COMPARATIVE EVALUATION OF PHYTOCHEMICALS AND ANTISICKLING ACTIVITIES OF FRUITS EXTRACTS OF PHOENIX DACTYLIFERA L. AND LEAVES OF TERMINALIA CATAPPA L.

ABSTRACT
In this study, dried and blended plant materials of fruits of Phoenix dactylifera L. and leaves of Terminalia catappa L. were percolated with methanol, the water soluble fractions were obtained by decoction while the methanol percolated portion was macerated and fractionated with n-hexane, chloroform and ethyl-acetate. The chemical screening of fractions revealed the presence of alkaloids, anthraquinones, cardiac glycosides, flavonoids, saponins and tannins. The plant fractions were evaluated against sickle cell using p-hydroxybenzoic acid and normal saline as positive and negative controls. The method involves inhibition of sodium metabisulphite induced sickling of HbSS red blood cells, collected from sickle cell patients. The methanol soluble fractions PDME and TCME (5mg/ml) showed antsickling activities of 78.2% and 82.8% at 180min of incubation respectively while the chloroform soluble fractions PDCE and TCCE (5mg/ml) demonstrated antisickling activities of 77.6% and 86.7% respectively at 180min incubation time. The n-hexane soluble fractions, PDHE and TCHE (5mg/ml) showed antisickling activities of 71.8% and 78.3% while that of the ethyl-acetate soluble fractions, PDEE and TCEE(5mg/ml) indicated antisickling activities of 70.2% and 83.5% respectively at 180min incubation time compared to p-hydroxybenzoic acid (5mg/ml) with 76.2% inhibition at 180min time of incubation and the water soluble fractions, PDWE and TCWE (5mg/ml) which showed 10.9% and 12.4% sickled red blood cells at 180min incubation time indicating antisickling activities of 89.1 and 87.6 respectively. Compared to the untreated SS suspension with 83.6% sickled red blood cells at 180mm incubation. These results showed that the fruits of date palm and leaves of Terminalia catappa are good candidates for sickle cell disease therapy.

CHAPTER ONE
1.0                                                     INTRODUCTION
Several diseases cause million deaths in the world, and particularly in Africa. Among these, is the sickle cell disease (SCD) that affects more than 50 million people. Each year, approximately 100,000 children are born worldwide with this hemoglobinopathy i.e. sickle cell disease which is a genetic disorder. This disease is considered as a public health problem in many countries, but with a major burden in Africa particularly in tropical regions in west and central Africa (Buchanan, et al., 2004).
Red blood cells (RBCs) or erythrocytes are made up of two main components, the cytoplasmic proteins and the membrane. Haemoglobin (Hb) constitutes 97.5% (by weight) of the protein system while the other 2.5% proteins provide energy and help regulate water and ionic composition of the cell. The membrane which constitutes a very small percentage of the cell mass, surrounds the intracellular concentrated solution of Hb (Mehanna, 2001). Membrane protein are involved in active ion transport which keeps the intracellular sodium ion (Na+) and calcium ion (Ca2+) concentration low and that of potassium ion (K+) and magnesium ion (Mg2+) high. The proteins provide also peculiar flexibility to the RBCs membrane (Elekwa et al., 2003; osuagwu and Mbeyi, 2007). In sickle cell anaemia, the shortened RBCs survival is due to increased  rigidity of cells and membrane damage caused by intracellular precipitation of Hbs. Hbs aggregation (polymerization) induces a panoply of cellular and tissue injuries (substantial loss of membrane flexibility, sickle shaped e.t.c). [Buchanan et al., 2004].
           Sickle cell anemia is a severe hereditary form of anemia in which a mutated form of hemoglobin distorts the red blood cells into a crescent shape at low oxygen levels. Up to date, there is no affordable solution for this disorder. Several therapeutic options were tried in order to fight against SCD without appropriate solution for poor African population. All of these therapeutic approaches are either expensive or toxic and are not accessible to the populations with low incomes (Mehanna, 2001).
