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1.0 INTRODUCTION

Nigeria is home to the largest population of sickle cell anemia patients, estimated to be in

excess of 4 million patients with more than 150 000 children born annually with the disease

(World Health Organisation, 2006). The condition is also incident in the African diaspora

population in North America and Europe. The SCA patient‟s quality of life and productivity

are significantly reduced; the condition causes episodes of pain that can last for hours or days

at a time (Steinberg, 2003; Todd et al., 2006; WHO, 2006; Hankins, 2009). There are few

effective therapies for SCA, a chronic blood disorder which interferes with the body‟s oxygen

transfer process and can lead to the formation of small blood clots, which over time deprive

organs and tissues of oxygen.

Despite decades of research, only one drug, hydroxyurea, approved by the American Food

and Drug Agency, is available for the treatment of sickle cell anaemia (Hankins and Aygun,

2009; Brandow et al., 2010). This chemotherapeutic agent stimulates healthy production of

fetal haemoglobin to counter the sickling process (Steinberg, 2003). Although long-term

clinical studies have shown the drug to improve survival, while concerns of genotoxic and

carcinogenic effects are yet to be ascertained (Lal and Ames, 2011). Developing novel drugs

from traditional medicinal plants can serve as a means to improve the treatment of this

disease.

Research on phytomedicine for the management of SCA has led to the development of a

herbal based drug called Niprisan which has been patented by the National Institute for

Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria. This development

indicates that more of such herbal based drugs could be produced consequent upon scientific

investigations on medicinal plants that are used in traditional African medicine.

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1-1 BACKGROUND OF STUDY

Sickle cell anemia is inherited in an autosomal recessive manner. A person heterozygous for

the disease is only a carrier with sickle cell trait (AS or HbAHb

S). In some cases there are

certain, mild symptoms but nothing close to life threatening. A person homozygous for the

disease - referred to as SS or HbSHb

S - has full fledged sickle cell anemia disease and exhibits

a myriad of symptoms. An affected individual will begin to show symptoms after six months

of age, this is when the fetal haemoglobin (HbF) subsides and the mutant HbS dominates.

Patients with sickle cell anemia suffer from episodic and painful vascular occlusions known

as crises that can result in organ damage and, ultimately, premature death. Currently, in

clinical practice, clotrimazole, hydroxyurea and erythropoietin are used in SCA management,

but the side effects of these drugs limit their clinical use (Rifai et al; 1995; Mehanna, 2001;

Elliott et al., 2006). In Nigeria, herbal medications are used as alternatives to orthodox

western medicine due to the perceived minimal side-effects, affordability and easy of

acquisition (Ogunkunle and Ladejobi, 2006; Okigbo et al., 2009).

Potent bioactive compounds formed during normal metabolic processes are found in specific

part(s) of medicinal plant(s), thus making such plant(s) components ideal in preparing

pharmaceutical products for medical treatments. Scientific investigation on such medicinal

plants could be of tremendous help in developing efficacious and safer drugs for SCA

treatment.

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1.2 STATEMENT OF PROBLEM

An affected (HbSHb

S) individual becomes symptomatic, after six months of age, when the

synthesis of fetal hemoglobin (HbF) subsides and the mutant HbS dominates. Under reduced

oxygen tension, HbS polymerises and deforms the RBC. The multifaceted clinical features of

this disease are the consequences of malformed properties of sickle cells due to a point

mutation in the gene coding for the beta globin moiety of hemoglobin. The deformed

structure of the red blood cells results in two problems that lead to a multitude of clinical

manifestations. These two problems are haemolysis, which is the premature destruction of the

red blood cells, and vaso-occlusion, which is the blockage of blood flow. Symptoms of sickle

cell anemia can be categorized as either acute, episodic such as the trademark crises or they

may be chronic. Many of the symptoms linked to haemolysis are due to the body trying to

compensate by increasing erythrocyte production, resulting in abnormality of bone shape or

size and heart dilation. The main clinical consequences of haemolysis include megaloblastic

erythropoiesis, aplastic crisis, clinical jaundice and gallstones.

Even though scientists have carried out researches on sickle cell disease (SCD) for over fifty

years they are yet to find a reliable therapy. Based on current knowledge of the disease, three

different approaches to therapy have been clinically and laboratory tested (Hankins and

Aygun, 2009). One approach is the chemical inhibition of HbS polymerization. This

approach has not yet been successful enough to warrant implementation, and to date, none of

the tested antisickling drug has an acceptable efficacy to toxicity ratio. The second approach

is through the reduction of the intracellular hemoglobin concentration. Progress has since

been made in the development of drugs that inhibit potassium and water loss from SS red

cells. The most successful approach is the induction of fetal hemoglobin (HbF). The objective

is to raise the levels of HbF which inhibits sickling. The antitumor drug hydroxyurea, which

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is also a diuretic, succeeded in increasing HbF in primates as well as patients with SCA

(Bunn, 1993), however not all patients respond to this drug and there are concerns over its

toxicity and carcinogenic effects (Weinfeld et al., 1994; IARC, 2000; Lal and Ames, 2011;

Latagliata et al., 2012).

1.3 AIMS AND OBJECTIVES

The aim of this study is to examine the efficacy of Senna alata and Senna podocarpa as

herbal remedies for SCA.

The specific objectives are as follows:

(i) To evaluate the human HbSS erythrocyte membrane stabilising effect of the

medicinal plant(s) extract and the influence of the medicinal plant(s) extract on

polymerisation/depolymerisation of mutant haemoglobin

(ii) To determine the antioxidant activity of the medicinal plant(s) extract

(iii) To evaluate the long term toxicological effect(s) of the medicinal plant(s) extract

using albino rats

(iv) To determine the effect of the medicinal plant(s) extract on rat RBC membrane

protein profile

(v) To evaluate the cytotoxic effect of the medicinal plant(s) extract on K562 Cells

1.4 SIGNIFICANCE OF STUDY

The study will help identify a herbal preparation that is safe and efficacious in protecting the

sickle cell membrane. This will help to reduce or eliminate RBC hemolysis, thereby

inhibiting haemoglobin polymerisation thus decreasing frequency of vaso-occlusive events.

Plant(s) / formulation(s) that can prevent dehydration, maintain the HbSS erythrocyte

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membrane integrity and have minimal or no toxicity would be of potential benefit to HbSS

patients.

1.5 HYPOTHESES

The working hypotheses are:

1. Increased RBC fragility, an hallmark of SCA may be reduced by protecting the

membrane from oxidative stress thus improving the clinical outcome / outlook of SCA

patients.

2. Senna alata and Senna podocarpa have been implicated in water homeostasis, and

since the pathophysiological root of SCA is RBC dehydration, they may mediate the

underlying process of dehydration.

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1.6 OPERATIONAL DEFINITION OF TERMS

Electrophoresis: A technique for separating the components of a mixture of molecules by

size or shape as a result of an electric field within a support gel.

Aqueous-methanolic extract: Freeze dried product of plant material(s) soluble in 80%

methanol.

Fetal haemoglobin (HbF): A type of haemoglobin made up of two alpha-globin chains and

two gamma-globin chains found majorly in the erythrocytes of neonates.

HbSS: The specific mutated allele composition of an individual with respect to the beta

globin gene located on chromosome 11, which codes for sickle haemoglobin.

K562 Cell line: Cell line originally established from a pleural effusion of a patient with

chronic myelogenous leukemia in terminal blast crisis.

Red Blood Cell (RBC): A type of cell in the blood that contains haemoglobin, which is the

oxygen carrying pigment that gives blood its characteristic red colour.

SDS-PAGE: Sodium Deodocyl Sulphate PolyAcrylamide Gel Electrophoresis - A type of

vertical gel electrophoresis used for separating mixture of molecules according to size.

Sickle Cell Anemia (SCA): A type of genetic disorder resulting from a point mutation (A-T)

on the sixth codon of the beta-globin gene located on chromosome 11, and often leading to

anemia.

Sickle haemoglobin (HbS): A type of haemoglobin made up of two alpha-globin chains and

two attenuated beta-globin chains found in the erythrocyte.

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2.0 LITERATURE REVIEW

Nigeria has the largest population of SCA patients, estimated to be in excess of 4 million

patients with more than 150,000 children born annually with the disease (WHO, 2006). The

homozygous state of SCA is a major health challenge in Nigeria and other developing

countries, because the condition is associated with complications and reduced life

expectancy. Carrier frequency of HbS varies significantly around the world, due to high

malaria occurrence, since carriers are somewhat protected against malaria.

The genetic basis of the disease has long been established, but it is the molecular remedy that

scientists are continually investigating. With advances such as the transgenic mice,

hydoxyurea and new herbal therapies being discovered, complications and premature death of

SCA patients may in the very near future, be a thing of the past. Hydroxyurea which is

currently the drug of choice is potentially mutagenic and carcinogenic (Latagliata, 2012).

Cancer and leukemia have been reported in hydroxyurea-treated sickle

cell disease patients,

but whether the incidence is higher than in the general population is not known (Steinberg et

al., 2003). The relative risk of leukemia in hydroxyurea-treated sickle cell anemia is much

less than the observed risk in myeloproliferative disorders. To put

this into perspective, the

risk of death from the complications of adult sickle cell disease appears to be at least 10-times

greater than the possible incidence of leukemia in hydroxyurea-treated sickle cell anemia

patients (Buchanan et al., 2004).

2.1 Historical Perspective

The acutely painful episodes that characterize sickle-cell anemia disease were described in

1872 by Africanus Horton, though the mechanism remained uncertain until, nearly thirty

years later, when James Herrick (1910) coined the term „sickle cell‟ to describe the peculiar

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morphology of the red blood cell of his patient, a dental student who presented with

pulmonary symptoms. He was not sure then, whether the blood condition was a new disease

or a manifestation of another disease, but over the next 15 years, several similar cases were

described, supporting the idea that this was a new disease and provided the platform for a

preliminary clinical and pathological description. Hahn and Gillespie (1927) thereafter,

suggested that anoxia caused RBC sickling by demonstrating that shape changes could be

induced by saturating a cell suspension with carbon dioxide. This concept was proven by

Scriver and Waugh (1930), in vivo by inducing venous stasis in a finger using rubber band in

experiments that would undoubtedly not receive institutional review board approval today.

They showed that stasis-induced hypoxia dramatically increased the proportion of sickle-

shaped cells from approximately 15% to more than 95%. These seminal studies were noted

by Linus Pauling, who was the first to hypothesize in 1945 that the disease might originate

from an abnormality in the hemoglobin molecule. His hypothesis was validated in 1949 by

the demonstration of the differential migration of sickle versus normal hemoglobin as

assessed by starch gel electrophoresis. Both hemoglobins migrated towards the positive end

but HbS migrated lesser, thus revealing that HbA had a greater net negative charge (Klug et

al., 1974). That same year, the autosomal recessive inheritance of the disease was elucidated

by James Neel and E.A. Beet through pedigree analysis, which revealed three phenotypes and

genotypes controlled by a single pair of alleles (HbA, Hb

S). Around the same time, Watson et

al. (1948) predicted the importance of fetal hemoglobin (HbF) by suggesting that its presence

could explain the longer period necessary for sickling of newborn RBC compared with those

from mothers who had “sicklemia”. Ingram and colleagues demonstrated shortly (1954)

thereafter that the mutant sickle hemoglobin (HbS) differed from normal hemoglobin A

(HbA) by a single amino acid in the primary structure of the globin portion. This finger

printing technique involved the enzymatic digestion of protein portion of hemoglobin into

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fragments which are then subjected to an electric field for initial migration and then placed in

a solvent where chromatographic action causes a second migration. These migrations result

in a two dimensional pattern. (Klug et al., 2010) This was followed by studies that analyzed

the structure and physical properties of HbS, which formed intracellular polymers upon

deoxygenation. These studies placed SCD at the leading edge of investigations to elucidate

the molecular basis of human diseases.

