2ml of the extract, 2ml of distilled water was added and agitated in a test tube for 5minutes. The formation of foams indicates the presence of saponin (Sofowora, 1989).
2.3.2. Test for Tannins
5 drops of 0.1% ferric chloride was added to 2ml of extract, a brownish green or blue black coloration indicates the presence of tannins (Sofowora, 1989).)
2.3.3. Test for Glycosides.
2ml of acetic acid was added to 2ml of the extract. The mixture was cooled in cold water bath. 2ml of concentrated H2S04 was then added, colour development from blue to bluish green indicates the presence of glycosides (Sofowora, 1989).
2.3.4. Test for Flavonoids
2ml of 10% Sodium hydroxide was added to 2ml of the extract in a test tube. An intense yellow colour was formed which turned colorless upon addition of 2ml of dilute hydrochloric acid indicating the presence of flavonoid (Sofowora, 1989).
2.3.5. Test for Antraquinones
2ml of the extract were boiled with 5ml of 10% hydrochloric acid for 3minutes. 5ml of chloroform was added. 5 drops of 10% ammonia was added. A rose pink coloration indicates the presence of Antraquinones (Harbourne, 1998).
2.3.6. Test for Carbohydrates
To 5ml of the extract, 1ml of benedict’s reagent was added and boiled for 5minutes. Apperance of red colour indicates the presence of carbohydrate (Harbourne, 1998).
2.3.7 Test for Alkaloids
1.27g of iodine and 2g of potassium iodide dissolved in distilled water (Wagner’s reagent), this was added to the plant extract. Appearance of reddish brown colour confirmed the presence of alkaloids (Harbourne, 1998).
2.3.8 Test for Steroids
To 1ml of the extract, 0.5ml of acetic anhydride and 0.5ml chloroform were added and concentrated sulphuric acid later added. Formation of a brownish green ring at the contact of the two liquids indicates the presence of steroids (Harbourne, 1998).
2.3.9 Determination of Quinones
Dilute sodium hydroxide was added to the extract in a test tube. The appearance of a red or blue-green color implies that Quinone’s are present.
2.2.4 Experimental Animals
About 20 young adult albino rats of both sexes, weighed between 150-250g, were purchased from the Animal House, Kaduna, Nigeria and were transported to the Department of Biochemistry Laboratory, Kebbi State University of Science and Technology, Aliero, Kebbi State, Nigeria and used for this study. They were fed with growers mash (Vital Feeds, Nigeria) and tap water. The albino rats were housed under standard laboratory environment, and allowed to acclimatize to the laboratory environment for 10 days before the commencement of the research (Harbourne, 1998).
2.2.5 Excision wound
The animals were anesthetized by using ketamine (100 mg/kg, im). An impression was made on the dorsal thoracic region 1 cm away from vertebral column and 5 cm away from ear on the anaesthetized rat. The particular skin area was shaved to a circular diameter of 40 mm by means of razor blades one day prior to the experiment and the shaved area was cleaned with 70% v/v ethanol before excision wounds were created. The skin of impressed area was excised to the full thickness to obtain a wound area of about 500 mm2 (Wemer et al., 1994).
2.2.6 Animal Groupings and Treatments
The rats were divided into five (5) groups of four (4) rats each for evaluation of wound healing activity of methanolic leaves extract of Piliostigma thonningii extract. Wound treatment commenced on the second day of wound excision.
Group one (1) served as untreated control,
Group two (2) was treated with normal base
Group three (3) served as standard group, treated with Penicillin
Group four (4) was treated with 10% w/w of methanolic leaves extract of Piliostigma thonningii.
Group five (5) was treated with 50% w/w of methanolic leaves extract of Piliostigma thonningii and the rats were observed daily.
The extract and reference drugs were then topically applied to the wounds 24 hourly for 15 days.