           Various approaches have been adopted in an effort to find agents that inhibit the polymerization of haemoglobin (Hb) and hence prevent or reduce the occurrence of crisis in SCD (Iyamu et al., 2002). Towards this goal, oxygen, carbon monoxide and sodium nitrite were used to reduced the amount of deoxy-Hb. The above approaches did not give the much-needed beneficial effects based on the reduction of painful crisis as the criterion for successful treatment (Iyamu et al., 2002). Bone marrow transplantation has in recent years been found to be an efficient but practically impossible method in developing countries in controlling the scourge. The cost implications, the availability of necessary expertise and the problems of finding suitable donors, however, constitute a major setback to this approach in third world countries with weak economies like Nigeria. While genetic counseling holds a prominent position in enlightening the population about this condition and have been found to be beneficial in guiding people with respect to the choice of a mate, its role in eradicating the condition is not feasible because of balanced polymorphism and the fact that providing the right counsel does not necessarily lead to the rational choice of a mate (Moody et al., 2003; Akinsulie et al., 2005).
           Furthermore, several current research activities are focused on identifying new drugs that are capable of preventing the loss of water from red blood cells (RBCs) or increasing the level of foetal haemoglobin, a variety of haemoglobin that prevents the sickling of RBCs. Clotrimazole, hydroxyurea and erythropoietin (a genetically engineered hormone that stimulates RBC production) were proposed in this regard. Unfortunately, these drugs are known for their serious side effects, hence, limiting their clinical use (Mehanna, 2001; Akinsulie et al., 2005).
           Presently, first-line clinical management of sickle cell anemia include use of folic acid, amino acids (as nutritional supplements), penicillin-prophylaxis (helps prevent infection) and anti-malarial prophylaxis (helps prevent malaria attack). The faulty ‘s’ gene is not eradicated in treatment, rather the condition is managed and synthesis of red blood cells induced to stabilize the patients hemoglobin level. Further management and treatment of this disorder with compounds or techniques which directly affect the hemoglobin (Hb) molecule (e.g. hydroxyurea, bone marrow transplantation and blood transfusion) are very expensive and out of reach for the low income earners and besides may expose the patient to mutagenicity, iron overload and fatal risks (Brittain and Han, 2004).
           Medicinal plants are plants containing inherent active ingredients used to cure disease or relieve pain. The use of traditional medicinal plants in most developing countries as therapeutic agents for the maintenance of good health has been widely observed (UNESCO, 1996). Medicinal plant is defined as any plant with one or more of its organs containing substance that can be used for therapeutic purpose or which can be used as precursors for the synthesis of drugs. The practice of traditional medicine using medicinal plants is as old as the origin of man. This type of health care was described as Herbalism or Botanical medicine. The growing sophistication in lifestyle among world populations makes it imperative to refer to herbal practice as alternative or complimentary medicine to appeal to a cross section of people  irrecspective of their cultural affiliation. These types of herbal practice that falls outside the mainstream of western or orthodox medicine and has remained largely unrecognized, have been described as traditional medicine, indigenous medicine or folk medicine. Two third of the world population (mainly in the developing countries) rely entirely on such traditional medical therapies as their primary form of health care. A review, reiterated that the use of traditional medicine can not fade out in the treatment and management of an array of diseases in the African continent. This was attributed to our socio-cultural heritage, lack of basic health care and personnel to take charge of every nook and cranny of our rural populations (Summer, 2000).
           The bioactive ingredients that have the therapeutic activity in plants used in traditional practice are mostly unidentified and traditional healers believe in the holistic nature of their treatment. Substances found in medicinal plants, containing the healing property of plants are known as the active principle. In recent years, these active principles have been extracted and used in different forms such as infusions, syrups, concoctions, infused oils, essential oils, ointments and creams (Sofowora, 1993).
           Medicinal plants are of great importance to the health of individual and communities. The medicinal value of these plants lies in some chemical active substances that produce a definite physiological action on the human body. The most important of these chemically active constituents of plants are; alkaloids, tannins, flavonoids and phenolic compounds. Many of these indigeneous medicinal plants are also used for medicinal purposes (Edeoga, 2005).