Sickle cell anemia (SCA), one of the most prevalent hereditary disorders with prominent

morbidity and mortality is an autosomal recessive disease caused by a missense point

mutation in the haemoglobin beta gene found on chromosome 11p15.4 (Harrison‟s, 1998).

This mutation results in the substitution of valine (a hydrophobic amino acid) for glutamic

acid (a hydrophilic amino acid) as the sixth amino acid residue on the beta globin chain of the

structurally abnormal haemoglobin (Hb), called sickle haemoglobin (HbS). The sickle cell

anemia pathology results from the acquisition of a novel property due to the change in amino

acid sequence, rather than from the loss of normal function (Presely et al., 2010).

Haemoglobin is an oxygen carrying protein that gives red blood cells their characteristic red

colour. Patients who are homozygous for the mutation in the gene encoding the chain of

hemoglobin (globin) that results in the substitution of valine for glutamic acid at position 6

(betaGlu6Val

) have hemoglobin S (HbS) and sickle cell anemia. Hemoglobin from these

patients polymerizes in the deoxy conformation into long fibers composed

of strands of HbS

tetramers. The betaGlu6Val

mutation in deoxy-HbS favors a hydrophobic interaction between

each strand and its neighbour. Other residues on the chain participate in the binding

of

adjacent tetramers within each strand and between strands. The interaction between the valine

at position 6 with the phenylalanine at position 85 and the leucine at position 88 on the

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partner strand is stereochemically unavailable in oxyhemoglobin. The

fibers of polymerized

deoxy-HbS are responsible for dehydration, rigidity, and lysis of red cells (Geva et al, 2004)

In persons who are heterozygous for the betaGlu6Val

mutation, hemoglobin can polymerize if

the non-HbS allele encodes permissive mutant hemoglobin (such as HbC, HbD, or HbO

Arab). In other words, patients with sickling disorders due to two heterozygous mutations

in

globin tend to have compound heterozygosity, such as HbS/C or HbS/D.

2.2 Red Blood Cells (RBC): Erythrocytes

During erythrocyte maturation, the blast forming unit-erythrocyte (BFU-E) matures into the

colony forming unit-erythrocyte (CFU-E), which finally evolves into a mature erythrocyte

after extrusion of the nucleus, acquisition of haemoglobin and a mature cytoskeletal support

system. Mature RBCs are discoid-shape and devoid of a nucleus and mitochondria. They are

rich in the heme-containing protein, haemoglobin.

Accelerated erythrocyte formation due to anemia may result in the release of RBCs prior to

the loss of the nucleus. These immature erythrocytes known as reticulocytes can be found

circulating in the blood in increased numbers (greater than 5% of the total RBCs). Once the

red blood cell is released into circulation, it has a normal life span of 120 days. Damaged or

senescent RBCs are sequestered in the spleen and destroyed by the splenic macrophages.

Pluripotent cells of the bone marrow differentiate by feedback inhibition mechanism into

various blood components. Body cells deprived of oxygen due to reduced level of circulating

oxygen transporting red blood cells triggers differentiation of bone marrow stem cells into

erythroblast which are eventually released into the blood stream.

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2.3 Hemoglobin

The biosynthesis of haemoglobin begins in the mitochondrion, continues in the cytoplasm

and is completed in the mitochondrion. A functional haemoglobin molecule consists of four

globin chains i.e two alpha and two non-alpha globin chains referred to as a tetramer, which

are the expression products of genes on the alpha globin and beta globin loci located on

chromosome 16 and 11 respectively. Transcriptional control elements positioned within

(Persons and Nienhuis, 2000) and upstream (Li et al., 2004) of this region coordinate the

sequential activation and silencing of these genes during three defined periods of human Hb-

Grower1 (δ2 ε2 ), Hb-Grower2 ( α2 ε2) and Hb- Portland (δ2 γ2) ( gestational weeks 4-8),

fetal globin – Hb-F (α2 γ2) (gestational week 8 through parturition) and adult globins – Hb-

A(α2 β2 ) and Hb-A2(α2 δ2) ( from birth onward) (He and Russels, 2002). The expression of

embryonic globin, like fetal haemoglobin (HbF), is normally silenced in adults despite the

fact that its encoding gene remains structurally intact (Bank, 2005). The red blood cells are

initially made in the liver and spleen, then in the long bones after which production in the

liver and spleen ceases.

Polymerisation of deoxy-HbS is greatly reduced in the presence of substantial fractions of

HbA or HbF within the RBC‟s (Steinberg et al., 2003). Erythrocytes from heterozygous AS

subjects, containing about 40% HbS do not sickle in vivo under normal oxygen tension,

except under conditions where their internal Hb concentrations is markedly increased by

osmotic shrinkage due to the hypertonic environment as of the renal medulla (Ataga and

Orringer, 2000). Individuals with the sickle cell trait are asymptomatic and have normal

haematological profiles, except under high stress of maximum exercise or low oxygen

tension (high altitude or sudden decompression in aircraft) (Bunn and Forget, 1986;

Gendreau and DeJohn, 2002; Lee et al., 2002), but individuals doubly heterozygous

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(HbSHb

F) for sickle Hb and for hereditary persistence fetal Hb, show no clinical evidence of

sickling and have normal haematological profiles. In sickle cell (SS) patients Hb conbinations

that prevent sickling invivo also prevent the development of the haematological and clinical

manifestations associated with dehydrated RBC‟s, despite substantial proportions of HbS

within the cells.

Human RBC‟s are water permeable sacs with the highest soluble protein concentration of any

cell approximately 7.2 mmol Hb/cell water. The RBC‟s characteristic biconcave shape and

flexibility is made possible due to its two dimensional spectrin-actin based meshlike

cytoskeleton, with a highly structured link to integral membrane proteins embedded in the

lipid bilayer. (Chasis et al., 1989; Palek and Lambert, 1990). Continued osmotic equilibrium

in the plasma is achieved by the RBCs‟ high water permeability so that they can swell or

shrink only by the loss or gain of a fluid isoosmotic with surrounding plasma. Since the

plasma protein concentration is less than 1mM, there is powerful pressure driving water into

the cells. Osmotic stability over the approximate 120 day circulatory life span of the mature

RBCs is actualised due to an evolved strategy of maintaining a nearly constant volume with

minimal energy consumption. Because of their low Na+ and K

+ permeabilities which require

relatively few metabolically fuelled Na+ pumps per cell to balance their cation gradient

driven leaks, RBCs have evolved an extremely efficient CO2 ferry between tissues and lungs,

based on the high Cl- and HCO3

- exchange (Lew and Bookchin,1986) and electrodiffusional

fluxes (Knauf et. al,. 1983). This homeostatic balance is markedly altered in sickle Hb

containing RBCs whose ion content regulation, ion fluxes and hydration state become highly

distrupted in the circulation. Enucleated erythroid cells termed reticulocytes released from the

bone marrow into the circulation differentiate into mature RBCs within 2-3 days. The

maturation process involves loss of residual RNA as well as protein synthesis with

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concomitant reduction or outright loss of many metabolic functions including a gradual

decline in monovalent cation transport activity and the reduction or inactivation of selected

transporters such as K+-Cl

- cotransporter and Na

+ pumps, via the formation of exosomes

(Johnstone, 1992).

2.4 Transport Proteins Involved In RBC Dehydration

Human RBC dehydration in vivo may arise from the single or synergistic activation of the

K+-Cl

- cotransporter (KCC), the Na

+ pump or the Gardos channel. These three transporters

expressed in the RBC plasma membrane differ considerably in their dehydrating potencies,

modalities and distribution.

2.4.1 K+-Cl

– Cotransporter (KCC)

The KCC, regulated by internal pH and cell volume, is functionally active in reticulocytes

and much less so in mature AA and SS RBCs. Most of the K+ traffic is mediated by this

transporter, which may be regulated by cell swelling, intracellular acidification, and

oxygenation state of Hb (Joiner and Franco, 2001) and to a lesser extent low intracellular

Mg2+

concentrations. Its functional state appears to be regulated by classical serine/threonine

and tyrosine residue (Merciris et al., 2003) phosphorylation and dephosphorylation (Lauf and

Adragna, 2000). Acidification in RBCs with active KCC may induce dehydration (Lauf et al.,

1992; Gillen et al, 1996), the extent of which may be limited by the inhibitory effect of cell

shrinkage, while in SS reticulocytes acid actification is abnormally exaggerated and may

therefore overcome the intensity of the volume-regulatory “brake”. Inhibitory agents may act

directly on the KCC or on its regulatory intermediates (Jennings and Schulz, 1991).

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2.4.2 The Na+ Pump

The Na+-K

+ flux ratio through the Na

+ pump is 3:2 (Lew et al., 1991). The high anion

permeability of reticulocytes and RBCs, require that electroneutrality be maintained, majorly

by anion efflux balancing the extra Na+ efflux. Elevated internal Na+/ K

+ concentration ratios

stimulate the Na+ pump, resulting in an increased NaCl flux, which if not compensated by

passive fluxes would induce dehydration (Joiner et al., 1986). Model simulations (Glynn and

Karlish, 1975; Brugnara et al., 1989; Skou and Esmann, 1992) confirms this effect to be

rather slow, moreover, in SS cells Na+ pumps have been found to be substantially inhibited

(Clark and Rossi, 1990; Ortiz et al., 1990), despite elevated internal Na+/K

+ . This inhibition

was due to elevated Mg/ATP ratio, because Mg2+

released from 2, 3-diphosphoglycerate (2,3-

DPG) during deoxygenation, pushed the ratio further away from the optimal (Ortiz et al.,

1990). Thus Na+ pump-mediated fluxes may contribute only marginally to SS cell

dehydratioin.

2.4.3 Gardos Channel

The Gardos channel is a Ca2+

-sensitive, small-conductance, K+-selective

channel which is

activated when the physiological intracellular RBC Ca2+

concentration levels of about 20-50

nM is increased above its activation threshold of 150 nM (Tiffert and Lew, 2001). A

maximally activated Gardos channel would ensure fast dehydration of the RBC. Unlike

erythroid cells from other mammalian species, whose Gardos channel activity

is lost during

maturation (Brown et al., 1978), mature human RBCs show persistent Gardos channel

activity. The capability of Gardos channel, when fully activated, to mediate rapid RBC

dehydration far exceeds that of the other transporters. Dehydration via Gardos channels may

be reduced by direct inhibition of the channels with charybdotoxin (IC50 1.2 nM) or

clotrimazole (IC50 51 nM) (Brugnara et al., 1993), or indirectly, by inhibition of the anion

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permeability (Bennekou et al., 2001). Mature RBCs, when compared with other cell types

lack specialised Ca2+

accumulating organelles, Ca 2+

signalling functions as well as minimal

cytoplasmic buffering capacity (Tiffert and Lew, 1997).