2.2.5 Wound size / contraction
Wound size measurement can be used to monitor the progress of healing through changes in the area of the wound with time. The size of the wound was measured at a regular interval of 72 hours. An excision wound area was measured by vernier calipers and millimeter ruler and expressed in percentage of healed wound area (Rashed, 2003). The evaluated surface area was then engaged to determine the percentantage of wound contraction, taking initial size of wound, 300mm2, as 100%, by using the following formula as:
Wound contraction (%) = initial wound size – specific day wound size x 100 Initial wound size
2.2.6 Epithelialization period
It was evaluated by noting the number of days requisite for the Escher to fall off from the wound surfaceexclusive of leaving a raw wound behind (Rashed et al., 2003).
2.7 Statistical Analysis
The values were calculated as mean ± S.E.M. The significance of the difference of the mean value with respect to control group was analyzed by one way ANOVA followed by Dunnet’s t-test.
CHAPTER THREE
3.0 RESULTS
3.1 Percentage Yield of extract
Methanolic extraction of piliostigma thonningii leaves yield 29%.
3.2 Preliminary phytochemical screening
The results for the phytochemical screening of Methanolic Leaves Extract of Piliostigma thonningii revealed the presence of saponin, tannin, glycoside, flavanoid, anthraquinones, carbonhydrates, alkaloid, steroids and quinones as shown in the Table 3.1 below.
Table 3.1: Phytochemical Constituents of Methanolic Leaves Extract of Piliostigma thonningii.
S/N Phytochemicals Results
1 Saponin +
2 Tannin +
3 Glycosides +
4 Flavanoids -
5 Anthrequinones +
6 Carbonhydrates +
7 Alkaloids +
8 Steroids +
9 Quinines +
Key: (+): detected, (-): Not detected
3.3 Wound Healing Studies
The results of percentage wound contraction and epitheliasation period are given in table 2 and table 3. The mean percentage of wound area were calculated for 15th post wounding days. At the 3rd day 50% (w/w) extract ointment of piliostigma thonningii leaves showed significant wound healing effect compared to control. It was also observed that 10% (w/w) extract ointment of piliostigma thonningii leaves treated groups showed significant wound healing effect from day 6th onwards which was comparable with standard drug (penicillin). The 50% (w/w) extract ointment showed complete epitheliasation in 10.750.75 days which was not significantly different from 10% (w/w) extract ointment (12.750.85 days), standard drug penicillin (13.250.63) but significantly different from control (16.001.16).
Table 3.2: Effect of methanolic leaves extract of Piliostigma thonningii on wound healing
Wound Area(mm2)SEM ( Wound contraction )
DAY Control Normal Base Penicillin 10% Extract 50% Extract
DAY 0 500.00±0.00
(0.00) 500.00±0.00
(0.00) 500.00±0.00
(0.00) 500.00±0.00
(0.00) 500.00±0.00
(0.00)
DAY 3 396.50±13.25
(20.70) 382.23±9.41
(23.53) 368.31±13.00
(26.34) 339.36±4.76*
(32.12) 313.86±16.25**
(37.22)
DAY 6 315.00±5.20
(37.00) 286.36±9.05
(42.72) 232.50±4.33**
(53.5) 228.75±7.18**
(54.25) 203.25±9.23**
(59.35)
DAY 9 186.33±26.91
( 62.73) 158.25±19.26
(68.35) 105±32.04*
(79.00) 90.5±6.19 **
(81.90) 71.75±17.60**
(85.65)
DAY 12 114.33±38.46
(77.13) 96.25±28.92
(80.75) 32.75±18.41*
(93.45) 22.25±5.11 *
(95.55) 12.25±7.88*
(97.55)
DAY 15 40.00±23.09
(92.00) 29.00±20
(94.20) 5.25±3.09
(99.00) 5.75±36
(98.85) 0.00±0.00
(100)
Each value is the mean ± S.E.M. of four rats; *P <0.05, **p<0.01 vs. control, One way ANOVA followed by Dunnet’s t-test.; % wound contraction is given within the bracket.