           Plants are used medicinally in different countries and are a source of many potent and powerful drugs. A wide range of medicinal plant parts are used as extract for raw drugs and they possess varied medicinal properties. The different parts used include root, stem, flower, fruit and twigs exudates While some of the these raw drugs are collected in smaller quantities by folk healers for local use, many other raw drugs are collected in larger quantities and traded in the market as raw materials for many herbal industries (Uniyal et al., 2006).
           Many pharmaceutical companies show interest in plant-derived drugs mainly due to the current widespread believe that “green medicine” is safe and more dependable than the costly synthetic drugs which may have adverse side effects. The world health organization (WHO) reported that 80% of the world population presently uses herbal medicine for some aspect of primary health care. About 42% of 25 top selling drugs marketed worldwide are either directly obtained from natural sources or entities derived from plant products. The role of traditional medicines in the solution of health problems is invaluable on a global level. Medicinal plants continue to provide valuable therapeutic agents, both in modern and in traditional medicine (Krentz and Bailey, 2005).
           The use of natural products in attempts at inhibiting sickling could be as old as when the sickle cell (SC) disease was discovered. Traditional history has indicated attempts made by inhabitants using plant derived recipes in parts of Nigeria to treat what they described as “fever of crises”, shifting joint pains, exacerbations and “constant abnormality of the blood, though relatively few have been validated scientifically. With the associated side effects of modern medicine, traditional medicines are gaining importance and are now being studied to find the scientific basis of their therapeutic actions (Thomas and Ajani, 1987).
           The plant kingdom has proven to be the most useful in the treatment of diseases and serves as an important source of the world’s pharmaceuticals. The most important of these bioactive constituents of plants are steroids, terpenoids, carotenoids, flavonoids , alkaloids tannins, saponins and glycosides. plants in all facet of life have served as valuable starting material for drug development (Edeoga, et al.,  2005).
           Phytochemicals are naturally occurring biologically active chemical compounds in plants. The presence of certain types of phytochemicals in some plants can act as a natural defence system providing protection such as attack from insects and grazing animals. In contrast, other plants produce phytochemicals that provide colour, aroma, and flavour, thus inviting attention from potential consumers. More than 4,000 of these compounds have been discovered, and it is expected that scientists will discover many more. Phytochemicals have been utilized as important sources of medicinal drugs and health products. They are important sources of potentially useful compounds for the development of new chemotherapeutic agents because they have a multiplicity of potentially useful actions. Phytochemistry and pharmacomodulation are some of the ways used to search for new drugs; research on the secondary metabolites of plants are desirable for the discovery of thier medicinal potential and to find the actual value of their therapeutic uses. Synthetic drugs are often the option for chemotherapy. Hoewever, most synthetic drugs kills not only targeted cells, but also normal cells, and most have severe side effects. There is, therefore, an urgent need for novel treatment options with improved features.
           Research on the phytomedicine for the treatment of SCD has led to the development of Niprisan (a herbal based drug) which has been patented by the National institute for pharmaceutical Research and development (NIPRID), Abuja, Nigeria and produced to meet increasing global demand by sufferers of SCD. This development indicates that more of such herbal based drugs could be consequent upon scientific investigations on plants that are used in folklore medicine (Ogunyemi et al., 2008).
           In the parlance of herbal therapy, Gillete et al., (2004) opined that all components of a therapeutic mixture of plants are necessary. However, the need for the role definition of each components of such traditional recipe could not be overemphasized on the roadmap of upgrading such traditional recipe.
           The use of crude herbal remedies in the form of water based extracts, tinctures, and concoctions is highest in individuals afflicted with chronic diseases like cancer, human immunodeficiency virus (HIV), diabetes and arthritis. This trend is aggravated by the poor socio-economic situation, ignorance, and exorbitant cost of most western  medicine. The search for new drugs has turned researchers to plant sources for the active molecules (Guzdek and Nizankowska, 1996).