2.5 Dehydration of RBC

The relationship between SS cell transport and sickling was investigated in 1955 by

Tosteeson and co-workers, who showed that upon deoxygenation of fresh, heparinised whole

blood obtained from HbSS patients their RBCs had a much larger gain of Na+ and loss of K

+

than RBCs from HbAA (normal) controls. But on reoxygenation, HbSS cells gained K+ and

lost Na+, affirming that the effects of deoxygenation were reversible. Further transport

experiments with tracers of Na

+, K

+, and Cs

+ indicated that sickling induced a reversible

and

poorly selective increase in the electrodiffusional cation permeability of the RBC membrane.

A sickling induced permeability pathway was proposed by Lew et al. (1997) When HbSS

RBCs was ultracentrifuged, a cell subpopulation accumulated at the bottom of the column,

these cells had normal Hb content, and their high density reflected a dehydrated state. The

distinct morphological features of this subpopulation earned the name irreversibly sickled

cells (ISCs), due to the fact that when fully oxygenated, they still retained the sickled shape.

Isotopic labelling techniques revealed that

ISCs were a relatively young HbSS cell

subpopulation which, after release from the bone marrow as reticulocytes, was rapidly

transformed into ISCs within 4–7 days, and then disappeared from the

circulation faster than

other mature, non-ISC RBCs. ISCs were therefore a subpopulation of SS RBCs in rapid

circulatory turnover. The exclusion of most high Hb F-containing HbSS cells (whose

polymerization

was reduced or inhibited) from the hyperdense, ISC-rich cell

fraction

suggested that sickling was necessary for ISC formation. Most research in the field was

dominated by the notion of gradual dehydration, with attention focused on the three transport

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systems potentially involved in HbSS RBC dehydration, notably, the Gardos channel, KCC

and Na+ pump.

2.6 Pathophysiology

The sickle phenotype results from the intracellular polymerization of deoxygenated α2βs2

heterotetramers (HbSS), into extended 14-strand fibres that disrupt both the shape and the

function of the mature erythrocyte (Bunn, 1997). The deformation of RBC causes changes in

their membrane structure (Ohnishi and Ohnishi, 2001), thereby enhancing the adhesion of the

cells to vascular endothelial cells (Hoover, 1979; Brittain et al., 2004). This, together with

elongated cell shapes, induces the obstruction of blood flow and causes the painful sickle cell

crisis. The red blood cell membranes of SCA patients are osmotically and mechanically more

fragile than those of normal subjects. For these reasons, sickle RBCs are easily destroyed and

removed from the circulation in the spleen, thus causing anemia. The average life span of

sickle erythrocytes is 10 - 20 days (Franco et al., 1998), as opposed the 120 days for the

normal RBC. Patients suffer from chronic anemia, frequent painful episodes (crisis) and

resultant malfunction of organs (especially the spleen) and degeneration of bone joints

(Serjeant, 1997).

The total cellular calcium content of sickle RBC is markedly abnormal (50µmol/L as against

5µmol/L for normal RBC), but oxygenated sickle RBC have moderately enhanced

permeability to external calcium and deoxygenation boosts calcium influx by about five fold

(Etzion et al.,1993). Though excess calcium is stored internally in vesicles without increase

in detectable ionized calcium, this influx activates a calcium-dependent potassium channel

called Gardos channel, through which potassium is extruded from the sickle RBC, thus

resulting in dehydration. Stimulation of sickle RBC membrane Ca2+

ATPase activity under

optimized conditions among patients have been found to be increased, deficient and normal,

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suggesting an insignificant effect on the calcium pumping capacity under physiological

conditions. Stimulation of the Na+K

+ATPase by deoxygenation of sickle RBC somewhat

increases passive K efflux and Na influx three to five fold, this leak sites (Lew et al., 1991),

areas of the membrane affected by speculation – a sickling effect – requires cell deformation

for induction of monovalent cation leak, though oxidative perturbation of the sickle

membrane may however not be ruled out. Some studies on RBC suggest that full homeostatic

capacity of the cell (- Quabain) leads to cellular dehydratioin while the passive component of

sickling-induced leak (+ Quabain) causes balanced leak. Cell swelling or low pH (<7.4)

activates in sickle reticulocytes, a potassium-chloride cotransport (KCC) pathway.

Irreversibly sickled cell (ISC) generated by repeated deoxygenation / oxygenation of sickle

RBC, regarded as the end-stage defects of highly damaged cells, can constitute anywhere

from a few percent to about 50% of RBC population. Formation of ISC in vitro is generally

prevented by an absence of external calcium, inhibition of Gardos channel, calcium channel

blockers and calmodulin-mediated processes. The calcium antagonist, zinc, appeared to

reduce ISC counts in patients consuming it, but formation of ISC formation in vitro was not

inhibited by in the presence of zinc. The implied role of calmodulin is interesting given its

interaction with the RBC cytoskeleton and the effects of calcium on certain protein / protein

interactions (Blackman et al., 2001; Chang and Low, 2001).

2.7 Cytoskeletal Dysfunction

The flexibility and strength of the normal RBC is derived from the protein component of its

membrane, the essential features of which is an underlying cytoskeleton ( spectrin, actin,

band 4.1 and other minor components) connected by linking units (ankyrin and band 4.1) to

proteins embedded in the lipid bilayer (band 3 and glycophorin). In sickle cell anemia, this

actin / spectrin lattice „locks‟, making red blood cells much less deformable, and causing

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them to obstruct the microcirculation. Sickle inside-out vescicles (IOV) have a diminished

capacity for ankyrin-dependent binding of spectrin (Platt et al., 1985) and

aminophospholipid, sickle IOV were found to have a slightly diminished quantity of band 4.1

(Schwartz et al., 1987) although artifacts due to protease action was not excluded. Sickle cell

patients administered vitamin E tend to have normalised band 3 anion transport activity and

high affinity ankyrin binding sites (Joiner et al., 1989). Careful analysis of sickle RBC

membranes showed a small amount of abnormal spectrin / globin adduct (Presley et al.,

2010), favoured by dehydration, reflecting an oxidative process possibly involving spectrin

(not Hb) thiols in the presence of peroxide (Rank et al., 1985)

Sickle RBC fragility has been observed to increase in patients undergoing vigorous exercise.

Invitro studies have documented notable reduction in the shear sensitivity of intact sickle

cells on rehydration and ghosts made from most dense sickle RBC are mechanically fragile.

This membrane instability reflects some protein/protein junctional association failure and

destabilization of the cytoskeleton maybe due to free heme found to be in excess in both

sickle membrane and sickle cytosol (Shalev and Hebbel, 1996).

Decreasing deformability of deoxygenating sickle RBC is highly dominated by development

of Hb polymer and cytoplasmic viscosity. It may be anticipated that abnormal microrheology

of sickle cell membrane would participate in vasoocclusion as increased static rigidity would

enhance mechanical trapping of RBC during their attempted passage through the

microvasculature, or capillaries of limiting diameter and flow velocity of cells therein

(Ferrone, 2004).

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2.8 Cell / Cell Interactions

Abnormal attachment of sickle RBC to mononuclear phagocytes or vascular endothelial cells

indicates relevance to haemolytic anemia and vasocclusive events respectively. Sickle RBC

have been found to be abnormally adherent to normal marrow, splenic or alveolar

macrophages and normal peripheral blood monocytes, after which they are phagocytosed

more readily than normal RBC. The ability of externalised phosphatidylserine (Yamaja Setty

and Betal, 2008) in promoting adherence to macrophages in vitro is enhanced by

deoxygenation, but exposure of normal RBC to peroxide or malondoaldehyde (peroxidation

by product) facilitates their phagocytosis. Abnormal adherence of sickle RBC to vascular

endothelial cells derived from bovine aorta, rat microcirculation and human umbilical vein is

enhanced by dehydration or calcium loading (Hoover et al., 1989; Kaul et al., 1993).

2.9 Pathophysiologic Role of Membrane Abnormalities

Emphases on HbS polymerization have dominated considerations of sickle cell disease

pathophysiology generally but it is now recognised widely that this disease is exceedingly

complex. Attempts to explain it based on a single feature would be artificial and the

intriguing heterogeneity amongst patients would be grossly underestimated. Severity of

anemia for sickle cell patients correlate directly with the magnitude of the very-dense cell

population.

2.10 Oxidant Levels

Increased oxidant susceptibility of sickle red blood cells (RBC) has been demonstrated to

play a major role in pathophysiology of the disease, in effect, reactive oxygen species (ROS)

produced during redox cycles appear to cause premature ageing and altered morphology of

RBCs, leading to their early removal from circulation. Another name for redox cycle

20

formation of reactive oxygen species and damage to the RBC is „oxidative stress‟. In other

cases toxicants can cause a disruption in oxidative metabolism within the cell, leading to

generation of reactive oxygen species. An important potential consequence of free-radical

formation is the occurrence of lipid peroxidation in the membranes within the cell. Lipid

peroxidation occurs when free radicals attack the unsaturated bonds of fatty acids,

particularly those in phospholipids. The free radical reacts with the fatty acid carbon chain,

abstracting one hydrogen atom. This causes a fatty acid carbon to become a radical, with

rearrangement of double bonds in the fatty acid carbon chain. This carbon radical in the fatty

acid reacts with oxygen in a series of steps to produce a lipid hydroperoxide and a lipid

radical that can then react with another fatty acid carbon. The peroxidation of the lipid

becomes a chain reaction, resulting in fragmentation and destruction of the lipid. Because of

the importance of lipids in membrane structure, the principal consequence of lipid

peroxidation for the cell is loss of membrane function. The reactive products generated by

lipid peroxidation can interact with other components of the cell as well, and this also could

contribute to cellular perturbation. Protecting the RBC membrane from oxidative stress may

thus improve the clinical manifestations of the patients (Ohnishi and Ohnishi, 2001).

2.11 Factors Contributing to Vasocclusion

HbS polymerization in vasocclusive events for most sickle RBC is never in equilibrium and

sickling is dominated by kinetics, so that clinical effects depend critically on the relationship

between microcirculatory transit time (short) and the unavoidable and relatively longer delay

time (about 15 seconds) for the onset of Hb polymerization (Bunn, 1997). It is controversial

whether or not the RBC membrane directly influences polymerization the occurrence of

painful crises actually correlates inversely with percentage dense cells and percentage ISC,

but correlates positively with adequate RBC deformability.

21

2.11.1 Environmental factors

The role of blood factors including plasma proteins (as modulators of RBC adhesivity), blood

pH (as stimulant for cation depletion, as well as Hb polymerization), osmolarity (as

determinant for dehydration) and zinc levels (as calcium antagonist) in vasocclusive crisis

cannot be underemphasized. Since cellular pathobiology is influenced by oxidative

phenomena, there is the possibility of accessibility to dietary factors like selenium, riboflavin,

vitamins E and C, exerting some beneficial influence. Presence of other globins like HbA or

HbF (levels varying enormously amongst patients), inhibit polymer formation while others

e.g. HbD and HbOArab

promote polymer formation.