Figure 3.1: Effect of methanolic leaves extract of piliostigma thonningii on wound contraction
TABLE; 3.3 Effect of Methanol Extract of piliostigma thonningii on Epithelisation Period
Groups Treatments (dose) Epitheliazation period (days)
1 Control (untreated) 16.00±1.16
2 Normal base (100% w/w) 15.50±0.65
3 Penicillin (10% w/w) 13.25±0.63
4 10% P.T (10%w/w) 12.75±0.85*
5 50% P.T (50% w/w) 10.75±0.75**
CHAPTER FOUR
4.0 DISCUSSION, CONCLUSION AND RECOMMENDATION
4.1 Discusion
Tannins act as free radical scavengers and terpenoids promote wound healing due to their astringent and antimicrobial property, and saponins due to their antioxidant and antimicrobial activity, which appear to be responsible for wound contraction and elevated rate of epithelialization. Steroids and polyphenols are also responsible for wound healing due to free radical-scavenging and antioxidant activity, which are known to reduce lipid peroxidation, thereby reduce cell necrosis and improving vascularity (Baravkar et al., 2008). Several types of injuries like burn, wounds, and skin ulcers usually generate superoxides and lipid peroxidation through the activation of neutrophils. Hence, any drug that inhibits lipid peroxidation is believed to increase the viability of collagen fibrils by increasing the strength of collagen fibres, increasing the circulation, preventing the cell damage, and by promoting the DNA synthesis. Better collagenation seen under the influence of some herbal extracts may be because of improved antioxidant status. Thus, an intervention into any one of these phases by drugs could eventually lead to either promotion or depression of the collagenation phase of healing (Umadevi et al., 2006).
In the present study, wound healing effect of Piliostima thonningii extract could be a function of either the individual or the synergistic effects of the phytochemical constituents. These active phytochemical constituents may promote the process of wound healing by increasing the viability of collagen fibrils, increasing the strength of collagen fibers either by increasing the circulation or by preventing the cell damage or by promoting the DNA synthesis (Majumdar et al., 2007).
4.2 Conclusion
P. thonningii leaves are used in many parts of West Africa including Kebbi State, Nigeria for management of wounds, chronic ulcer, gastric heart pain and headache. The methanolic leaves extract of Piliostigma thonningii exhibited remarkable wound healing effect via rapid wound contraction and epitheliazation period respectively which was comparable to the standard drug penicillin. These findings may justify the medicinal uses of P. thonningii for management of wound.
4.3 Recommendation
This work is preliminary work. Further research necessary for isolation, purification and characterization of the bioactive compounds in the extract responsible for it wound healing effect as well as its mechanism of wound healing action. In vivo toxicological studies of the extract should be done to evaluate its safety and therefore, in order to unravel the possible ingredient(s) further isolation study is warranted
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APPENDIX
APPENDIX I
PERCENTAGE YIELD OF EXTRACT
The percentage yield of extract is calculated using the formula below:
% yield = Weight of dried extract
Weight of powdered leaves soaked
% yield = 29g x100
100g
= 29%
APPENDIX II
PREPARATION AND APPLICATION OF EXTRACT OINTMENT
⦁ Preparation of 10% (W/W) Extract Ointment
4g of extract was added in 36g of normal base and was thoroughly mixed.
⦁ Preparation of 50% (W/W) Extract Ointment
21.5g of extract was added in 21.5g of normal base and was thoroughly mixed.
Dermal application of extract
APPENDIX III
Wound Healing Effect
The length and width of wound of each rat were measured in mm and multiply to give the total wound area.