           Alternative strategy in the management of SCD is now focusing on the identification of the novel antisickling agents mainly from medicinal plants. Indeed traditional medicine continues to play a very significant role in the medicinal primary health care implementation in developing countries as research work has intensified and informations on these plants exchanged ( Mapiana, et al., 2012; Imaga, 2013). Natural substances are employed, either directly or indirectly by a large number of industries. Natural plant products (phytochemicals) figure prominently in several of these (Balandrin and Wocke, 1988). The world health organization supports the use of traditional medicine provided they are proven to be efficacious and safe (W.H.O., 1985).
           Ever since the dawn of civilization, medicinal plants have been of great importance to the health of individuals. Man has continually investigated tropical and subtropical medicinal plants in order to assess the importance of developing natural, sustainable and affordable drugs and cosmetics (Iwu, 2000). Medicinal plants have been a source of succor in the control of many diseases in developing countries and sickled cell disease is no exception. The lower strata of the population living in developing countries rely heavily on traditional medicine due to their cultural alignment as well as their inability to afford the cost of treatment offered by orthodox medical practitioners.
Some medicinal plants have these last years, shown an antisickling activity. What indicates a  new therapeutic way to the range of the poor African populations which are affected by this hemoglobinopathy. The fact that the seeds of Cajanus cajan accumulates phenylalanine, an aromatic amino acid known to possess sickling activity suggest that other plants parts could contain this acid or other amino acids which are known to have antisickling activity. The amino acid “tyrosine and “tryptophan”, as well as small peptides containing these amino acids, have antisickling activity. Other plants reported to be used in the management of sickle cell anemia includes aqueous extracts of Lawsonia inermis which was found to inhibit sickling and increase the oxygen affinity of sickled red blood cells (Hbss) during a screen or study of substances known to bind proteins (Dean and Schechter, 1978; Ekeke and Shode, 1990).
Since Terminalia catappa (almond) and Phoenix dactylifera (date palm)  thrives in  hostile environment, it is clear that secondary metabolites (photochemicals) play an important role in conferring a protective effect against high temperature, Uv-damage, and tolerance to drought and salinity; Proven deleterious to plant pathogens and pests. (Balandrin and Wocke, 1988).
           This research work is therefore justified since all the therapeutic approaches are either expensive or toxic and are not accessible to the populations with low incomes, coupled with the fact that very few ethnomedicinal remedies for the treatment of sickle cell anemia have been reported due to secrecy attached to the treatments of the disease.
           Furthermore, research into the phytotherapy of diseases is a current trend in the management of tropical and genetic disorders like sickle cell anemia, with a view to finding cheaper, alternative and less toxic therapies the poor and teeming population can have immediate access to (Sofowora, 1975; Ekeke and Shode, 1985).
This research will therefore, go a long way in the scientific exploration of medicinal plants for the benefit of man and is likely to decrease the dependence on synthetic drugs.
 OBJECTIVES OF THE STUDY
           The overall aim of this research work is to collect and identify two plants part; leaves of Terminalia catappa (almond) and fruits of Phoenix dactylifera (date palm) and comparatively evaluate them in vitro for antisickling activities by testing their extracts on blood samples from sickle cell patients, and carrying out phytochemical tests on them.
The specific objectives of the study are:
i.        To collect an identify two plant parts; leaves of Terminalia catappa and fruits of Phoenix dactylifera .
ii.      To subject the two plant materials (Almond and date palm ) to different extractions.
iii.     To subject the different extracts to photochemical screening.
iv.    To evaluate the antisickling activities of the different extracts in vitro on blood samples from sickle cell patients.
v.      To compare the antisickling activity of the extracts of Phoenix dactylifera to that of Terminalia Catappa.
vi.    To find which fraction of the extracts will comparatively be most effective at inhibiting sickle cell.
vii.  To create possible opportunities for alternative, cheaper and less toxic therapies for sickle cell disease management thereby decreasing the dependence on synthetic drugs.

viii.To verify claims by traditional healers and validating the ethno-medicinal use of these plant species.

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