2.11.2 Internal factors

Proper functioning of various RBC enzymes [(Gluthathione-S-transferase (GST),

superoxidedismutase (SOD), Catalase (CAT), Methemoglobin reductase (NADHmr),

Diaphorase (NADPHd) and Glucose-6-phosphate dehydrogenase (G-6PD)] systems play

crucial roles in sickling. The SODs are a group of metalloenzymes that catalyze the

conversion of reactive superoxide anions (O2-) to yield H2O2, which is in itself, is an

important ROS as well. Hydrogen peroxide is subsequently detoxified by two types of

enzymes; catalases and glutathione-dependent peroxidases (GPOXs). Superoxide dismutases

are considered to play a pivotal antioxidant role. Their importance is indicated by their

presence in all aerobic organisims examined (Stegeman et.al., 1992). The SOD-CAT system

provides the first defence against oxygen toxicity. Superoxide dismutase catalyses the

dismutation of the O2- to molecular oxygen and hydrogen peroxide which is detoxified by

CAT activity to water and oxygen. (see equation below)

Equation. 2O2- + SOD → H2O2 + O2 + CAT → H2O + O2

22

Fig.1 A schematic diagram showing the inter relatioship between the possible role of Hb

polymerization, membrane defects and abnormal behaviour of HbS in sickle cell anemia disease

pathophysiology

Sickle

Hemoglobin

Hb polymerization

Decreased solubility

Vaso-occlusion and

Hemolytic anemia

RBC sickling

Oxidant generation

Iron compartmentalization

Hb/membrane interaction

Abnormal microrrheology

Endothelial adhesivity

Marcrophage interaction

Phospholipid destabilization

Protein defect

Decreased stability

Cellular dehydration

23

2.12 Histopathology

Indicators of stress at several levels of biological organisation have been used to evaluate

effects of contaminants / toxicants at organizational level. These approaches vary from

measures of genetic integrity to growth and reproductive competence of the individual (Teh

et. al., 1996). As far as the individual organism is concerned, manifestations of stress at the

tissue level represent an intermediate effect between the biochemical and reproductive levels

(Hinton, 1990). Histopathological characteristics of specific organs express condition and

represent time-intergrated endogenous and exogenous impact on the organism stemming

from alterations at lower levels of biological organisation (Chavin, 1973). According to

Segner and Braunbeck (1998), histological changes occur earlier than reproductive changes

and are more sensitive than growth or reproductive parameters and, as an integrative

parameter, provide a better evaluation of organism health than a single biochemical

parameter. Many toxicants can damage specific cell types or organ systems.

2.13 Orthodox Treatment

The main drug therapy for sickle cell anemia, hydroxyurea, is thought to reduce the formation

of sickle haemoglobin and hence ameliorate the structural consequences, while also

decreasing neutrophil numbers which promote adhesion of sickled cells to blood vessel walls.

However, a recent study suggests that hydroxyurea acts directly on the plasma membrane.

The drug is known to decrease expression of adhesion molecules on the red blood cells, one

of which is phosphatidylserine, usually expressed on the outer surface of some RBCs in

sickle cell anemia. In a study by Covas et al., 2004, sickle cell patients receiving 12 months

of hydroxyurea treatment had significantly reduced phosphatidylserine levels in their blood

samples.

24

The multifaceted clinical features of this disease are the consequences of malformed

properties of sickle cells. The deformed structure of the red blood cells results in two

problems that lead to a multitude of clinical manifestations. These two problem mechanisms

are haemolysis, which is the premature destruction of the red blood cells, and vaso-occlusion,

which is the blockage of blood flow. Symptoms of sickle cell anemia can be categorized as

either acute, episodic such as the trademark crises or they may be chronic. Many of the

symptoms linked to haemolysis are the results of the body trying to compensate. The body

compensates by increasing erythrocyte production, resulting in abnormality of bone shape or

size and heart dilation due to increased heart action. The main clinical consequences of

haemolysis include megaloblastic erythropoiesis, aplastic crisis, clinical jaundice and

gallstones (Issa and Al-Salem, 2010).

The life span of erythrocyte in people with sickle cell disease is ten to twenty days as

opposed to a normal 120 day span (Franco, 1998). This increased rate of destruction demands

a significant increase in bone marrow activity. Because of the raised level of bone marrow

activity a greater amount of folic acid is required. The shortage of this acid results in

megaloblastic erythropoiesis with low reticulocyte counts, increasing mean cell volume, and

reduced haemoglobin. The increased metabolic demand of the bone marrow competes with

the demands of growth plates, usually resulting in impaired growth. Aplastic crisis, from

bone marrow depression may occur and its characteristic symptoms include lethargy, dyspnea

and possibly coma. The rapid haemolysis means increased bilirubin excretion which is

associated with clinical jaundice and the formation of gallstones (Alexander-Reindorf et

al.,1990; Issa and Al-Salem, 2010).

The second problematic mechanism, of the deformed red blood cells is the blockage of

venous microcapillary, resulting in impeded blood flow. Tangled, sickled cells obstruct

25

vessels, causing tissue anoxia and possible necrosis. Manifestation of symptoms depends on

which vessels that are being blocked. Damage to the splenic vasculature via obstruction, is an

early pathological signature of sickle cell anemia. The spleen normally acts like a filter,

removing damaged red blood cells and bacteria from the blood stream; however, in sickle cell

patients the damaged red blood cells obstruct this filter, predisposing the individual to

infection. The most common crisis, vaso-occlusive crisis, is caused by this blockage

mechanism. Vaso-occlusive crisis (painful episode) is the term used for the excruciatingly

painful period while the vessels are occluded by cells and tissue is being deprived of oxygen.

It is characterized by severe thoracic, abdominal, muscular or bone pain. This form of crisis

may last from a few hours to several weeks (Okpala and Tawil, 2002; Wright and Ahmedzai,

2010). Vessel blockage leads to extra complications in pregnant women, where mother and

foetus face increased risk due to oxygen variability and children also face the devastating risk

of stroke due to the occlusion of major cerebral vessels.

Sickle cell patients may develop chronic complications such as leg ulcers, below average

growth rate and complications from organ infarctions such as retinopathy, nephropathy and

possibly auto spleenectomy (Hassell, 1994).

Diminished solubility of HbS in its deoxygenated state results in the formation of a network

of fibrous polymers, which stiffen and distorts the red blood cell‟s shape. This polymerization

produces the classic sickle shape. The kinetic feature of this polymerization is critical. When

rapid deoxygenation occurs, polymerization doesn‟t alter the cell‟s disk-like shape. However,

when HbS red cells are slowly deoxygenated a nucleus of HbS molecules combine, grow the

fibers and transform into the sickle shape (Bunn, 1997).

The rate and extent of polymer formation in the SS red cells depends on three variables that

dictate the extent of polymerization and thus, to an uncertain degree, the severity of sickle

26

cell disease. Those variables are the degree of deoxygenation, intracellular hemoglobin

concentration, and presence of fetal hemoglobin. Deoxygenation causes polymerization

induced damage which leads to dehydration resulting in formation of dense cells which cause

sickling. This appears to be the basic domino effect. The polymerization rate of deoxygenated

HbS, is dependent on the hemoglobin concentration. This translates into the fact that dense

HbS cells are more likely to sickle. Dense cells are partly created by dehydration. There are

two main contributors to dehydration in these mutant red blood cells. First is the greatly

increased rate of potassium chloride co-transport in the erythrocytes. The second contributing

factor results from the increased calcium triggering a potassium channel, thus providing

another pathway for the loss of water (Bunn, 1997). The third variable in the severity of the

expression of the disease is the presence of fetal hemoglobin, which until approximately the

sixth month of life is the predominant type of haemoglobin (Stamatoyannopoulos, 2005;

Oneal et al., 2006). Thus affected infants are more or less symptom free. After this time

period the HbS is at normal levels of production and symptoms are revealed. In adults, the

level of HbF correlates with the parental HbF levels in a genetically dominant pattern (Milner

et al., 1984). HbF inhibits polymerization which halts the domino effect that generally results

in sickled cells.

Research on sickle cell disease has been on for over fifty years and the current knowledge of

the disease dictates three different approaches to therapy which have been clinically and

laboratory tested. One approach is the chemical inhibition of HbS polymerization. The

challenge faced is a formidable one, since the drug must be absorbed, circulate in the plasma

without binding strongly to plasma proteins, penetrate the erythrocyte membrane and bind

specifically to the HbS. To date, no antisickling drug has been tested with an acceptable

efficacy to toxicity ratio (Bunn, 1993).

27

A second plan of attack is the reduction of the intracellular hemoglobin concentration. This

approach relies on the dependency of the rate of polymerization of HbS on the HbS

concentration. Progress has seen made in the development of drugs that inhibit potassium and

water loss from SS red cells. This in turn reduces the intracellular hemoglobin concentration.

The drug Clotrimazole inhibits potassium loss at a specific channel and has proven effective

in transgenic mice and patients with sickle cell anemia (Bunn, 1993).

The other mode of cell dehydration, potassium-chloride co-transport, has not yet fully

conformed to pharmacological manipulation, despite it‟s inhibition by high intracellular mg2+

(Buchanan et al., 2004)

The most successful of the three approaches is the induction of fetal hemoglobin (HbF). The

objective is to raise the HbF levels because it inhibits sickling. The antitumor drug

hydroxyurea succeeded in increasing HbF in primates as well as patients with SS disease

(Bunn, 1993).

Jayabose and associates (1996) ran a study on treatment with hydroxyurea of children with

severe sickle cell anemia. Their aim was to determine the effect on the hemoglobin levels, to

evaluate the toxicity of the drug and to detect any change in the frequency of the vaso-

occlusive crises. Group one was made up of children with sickle cell anemia who suffered

frequent vaso-occlusive crises. The second group consisted of children with sickle cell and

severe anemia. Each group was given 20-35mg/kg/day. The frequency of vaso-occlusive

crises before and after treatment and the peak hemoglobin levels before and during treatment

were recorded. The trial resulted in a statistically significant drop in the number of crises,

hospital days and transfusions per year. The hemoglobin levels increased from 8-50% over

baseline values. Multiple factors such as increasing mean corpuscular volume and decreasing

28

adhesion of red blood cells to the endothelium, are probably responsible for the clinical

benefits of hydroxyurea.

One of the most important breakthroughs dealing with this disease did not even involve a

human patient, but rather mice were in the limelight. Two separate research teams developed

mutant mice that mimic human sickle cell anemia. The mice globins were completely

eliminated and complement human genes, including the sickling mutant form of beta globin,

were introduced. These mice make only the disease causing, mutant form of hemoglobin that

clumps when deoxygenated and sickles. The mice suffer the same array of symptoms that a

human with sickle cell disease endures. The creation of this genetically unique mouse is so

valuable because now drugs and therapies can be tested just as they would in a person with

the disease. The mice have exactly the same mutant beta globin that the majority of affected

humans possess. Until the development of these mice, experiments were conducted in test

tubes or on patients where a full range of possibilities couldn‟t be tested (Barinaga, 1997).

The effectiveness of the drug clotrimazole in reducing the intracellular hemoglobin

concentration was also tested on these transgenic mice.

Prevention is the best form of treatment to prevent the intravascular sickling. The trademark

crises of sickle cell are precipitated by fever, dehydration and cold exposure. To prevent these

is an effort to prevent the extremely dangerous crises. Sudden transition to high altitudes and

exposure to freezing temperatures should be avoided. Hydration is essential, as are

immunizations.