Raw Data for Wound Healing Effect of methanolic leaves extract
⦁ Raw Data of Control (Untreated) group
Groups Day 0
L x W Day 3
L x W Day 6
L x W Day 9
L x W Day 12
L x W Day 15
L x W
R1 22.36x22.36=
500mm2 20.5x20=
410mm2 18x17.5=
315 mm2 16x15= 240mm2 14x13= 183mm2 10x8= 80mm2
R2 22.36x22.36=
500mm2 21x19.5=
409.5mm2 18x18= 324mm2
14x12= 163mm2 10x11=
110mm2 5x8= 45mm2
R3 22.36x22.36=
500mm2 18.5x20=
370mm2
17x18= 306mm2 13x12=
156mm2 5x10= 50mm2 Healed
⦁ Raw Data of Normal Base group
Groups Day 0
L x W Day 3
L x W Day 6
L x W Day 9
L x W Day 12
L x W Day 15
L x W
R1 22.36x22.36=
500mm2 20x19= 380mm2 16x17= 272mm2 15x14= 210mm2 13x14= 182mm2 9x9= 81mm2
R2 22.36x22.36=
500mm2 19x19.5=
370.5mm2 17x17.5= 297.5mm2 15x10= 150mm2 10x8= 80mm2 7x5= 35mm2
R3 22.36x22.36=
500mm2 18.5x20= 370mm2 15x18= 270mm2 9x13= 117mm2 10x6= 60mm2 Healed
R4 22.36x22.36=
500mm2 20.5x20=
410mm2 18x17= 306mm2 12x13=
156mm2 7x9= 63mm2 Healed
⦁ Raw Data of Penicillin treated group
Groups Day 0
L x W Day 3
L x W Day 6
L x W Day 9
L x W Day 12
L x W Day 15
L x W
R1 22.36x22.36=
500mm2 18x19= 342 mm2 15x16= 240 mm2 12x10= 120 mm2 8x7=
56 mm2 3x4= 12 mm2
R2 22.36x22.36=
500mm2 19.5x19.5= 380.5 mm2 15x16= 240 mm2 10x11= 110 mm2 6x5=
30 mm2 3x3= 9 mm2
R3 22.36x22.36=
500mm2 20x20= 400 mm2 15x15= 225 mm2 10x8=
80 mm2 5x4= 20 mm2 Healed
R4 22.36x22.36=
500mm2 18x19.5= 351 mm2 15x15= 225 mm2 10x11= 110 mm2 5x5=
25 mm2 Healed
⦁ Raw Data of 10% (w/w) extract ointment treated group
Groups Day 0
L x W Day 3
L x W Day 6
L x W Day 9
L x W Day 12
L x W Day 15
L x W
R1 22.36x22.36=
500mm2 18.5x18.5= 342.25mm2 15x15= 225 mm2 8x10.5= 84 mm2 4x8=
32 mm2 3x5=15mm2
R2 22.36x22.36=
500mm2 18x18.5= 333 mm2 15x16= 240 mm2 12x9= 108 mm2 5x6=
30 mm2 2x4=8mm2
R3 22.36x22.36=
500mm2 18.9x17.5= 330.75mm2 14x15= 210 mm2 8x10= 80 mm2 3x5=
15 mm2 Healed
R4 22.36x22.36=
500mm2 19x18.5= 351.5 mm2 16x15= 240 mm2 9x10=
90 mm2 2x6=
12 mm2 Healed
⦁ Raw Data of 50% (w/w) extract ointment treated group
Groups Day 0
L x W Day 3
L x W Day 6
L x W Day 9
L x W Day 12
L x W Day 15
L x W
R1 22.36x22.36=
500mm2 18x17= 306 mm2 14x15= 210 mm2 8x10= 80 mm2 3x5=
15 mm2 Healed
R2 22.36x22.36=
500mm2 18x18= 324 mm2 13x14= 182 mm2 5x5= 25 mm2 2x3=
6 mm2 Healed
R3 22.36x22.36=
500mm2 17x16= 274 mm2 15x15= 225 mm2 11x10= 110 mm2 5x4=
20 mm2 Healed
R4 22.36x22.36=
500mm2 19x18.5= 351.5 mm2 14x14= 196 mm2 8x9= 72 mm2 4x2=
8 mm2 Healed
⦁ Raw Data for Epitheliazation Periods
Groups Control (untreated) Normal base Penicillin 10% (w/w) extract ointment 50% (w/w) extract ointment
R1 18 17 15 13 12
R2 16 16 12 15 9
R3 14 15 13 11 12
R4 14 13 12 10
Day 1 (Control) Day 15 (Control)
Day 1 (50% extract) Day 15 (50% extract)
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