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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