The most effective of the non-controversial therapies available is the blood transfusion. This

therapy includes packed red cell transfusions at three week intervals. This is generally enough

of an effort to maintain the donor cell (HbA) circulation above fifty percent. The transfusions

reduce the tendency for sickling by diluting the mutant host cells, temporarily suppressing the

29

production of erythrocytes containing HbS, thus improving blood and tissue oxygenation.

However this therapy does have its downsides. It requires a tremendous blood resource and

the risks of hepatitis and alloimmunization are potentially life threatening (Sandler, et al.,

1997).

Bone marrow transplants have the potential to regulate the hemoglobin synthesis. This

procedure causes life threatening complications in itself. Once these complications are

improved, it will most likely become an option for severe sickle cell anemia patients (Ferster

et al., 1992).

Since it is apparent that prevention of complications is the best form of treatment for this

disease, it is helpful to detect the mutation early on. When it comes to genetic screening,

sickle cell disease has had a rocky past. In the early 1970‟s efforts were made to identify

carriers of the recessive allele for the disease. The goal of this search was to advise the carrier

which would help in family planning which would hopefully lower the disease incidence.

However this plan backfired for several reasons. The counselling was not adequate and

resulted in people confusing the harmless trait for the deadly disease. People were

discriminated against at jobs and in society in general. It was even beleived that this was the

white man‟s way of medically reducing the reproduction of African Americans. Needless to

say the screenings were stopped. Several key ideas were learned from this fiasco which is

now been implemented in screenings for diseases from HIV to breast cancer. The test must

have a direct benefit to the person tested. The results must be kept in the strictest confidence

and the screening must be followed by counseling (Klug et al, 2010).

Prenatal diagnosis allows for preventative measures to begin while the child is still in the

mother‟s womb. Fetal cells may be acquired by amniocentesis. The DNA is extracted from

the cells and digested with restriction enzymes. The enzyme would cut twice within the

30

normal beta globin gene, once at each site. Thus a normal gene produces two small DNA

fragments. However, in the mutant alleles the second site has been destroyed by the mutation

resulting in the production of one long restriction fragment. When run on a gel, one long band

with two short fragments identifies a heterozygous state. This person will be a carrier. A gel

showing one long band identifies a person homozygous for the sickle cell mutation (Klug et

al, 2010).

With the aforementioned advances such as the transgenic mice and discovery of hydoxyurea,

that positive headline may be in the very near future. Due to the genotoxicity, carcinogenicity

of hydroxyurea as well as the non-responsiveness of some patients to hydroxyurea therapy,

other therapeutic options are being considered.

2.13.1 Therapeutic options based on disease pathophysiology

2.13.1.1 HbS Polymerization

Deoxygenation induced polymerisation of HbS (α2ß2S) is the

proximate cause of sickle cell

anemia and a necessary but insufficient precursor of the disease phenotype. HbS and its

polymer induce a variety of cellular and tissue injuries, but neither the fetal

hemoglobin

(HbF) tetramer (α2γ2) nor the α2ßSγ hybrid

tetramer is incorporated into the HbS polymer,

thus providing the rationale for treatments aimed at increasing HbF concentration.

2.13.1.2 Sickle Erythrocyte Damage and Dehydration

The presence of dense, dehydrated erythrocytes and abnormal reticulocytes is a distinguishing

feature of sickle cell anemia. Since polymerization of HbS is uniquely dependent on the

cellular concentration of HbS, the tendency

for HbS polymerization and cell sickling is

markedly enhanced by the increased cellular hemoglobin concentration of dehydrated sickle

31

erythrocytes. Of the four pathways that have been implicated in the dehydration of sickle

erythrocytes, and modulation of these pathways, especially the Gardos channel

and K

+-Cl

– co-

transport, is a potential means of treatment.

2.13.1.3 Endothelial damage

Cellular damage enables adhesive interactions between sickle cells, endothelial cells and

leukocytes. Vasoconstriction may be favoured as nitric oxide (NO) production is impaired in

a perturbed endothelium. Several agents directed at the endothelial receptors for sickle

erythrocytes or leukocytes may interrupt cell-cell interactions.

2.13.1.4 Inflammation, reperfusion injury, oxidant radical production

Neutrophils mediate inflammation and tissue damage. Neutrophil numbers are increased and

they may be abnormally activated and adherent. An "oxidant" environment may also be

present in the sickle erythrocyte and endothelium. Novel ways of countering

oxidant-induced

injury have been proposed, chiefly, via exogenous antioxidant intake

2.13.2 HbS Polymerization

Several classes of drug, when titrated optimally, can increase levels of HbF in most patients

with sickle cell anemia. Only one is approved by the American Food and Drug Agency for

treating sickle cell anemia.

2.13.2.1 Hydroxyurea

Hydroxyurea, the sole US Food and Drug Administration (FDA)–approved drug for treating

sickle cell anemia, could be used in all adults where indications for this treatment are present

(Steinberg, 1999). Unfortunately, for complex reasons, only a fraction of patients who might

benefit from treatment receive it. Hydroxyurea increases HbF in sickle

cell anemia because its

32

cytotoxicity causes erythroid regeneration and perhaps because its metabolism leads to NO–

related increases in soluble guanylate cyclase (sGC) with an increase

of cGMP that augments

gamma-globin gene expression (Cokic et al., 2003). A multicenter trial of hydroxyurea in

adults with sickle cell anemia, where the drug was given at sub-toxic doses, showed that

hydroxyurea reduced the incidence of pain and acute chest syndrome by nearly

half, with

little risk seen during more than 9 years of observation. Cumulative mortality was reduced

nearly 40%, and a favourable result was related to the ability of the drug to increase HbF

and

reduce painful episodes and acute chest syndrome (Steinberg et al., 2003) No relationship

between decrements in neutrophil counts and mortality was found. In infants, children and

adolescents with sickle cell anemia, their HbF response to hydroxyurea is more robust than in

adults. In a study of more than 100 children who received maximal drug

doses, HbF increased

to almost 20% and the treatment effects were sustained for 7 years without clinically

important toxicity (Zimmerman et al., 2004). HbF levels achieved during treatment were

associated with baseline HbF level, hemoglobin level, reticulocyte count, and leukocyte

count

and with compliance to treatment. According to some experts, pushing the drug dose to near

toxic levels is not necessary for a clinically beneficial result. Some have proposed using

hydroxuyrea for secondary prevention of stroke in children (Ware et al., 1999). Hydroxyurea

may also conserve resting energy expenditure by curbing the hypermetabolic state observed

in children with sickle cell disease (Fung et al., 2001)

Hydroxyurea is potentially mutagenic and carcinogenic (Latagliata, 2010). Cancer

and

leukemia have been reported in hydroxyurea-treated sickle cell disease patients, but whether

the incidence is higher than in the general population is not known (Steinberg et al., 2003).

The relative risk of leukemia in hydroxyurea-treated sickle cell anemia is much less

than the

observed risk in myeloproliferative disorders. To put this into perspective, the risk of death

33

from the complications of adult sickle cell disease appears to be at least 10-times

greater than

the possible incidence of leukemia in hydroxyurea-treated

sickle cell anemia patients

(Buchanan et al., 2004).

2.13.2.2 Decitibine

A less-toxic analog of 5-azacytidine, 5-aza-2'-deoxycytidine (decitibine), may affect HbF

levels by causing hypomethylation of the gamma-globin genes. In 8 symptomatic sickle cell

anemia patients who failed to respond to hydroxyurea, decitibine treatment (0.2

mg/kg

subcutaneously 1–3 times per week for 2 6-week cycles) led to an increase in HbF from 6.5%

to 20.4%, with an increase in hemoglobin concentration from 7.6 to 9.6 g/dL and a fall

in

reticulocytes from 231 to 163 x 109/L (Saunthararajah et al., 2003).

2.13.2.3 Short chain fatty acids

By acting as inhibitors of histone deacetylase (HDAC) and causing histone hyperacetylation

and changes in chromatin structure, short-chain fatty acids, their derivatives, and other

compounds with HDAC activity can enhance gamma-globin gene expression in erythroid

cells of patients with sickle cell anemia and ß thalassemia. Very low concentrations of one

HDAC inhibitor, an analog of trichostatin A, induced gamma-globin gene expression in an

erythroleukemia cell line transfected with a reporter construct and in erythroid

colonies of

normal adults (Cao, 2004)

In the most advanced clinical trials of HDAC inhibitors, still only Phase II studies, arginine

butyrate given by infusion once or twice a month was associated with a mean increase in HbF

from 7% to 21% in 11 of 15 patients with sickle cell anemia. In some individuals, this level

was maintained for 1–2 years (Atweh et al., 1999). No HDAC inhibitor of any class other

than butyrate and phenylbutyrate has yet been used clinically.

34

2.13.3 Sickle Erythrocyte Dehydration

2.13.3.1 Inhibition of K+-Cl

– co-transport

Oral magnesium supplementation inhibits erythrocyte K+-Cl

– co-transport in vivo. Following

studies that showed a beneficial effect on the erythrocyte membrane of transgenic sickle

mice, a 6-month clinical trial of oral Mg pidolate improved erythrocyte

hydration and was

associated with a reduction in the number of painful days (De Franceschi et al., 2000).

Studies to evaluate the effects of long-term magnesium supplementation in adult and pediatric

patients with sickle cell anemia are ongoing, and additional studies are planned

in HbSC

disease patients where activated K+-Cl

– co-transport

and cell dehydration are likely to play a

major pathophysiological role (Buchanan et al., 2004)

2.13.3.2 Inhibition of Gardos channel

Clotrimazole is an inhibitor of the human red cell Gardos channel but its use was associated

with dysuria and reversible hepatocellular toxicity. ICA 17043, a clotrimazole derivative

lacking the toxic imidazole residue, was a 10-fold more potent blocker of the

Gardos channel

than the native drug. In a recently completed Phase II trial of this agent in patients with sickle

cell disease, cell density and hemolysis were decreased while hemoglobin concentration

was

increased. To see whether clinical benefit accrues from these erythrocyte and hematological

changes will require a Phase III clinical trial (Ataga et al., 2008).

2.13.3.3 Inhibition of other channels

Movement of K+ via the Gardos channel requires the parallel

movement of Cl

– anions to

maintain electroneutrality. High-affinity blockers of Cl

– conductance can reversibly

block

human erythrocyte chloride conductance in vitro without directly affecting the Gardos

channel or the K+-Cl

– co-transport.

In sickle mice treated with a Cl

– conductance inhibitor,

35

packed cell volume (PCV) increased and mean corpuscular hemoglobin

concentration

(MCHC) decreased with an increase in cell K+. A selective loss of the densest erythrocytes,

with a shift from sickled to well-hydrated discoid erythrocytes, was seen.

Clinical trials of this

class of agent have not been done (Buchanan et al., 2004).

2.13.4 Anti-Adherence Therapy

Anti-adherence therapy for sickle cell disease targets the abnormal interactions among

erythrocytes, endothelial cells, leukocytes and platelets that are part of the pathophysiology of

the disease process. Potential anti-adherence agents have been studied in

acute painful events,

where, through poorly understood mechanisms, they restore microvascular circulation and

improve tissue ischemia. In Phase II studies of a non-ionic surfactant copolymer, poloxamer

188, this agent reduced the duration and increased the resolution of acute painful episodes, an

effect especially notable in children

less than 15 years old and in patients receiving

hydroxyurea (Orringer et al., 2001). Whether poloxamer 188 exerts its effects by modifying

interactions of sickle cells or other blood cells to endothelium is not known.

Its clinical

effectiveness as a single agent to treat or prevent vasoocclusive complications is, at best,

modest, and currently no additional trials of this agent are on-going.

Endothelium-dependent vasodilation is disturbed in sickle cell disease (Eberhardt et al., 2003)

and superoxide generation induces chronic inflammation

that may be inhibited by

apolipoprotein (apo) A-1 mimetics.

L-4F, an apoA-1 mimetic, inhibited superoxide

production and improved vasodilation in sickle mice (Ou et al., 2003) While a mechanism of

action is not yet totally clear, this agent may preserve endothelial function and endothelial

nitric oxide synthase (eNOS) activity. This type of drug may have widespread application in

vascular diseases including sickle cell anemia (Ansell et al., 2003). Most agents that might

36

disrupt the adhesive interactions and inflammation

hypothesized to presage sickle

vasoocclusion have not been studied clinically.

2.13.5 Nitric Oxide

A potent vasodilator, NO is an important regulator of vascular tone and, because of its

interaction with hemoglobin, blood vessels and blood cells, has been hypothesized to have

several advantageous effects in sickle cell disease. Generated from

L-arginine by NO

synthases, NO activates soluble guanylate cyclise (sGC), to produce the second messenger,

cGMP (Buchanan et al., 2004). Nitric oxide inhibits the adhesive interactions

among

platelets, leukocytes and sickle erythrocytes (Hankins and Aygun, 2009). It also decreases

vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells.

While a

biologically sound rationale exists for using NO in the acute chest syndrome and pulmonary

hypertension of sickle cell disease, a controlled trial of this treatment has not been reported. It

should be remembered that this approach could be deleterious as NO can be metabolized

to

damaging oxidants like nitrite (NO2–) and peroxynitrite

(ONOO

–). In a clinical trial, inhaled

NO was associated with a small reduction in pain score and opioid use in children

with acute

painful episodes (Weiner et al., 2003).

2.14 Unorthodox Treatment

In Nigeria, herbal medications are used as alternatives to orthodox western medicine due to

the perceived minimal side-effects, affordability and easy of acquisition (Ogunkunle and

Ladejobi, 2006; Okigbo et al., 2009). Medicinal plants can be described as plants whose part

/ organ contains a substance that can be used for therapeutic purposes or as a precursor for the

synthesis of antimicrobials or other useful drugs (Sofowora, 1982). World Health

Organization (W.H.O.) as cited by Okigbo et al. (2009) defined medicinal plants as herbal

37

preparations produced from chemical, physical, or biological processes which may be used

for immediate consumption or as a basis of herbal products. Medicinal and aromatic plants

used in such herbal medications contain biologically active substances (secondary

metabolites) such as saponins, tannins, flavonoids, alkaloids and other chemical substances

which have curative properties (Okigbo et al., 2009). Most of these phytochemical

constituents formed during normal metabolic processes are potent bioactive compounds

found in specific plant parts, thus many plant components are used in preparing

pharmaceutical products for medical treatments. It has also been noted that most treatments

for diseases and infections in developing regions such as Africa largely depend on herbal

preparations (Okigbo et al., 2009). WHO estimates that 80% of the world population,

approximately 4 billion people, uses herbal medicine for primary health care (Macedo et al.,

2008). Plant materials thus play a major role in primary health care as therapeutics in many

developing countries. Herbal medicine has a long and respected history as the oldest form of

healthcare to mankind being used worldwide in various forms.

The use of phytomaterials such as Piper guinensis, Pterocarpa osun, Eugenia caryophylla

and Sorghum bicolor extracts for the treatment of SCA has been reported by Wabembe et al.

(2001). The extracts of Pterocarpus santolinoides and Aloe vera were reported to increase the

polymerisation time of Hbf and inhibit sickling in vitro (Ugbor, 2006). Sofowora and Isaac-

Sodeye (1971) reported the reversal of sickling by root extracts of Fagara zanthoxylloides.

Terminalia catappa could be effective antisickling agents that inhibit osmotically induced

haemolysis of human erythrocytes (Mgbemene and Ohiri, 1999). The aqueous extract of

Gracinia kola has been described by Elekwa et al.(2003) as having a higher membrane

stabilising effect than phenylalanine, while 2 – hydroxybenzoic acid has been identified as

the antisickling agent in aqeous extract of Zanthoxylum macrophylla roots (Elekwa et al.,

38

2005). The in vitro HbS gelling time was reportedly increased by the extract of Pterocarpus

santolinoides and Aloe vera (Mohanty et al., 2012). Antisickling activity of twelve Congolese

plants namely Alchornea cordifolia, Afromomum albo violaceum, Annona senegalensis,

Cymbopogon densiflorus, Bridelia ferruginea, Ceiba pentandra, Morinda lucida,

Hymenocardia acida, Coleus kilimandcharis, Dacryodes edulis, Caloncoba welwithsii, and

Vigna unguiculata have been reported (Mpiana et al., 2007). It has been suggested that

Cajanus cajan seeds (Onah et al., 2002), aged garlic (Ohnishi and Ohnishi, 2001; Moriguchi

et al., 2001), unripe Carica papaya fruit (Oduola et al., 2006; Thomas and Ajani, 1987),

leaves (Imaga et al., 2009), the roots of Cssus populnea L. CPK (Moody et al., 2003), Khaya

senegalensis (Fall et al., 1999 ), Scoparia dulcis (Ahmed and Jakupovic, 1990) as well as

Parquetina nigrescens (Wambabe et al., 2005) have various degree of antisickling activities.

The Nigerian Zanthoxylum has been extensively studied (Sofowora and Isaacs-Sodeye, 1971;

Sofowora, 1974; Sofowora, 1975a; 1975b; Adesanya and Sofowora, 1983). The bioactive

compounds responsible for anti-sickling properties were identified as vanillic acid, p-hydroxy

benzoic acid and p-fluoro benzoic acid. There are some other drugs prepared from medicinal

plants in the market today under various names. The most prominent and widely used of them

all is Ciklavit developed by Prof G Ekeke after eighteen years of intensive research in

collaboration with Neimeth Pharmaceuticals, Lagos, Nigeria. (Okpuzor, et al., 2008)

Research on phytomedicine for the treatment of SCD has led to the development of Nicosan

formerly called Niprisan (herbal based drug) which has been patented by the National

Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria and

produced to meet increasing global demand by sufferers of SCA. This development indicates

that more of such herbal based drugs could be consequent upon scientific investigations on

plants that are used in folklore medicine.

39

On the understanding that herbal remedies and medicinal plants products from indigenous

flora have long been used in folk medicine in the management of SCA, it appears that proper

and in-depth scientific investigation on such medicinal plants could be of tremendous help in

developing efficacious and safer drugs for SCA treatment.

The species Senna alata (Fabaceae) and Senna podocarpa (Fabaceae) are examples of plants

commonly used in popular Nigerian folk medicine. Both S. alata and S. podocarpa bear a

common Yoruba name „asunwon‟ while S. alata is referred to as „asunwon oyinbo‟, the

suffix meaning exotic or introduced species and S. podocarpa is known as „asunwon gidi‟

the suffix meaning indigenous or native species (Odugbemi, 2006; Ogunkule and Ladejobi,

2006).

2.14.1 Physical Description of Senna alata

Senna alata (Fabaceae) is a plant growing 6-12 feet as a shrub with erect waxy yellow spikes

resembling fat candles before individual blossoms open. The leaves are lanceolate in shape,

bilaterally symmetrical opposed with smooth margins, folding together at night. The leaflets

are 8-20 in number arranged in four pairs. The buds of the plant are rounded with five

overlapping sepals, five free or less equal petals narrowed at the base. Fruits are winged pods

with seeds small and square. It is found in the tropics (West Africa, Islands of Central and

South Pacific, South America and Australia) in secondary vegetations or along riverbanks. It

is commonly referred to as Ringworm Cassia, Candle Bush, Candelabra Bush, Empress

Candle Plant, Ringworm Tree or “Candletree”. Its conservation status is secure. In Southwest

Nigeria it is referred to as “Asunwon oyingbo” in the Yoruba language for its exotic origin;

Crawcraw or Ringworm plant for its use in skin diseases (Odugbemi, 2006; Ogunkunle and

Ladejobi, 2006).

40

2.14.1.1 Medicinal Attributes of Senna alata

Senna alata is a widely recognized medicinal plant amongst herbal traditional practitioners

(Ogunkunle and Ladejobi, 2006). It is used in form of poultice, decoctions with plant parts

such as leaves, stem, bark and pods used in preparations and are widely believed to be

applicable in skin diseases, dysentery, abortifacient, antihelminthic, itch, ringworm, measles,

eczema, bronchitis, black menstruation, venereal disease and stomach ache (Odugbemi,

2006). Other members of the Caesalpinioideae subfamily are recognized as well for their

medicinal properties-Senna podocarpa, Cassia fistula, S. tora, C. obtustifolia, C. nigricans,

C.acutifolia, C. sieberiana, C. occidentalis, C. angustifolia, C. acutifolia, C. spectabilis (Ayo

and Amupitan, 2007). For example, S. tora has been noted for its anti-microbial and anti-

oxidant abilities (Uddin et al., 2008). Out of the thirty-three species found in Nigeria, Senna

alata has been noted for its use in the treatment of skin ailments and its use as a laxative or

purgative with anti-microbial action against dermatophyte bacterial and fungal infections

(Owoyale et al., 2005; Ayo and Amupitan, 2007). It has been stated that all the plant parts

can be used as an oral laxative due to its anthraquinone content (Owoyale et al., 2005).

The leaves of Senna alata are used as infusions to treat constipation or poultice to treat

oedema and skin disease (Okigbo et al., 2009). The leaf sap has been used to treat ringworm,

scabies, ulcers, swellings, inflammation and skin parasites (Owoyale et al., 2005; Pieme et

al., 2006). The decoction of the leaf, flower bark and wood has been used in treating eczema

and allergy and the decoction of the leaf flower only has been used as an expectorant in

bronchitis, dyspnoea and as an astringent and mouthwash (Pieme et al., 2006; Idu et al.,

2006). The methanolic extract has been shown to have a strong growth inhibition against

bacteria and fungi- Mucor sp., Rhizopus sp., Aspergillus niger, Escherichia coli, Bacillus

41

subtilis, Salmonella typhii, Pseudomonas aeruginosa and Staphylococcus aureus (Owoyale et

al., 2005).

Further research has shown that the methanolic extract of S. alata also inhibited the growth of

Microsporum canis, Trichophyton simii, Fusarium solani, Staphylococcus pyogenes,

Corynebacterium diptheriae, Dermatophilus congolensis, Actinomyces bovis, Blastomyces

dermatitidis, Trichophyton mentagrophytes, Candida albicans, Aspergillus flavus (Makinde

et al., 2007). Plants possess primary metabolites and secondary metabolites; some secondary

metabolites formed during normal metabolic processes may be responsible for their

therapeutic ability. Phytochemical analysis of S. alata leaves shows the presence of phenolic

compounds, eugenol, glycosides, saponin, flavonoids, alkaloids, tannins and anthroquinone.

Anti-microbial activity of the methanolic extract of the leaves on the isolates of

Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa,

Bacillus subtilis, Microsporum gypseum and Trichophyton rubrum has been established

(Phongpaichit et al., 2004; Ogunkunle and Ladejobi, 2006; Idu et al., 2006). S. alata has been

found to contain azulene, guiane, 1, 5, 7-trihydroxy-3-methylanthraquinone, rhein (1, 8-

dihydroxyanthraquinone), chrysophanic acid, chrysophenol and aloe-emodin (Hauptmann

and Nazario, 1950; Phongpaichit et al., 2004; Ayo and Amupitan, 2007). Kaempferol is a

flavonoid compound isolated from the methanolic extract of leaves of S. alata plant found to

be responsible for the antioxidant effect of the extract (Panichayupakaranat and Kaewsuwan,

2004). Ethanolic extract of Cassia alata was found to exhibit protective antioxidant activities

in rats exposed to carbon tetrachloride (Wegwu, 2005). The methanolic extract of S. alata

leaves was found to be more effective than the chloroform, petroleum ether or water/aqueous

extracts in anti-microbial activity (Owoyale et al., 2005; Idu et al., 2006; Makinde et al.,

42

2007). Pieme et al. (2006) concluded in their in vivo experiments on mice and rats that the

aqueous ethanolic extract of the leaves of S. alata had low toxicity and was safe to use.

2.14.2 Physical Description of Senna podocarpa

Senna podocarpa (Fabaceae) formally known as Cassia podocarpa is an herbal shrub that

grows up to 5m high with dense racemes of yellow flowers and thin flat pods. It has a

perennial life span and is widely distributed in Africa, and in some part of Asia. S. podocarpa

is found in the savanna forest area of West Africa (Akanmu et al., 2005). The plant is found

locally, on old farmlands in both western and northern parts of Nigeria (Dalziel and

Hutchinson, 1958).

2.14.2.1 Medicinal Attributes of Senna podocarpa

This plant has been used extensively in the folklore medicines for the treatment of a variety

of skin diseases such as scabies, ringworm and eczema (Sofowora, 1986) especially amongst

the Igbo and Yoruba speaking tribes of Nigeria where it is locally known as Ageloo-ogala

and Asunwon gidi, respectively (Adefemi et al., 1998). The leaves have been extensively

used based on their antigonorrehoeal and purgative properties as well as sore-healing remedy

(Elujoba et al., 1994; Akanmu, 1999; WHO, 1999). The virucidal property against Herpes

simplex virus and African swine fever virus and their infections was established by Silva et

al. (1997) Decoction of the leaves is given as a mild laxative and in large doses, it acts as a

purgative (Trease and Evans, 1996). In Liberia folk medicine, amongst the Kpelle people, the

effects of stem bark extracts in vitro and in vivo on microfilaria were examined and found to

possess curative effect (Kilian et al., 1990). According to Gomes et al. (1997) S. podocarpa

is widely used by Fulani traditional healers living in Guinea Bissau to treat veneral diseases.

43

Ehtnobotanical and phytochemical studies of the leaves and pods of S. podocarpa have

identified some of the plant constituents, which include rhein, emodin, chrsophanol, free or

combined anthraquinones (Rai and Obayemi, 1973; Rai and Abdulahi, 1978; Elujoba et al.,

1994). Anthraquinones derivatives (Akinyemi et al., 2000) obtained from the pods and leaves

have been held responsible for the plants‟ laxative as well as purgative activites. Other

phytochemical groups in the leaf extracts of the plants include flavonoids, tannins,

phlobatannins and saponins (Ogunkunle and Ladejobi, 2006)

2.15 Cytotoxicity testing

Sickle cell anemia (SCA) can potentially be cured by gene therapy (Perumbeti and Malik,

2010) and hematopoietic stem cell transplantation (Bernaudin et al., 2007) but neither is

currently applicable to most patients with this disease due to technical reasons, cost and lack

of highly sophisticated medical care necessary to provide these therapies in Nigeria. Over 50

agents have been described that increase γ-globin expression and/or HbF in primary human

erythroid cell cultures or when administered to humans. But most inducing agents, including

histone deacetylase inhibitors, DNA methyltransferase inhibitors and cancer chemotheraphy

drugs may be cytotoxic, and may alter epigenic marks on a genome-wide basis, damage DNA

or suppress erythropoiesis (Mabaera et al., 2008). Hydroxyurea is the only approved drug for

use in humans, though it has provided a major advance in the care of people with SCA, it is

only effective in one – half of SCA patients (Steinberg et al. 1997a) and it produces

significant suppression of blood counts. These and other issues have led to the low rate of

compliance in Africa (Aliyu and Babdok, 2007) and United States (Hampton, 2008).

In view of the shortcomings of current inducing agents, there are no drugs that are effective,

safe and have the ease of use that would make them applicable to most patients with SCA.

There is therefore an urgent need to screen for natural inducers of HbF and identify new types

44

of agents that can induce HbF production with greater efficiency and lower toxicity (Lal and

Vichinsky, 2004). In developing countries, so many constituents of plants capable of diseases

treatment have been investigated by scientists with a view to contributing to the search for

substances that would be effective in the management of the diseases. Crude extracts from

plants have been used in treating many diseases since ancient times, although, the biological

concentration and toxicity of such plant extracts are seldom known. The safety of any

therapeutic agent is of paramount importance and appropriate toxicity assays to evaluate

biological safety of biomaterials are essential. In vitro cytotoxicity assays with cultured cells

are very common because they are rapid, inexpensive and do not have ethical implications

(Ishiyama et al., 1996; Harbell et al., 1997; Fernandes et al., 2005). Several methods for

determining cellular viability and/or growth following in vitro exposure to compounds have

been reported. These methods used well know dyes that develop a colour, allowing a

colorimetric measurement of cell viability.

Cytotoxicity is a technique for studying the effectiveness of substances on the survival and

proliferation of cells before testing them in patients (Carney and Winkler, 1985). The assay

measures the toxicity of a treatment on cells at a wide range of doses and it is frequently used

in cancer research laboratories to determine the effect of drugs on proliferating tumor cells

(Hoffman and Robert, 1991). In vitro cytotoxicity tests are based on the idea of „basal‟

cytotoxicity – that toxic substances affect basic functions of cells which are common to all

cells, and that the toxicity can be measured by assessing cellular damage. The development of

in vitro cytotoxicity assays has been driven by the need to rapidly evaluate the potential

toxicity of large numbers of compounds, to limit animal experimentation whenever possible,

and to carry out tests with small quantities of compound (Barile et al., 1994). Evidence for

the use of in vitro cytotoxicity tests has led many pharmaceutical companies to screen

45

compound libraries to remove potentially toxic compounds early in the drug discovery

process (Davila et al., 1990; Barile et al., 1994 and Todd et al., 1999). Early identification of

toxic effects can help project between chemical series and identify toxicity relationships that

may exist between the chemicals and the cells (Todd et al., 1999).

A reduction in cellular ATP levels or mitochondrial activity is an early indication of cellular

damage. It has been shown that changes in metabolic activity are better indicators of early

cell injury, and that effects on membrane integrity are indicative of more serious injury,

leading to cell death (Davila et al., 1990). The key features of cytotoxicity assay include low

compound consumption and multiple assays. Mitochondrial enzyme, succinate-tetrazolium

reductase, present in viable cells reduces tetrazolium salts to soluble or insoluble coloured

formazan compounds that can be quantified spectrophotometrically. The most commonly

used tetrazolim salt is MTT [3-(4, 5-Dimethylazol-2-yl)-2,5-diphenyltetrazolium bromide],

however, the disadvantage is that the formazan dye produced from MTT is extremely water

insoluble, so an additional extraction step is needed for spectrophotometric quantification,

which can be avoided with the use of WST-1 [2,(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-

disulfophenyl)-2H-tetrazolium]. Since the bio reduction of tetrazolium salt, is a sensitive

chromogenic indicator for NADH produced by metabolically active cells in culture, the

amount of formazan dye formed directly correlates to the number of metabolically active

cells in the culture (Riss and Moravec, 2004).

One parameter for cell death is the integrity of the cell membrane, which can be measured by

the cytoplasmic enzyme activity released by damaged cells (Riss and Moravec, 2004).

Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme present in all cells. It is rapidly

released into the cell culture supernatant upon damage of the plasma membrane

(Korzeniewski and Callewaert, 1983).The LDH activity is determined in an enzymatic test.

46

The first step is the reduction of NAD+ to NADH/H+ by the LDH catalyzed conversion of

lactate to pyruvate. In a second step, the catalyst (diaphorase) transfers H/H+ from

NADH/H+ to the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium

chloride (INT), which is reduced to a red formazan (Nachlas et al., 1960; Decker and

Lohmann-Matthes, 1988; Lappalainen et al., 1994). Neutral red (3-amino-m-dimethylamino-

2-methylphenazine hydrochloride) has been used previously for the identification of vital

cells in cultures (DeRenzis and Schechtman, 1973). This assay quantifies the number of

viable, uninjured cells after their exposure to toxicants; it is based on the uptake and

subsequent lysosomal accumulation of the supravital dye, neutral red. Quantification of the

dye extracted from the cells has been shown to be linear with cell numbers, both by direct cell

counts and by protein determinations of cell populations (Borenfreund and Puerner, 1985,

1986).

Cell lines are often used for in-vitro cytotoxicity testing, and an experimental model system

employing the human erythroid-like K562 cell line, isolated and characterized by Lozzio and

Lozzio (1975) from a patient with chronic myelogenous leukemia in blast crisis, has been

extensively employed as a very useful in vitro model to study the molecular mechanism(s)

regulating the expression of embryonic and fetal human globin genes (Bianchi et al., 2000),

as well as to determine the therapeutic potential of new differentiation-inducing compounds

(Osit et al., 1997;Bianchi et al., 2001). K562 cells grow in culture as single, undifferentiated,

cells in suspension, with low production of hemoglobins (Bianchi et al., 2007) but when

stimulated by various agents, they respond within few days with a significant increase in the

production of hemoglobins and -globin mRNA.

47

3.0 MATERIALS AND METHODS

3.1.1 Plant Materials

The Cajanus cajan (beans and leaves), Smilax kraussiana (roots), Alchornea cordifolia

(leaves), Senna alata (leaves), Senna podocarpa (leaves) used in this study were sourced,

during the rainy season (July – September) from 2006 - 2007, and authenticated by herbarium

curators at the Forestry Research Institute of Nigeria (FRIN), Ibadan. The voucher specimens

of Senna alata leaves (No.FHI.106929) and Senna podocarpa leaves (No.FHI.106995) were

deposited at FRIN herbarium. “Jana”, an herbal formulation, was graciously provided by

Prof. (Mrs.) Okpuzor of the Department of Cell Biology and Genetics, University of Lagos

3.1.2 Participants

The informed consent of the volunteers (SCA patients) was obtained after counseling in

accordance with the approved University protocols (Apendix 1). Blood was collected from

HbSS patients in steady states who had not been on any herbal therapy for at least six months,

but were on sustainance therapy (folic acid and paludrin). Blood (3ml) from donors was

taken into heparin or EDTA tubes and used within two hours post collection.

3.1.3 Experimental Animals

Male albino rats (94 -110 g) were purchased from the University of Ibadan animal house. The

rats were allowed to acclimatize for 2 weeks at the University of Lagos, Department of Cell

Biology and Genetics animal house experimental facility, provided standard feed for

laboratory rodent, regular tap water ad libitum. All animals had free access to water and

standard rat pellets (Pfizer). Rats were fasted (12-15hrs) prior to the administration of extract.

48

3.2 Preparation of Extracts

The plant materials (200g) were extracted with either 80% ethanol or 80% methanol using a

Soxhlet apparatus, concentrated under reduced pressure to a viscous liquid, freeze dried,

weighed and stored at 4 oC until use. For water extract, 10 g of powdered leaf material was

mixed with boiling water (500 ml) for 15 min. The extract was filtered and evaporated under

vacuum below 70 °C on a rotary evaporator according to Duh and Yen, (1997).

3.3 Preliminary Screening

The hydroethanolic extract of Cajanus cajan (beans and leaves), Smilax kraussiana (roots),

Alchornea cordifolia (leaves), Senna alata (leaves), Senna podocarpa (leaves) as well as Jana

were evaluated for their HBSS erythrocyte membrane stabilizing activity using the osmotic

fragility test (Dacie and Lewis, 1991)

3.4 Phytochemical Screening

The phytochemical compositions of the extracts were evaluated according to the methods of

Trease and Evans (2002) and Harborne (1991).

3.5 Proximate Analysis

3.5.1 Preparation of the plant materials for chemical analyses:

Senna alata leaves and Senna podocarpa leaves were washed with distilled water dried at

room temperature to remove residual moisture, and oven-dried at 55 ºC for 24 hours (Aletor

and Adeogun, 1995; Abuye et al., 2003). The dried leaves were ground into powder using a

blender (MARLEX; CM/L 7371373), and sieved through 20-mesh sieve. The leaf powder

was used for the chemical composition analyses.

49

The methods recommended by the Association of Official Analytical Chemists were used to

determine ash (#942.05), crude lipid (#920.39), crude fibre (#962.09) and nitrogen content

(#984.13). All the proximate values were reported in % (AOAC, 1990).

3.5.2 Determination of Moisture Content

Moisture content of the leaf of the two Senna species were determined by drying leaf samples

at 105oC in a hot air oven to constant weight (3 hrs) (AOAC, 1990).

3.5.3 Determination of Ash Content

Two gram aliquot of the sample was placed in a pre-weighed crucible; this was heated on a

hot plate until no smoke was visible. The residue containing crucible was transferred into a

muffle furnace set at 550oC for 3 hours after which the ash containing crucible was cooled in

a dessicator and weighed. The weight of the ash containing crucible was subtracted from that

of the pre-weighed crucible to obtain the ash content. (AOAC, 1990)

3.5.4 Determination of crude lipid and crude fibre content:

Two grams of dried leaves were weighed in a porous thimble of a Soxhlet apparatus. The

thimble was placed in an extraction chamber, which was suspended above a pre-weighed

receiving flask containing petroleum ether (b.p.40-60ºC). The flask was heated on a heating

mantle for eight hours to extract the crude lipid. After the extraction, the thimble was

removed from the Soxhlet apparatus and the solvent distilled off. The flask containing the

crude lipid was heated in the oven at 100ºC for 30 min to evaporate the solvent, then cooled

in a desiccator, and reweighed. The difference in weight was expressed as percentage crude

lipid content. Crude fibre was estimated by acid-base digestion with 1.25 % H2SO4 (prepared

by diluting 7.2 ml of 94 % conc. acid of specific gravity 1.835 g ml-1

per 1000 ml distilled

water) and 1.25 % NaOH (12.5 g per 1000 ml distilled water) solutions. The residue after

50

crude lipid extraction was placed in a 600 ml beaker and 200 ml of boiling 1.25 % H2SO4

added. The contents were boiled for 30 minutes, cooled, filtered through a filter paper and the

residue washed three times with 50 ml aliquots of boiling water. The washed residue was

returned to the original beaker and further digested by boiling in 200 ml of 1.25 % NaOH for

30 minutes. The digest was filtered to obtain the residue. This was washed three times with

50 ml aliquots of boiling water and finally with 25 ml ethanol. The washed residue was dried

in an oven at 130ºC to constant weight and cooled in a dessicator. The residue was scraped

into a pre–weighed porcelain crucible, weighed, ashed at 550ºC for two hours, cooled in a

dessicator and reweighed. Crude fibre content was expressed as percentage loss in weight on

ignition, (AOAC, 1990; Nesamvuni et al., 2001).

3.5.5 Determination of nitrogen content and estimation of crude protein:

Macro-Kjeldahl method was used to determine the nitrogen content, which is the major

component of amino acid, a precursor for protein. Two grams of dried leaves were digested

in a 100 ml Kjeldahl digestion flask by boiling with 10 ml of concentrated tetraoxosulphate

(VI) acid (H2SO4) and a Kjeldahl digestion tablet (a catalyst) until the mixture was clear. The

digest was filtered into a 100 ml volumetric flask and the solution made up to 100 ml with

distilled water. Ammonia in the digest was steam distilled from 10ml of the digest to which

had been added 20 ml of 45 % sodium hydroxide solution. The ammonia liberated was

collected in 50 ml of 20 % boric acid solution containing a mixed indicator. Ammonia was

estimated by titrating with standard 0.01 M HCl solution. Blank determination was carried

out in a similar manner. Crude protein was estimated using the following formula:

% Crude protein = % Nitrogen content X 6.25 (AOAC, 1990).

51

3.5.6 Determination of carbohydrate and energy values:

Available carbohydrate was estimated, by subtracting the total sum of percent crude protein,

crude lipid, crude fibre and ash from 100 % dry weight of the plant. The plant calorific value

(kJ) was estimated by multiplying the percentages of crude protein, crude lipid and

carbohydrate by the factors 16.7, 37.7 and 16.7 respectively and adding up the products.

(AOAC, 1990).

3.6 Invitro Studies

3.6.1 Effect of Extract on HbSS Erythrocytes

3.6.1.1 Determination of Osmotic Fragility

Osmotic fragility of red cells was determined according to the modified method of Dacie and

Lewis (1991). A 10% buffered saline solution was prepared by dissolving 45g NaCl, 6.828g

Na2HPO4 and 0.935g NaH2PO4 in 500 ml of distilled water. A 1 % buffered saline solution

was then prepared and its pH adjusted to 7.4 using a pH meter. Graded concentrations of

buffered saline solution (0.00 - 0.85 %) were prepared and 5.0 ml of each concentration was

dispensed into prelabelled tubes (control), while 4.0 ml of each concentration was dispensed

into another set of eight tubes and 1.0 ml of buffered extract was added to each of the tubes

(experimental). A drop (0.02 ml) of heparinised whole blood was then added to each tube and

mixed thoroughly by five gently inversions, then incubated at room temperature for 30 mins

after which the contents of the tubes was again mixed and centrifuged for five minutes at

2500 rpm. The supernatant was decanted into cuvettes and the optical density read at a

wavelength of 540 nm using the supernatant from the 0.85 % tube as blank for each set of

tubes. Lysis was recorded as a percentage (optical density of the solution of tube without

NaCl was taken as 100 % hemolysis), and plotted against percentage sodium chloride to

52

obtain a fragility curve. The mean corpuscular fragility (MCF) index, which is the

concentration of saline solution (NaCl g/L) that caused 50% lysis of erythrocytes, was

interpolated from the plot and the % membrane stabilisation was calculated using the

following formula : % Membrane Stability = (MCFcontrol – MCFtest / MCFcontrol)*100.

(Okpuzor and Adebesin, 2006; Chikezie and Uwakwe, 2011)

3.6.1.2 Activity of the Extracts on Haemoglobin Polymerisation

The venous blood was washed with incubation media and collected by centrifugation at 2500

rpm for five minutes, until the supernatant was clear. A 10 % or 35% hematocrit was

prepared for antisickling or desickling experiment respectively. All prepared blood samples

were stored at 4oC until use and allowed to attain room temperature before use.

3.6.1.2.1 Antisickling Activity of the Extracts

A 2000 µl aliquot of washed red blood cells (10 % hematocrit) was mixed with 2000 µl of

buffered plant extract (5 mg ml-1

) or control. The mixture was gently shaken and at 30 mins

interval, a 400 µl aliquot was withdrawn and mixed with 600 µl of freshly prepared 2 %

sodium metabisulphite solution in a fresh tube. The resulting mixture was layered with 600µl

of liquid paraffin and incubated at 37oC. A 100 µl aliquot was withdrawn every 35 mins into

a fresh Eppendorf tube, mixed thoroughly with 200 µl of 5 % buffered formalin and then

incubated at room temperature until ready to prepare slides. For the positive controls, namely

NICOSAN (an herbal formulation currently used by SCA patients) (5 mg ml-1

),

parahydroxybenzoic acid (synthetic drug) (5 mg ml-1

) and negative control (sodium chloride)

a 1000 µl aliquot of the washed RBC and 1000 µl of control (NICOSAN /

parahydroxybenzoic acid / sodium chloride) were thoroughly mixed at 37oC and immediately

(zero time) a 200 µl aliquot was withdrawn and at intervals of 30mins thereafter, into fresh

53

Eppendorf tubes, mixed with 300 µl of freshly prepared 2 % sodium metabisulphite solution

and the resulting mixture was further diluted with 500 µl of buffered formalin.

3.6.1.2.2 Desickling Activity of the Extracts

A 2000 µl aliquot of washed red blood cells (35 % hematocrit) was layered with liquid

paraffin and 2000 µl of freshly prepared 2 % sodium metabisulphite solution was added with

the aid of a needle and syringe. The mixture was gently rolled between the palms and

incubated for 2 ½

hours at 37oC with occasional shaking. After incubation a 500 µl aliquot of

the mixture was withdrawn and mixed with 500 µl of Senna alata (5 mg mlˉ¹), Senna

podocarpa (5 mg mlˉ¹), parahydroxybenzoic acid (5 mg mlˉ¹) or physiological saline

solution in a fresh tubes layered with liquid paraffin and preincubated at 37oC. A 100 µl

aliquot was withdrawn every 30 mins into a fresh Eppendorf tube, mixed thoroughly with

200 µl of 5 % buffered formalin and then incubated at room temperature until ready to

prepare slides.

3.6.1.2.3 Preparation of Microscopic Slides

The mixture containing buffered formalin and control was centrifuged at 2000 rpm for 5

minutes and 250 µL of the supernatant was removed. With the aid of a capillary tube, a drop

was taken and placed on a microscope slide. A smear was made and fixed with 95 %

methanol. The slide was stained with Giemsa stain and excess stain was washed off. The

slide was air dried and examined under light microscope. About 100 RBCs were counted

from five different fields.

54

3.6.2 Antioxidant Potential of Extracts

3.6.2.1 Total Antioxidant Capacity

The total antioxidant capacity of the plant extract(s) was determined using the method of

Prieto et al. (1999) with slight modifications. The assay is based on the formation of a green

phosphate- molybdenum (V) complex by the reduction of molybdenum (VI) at acidic pH.

Aliquots of 0.1 ml of each extract or the standard - ascorbic acid (100 µg ml-1

) was mixed

with 3 ml of working reagent (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM

ammonium molybdate). The tubes were incubated at 95oC for 90 minutes, cooled to room

temperature and the absorbance of each solution was measured at 695 nm against a blank

(working reagent plus solvent-methanol/water) using a spectrophotometer. The experiment

was performed in triplicates. The total antioxidant capacity was expressed as milligram

equivalent of ascorbic acid.

3.6.2.2 Total Reducing Power

The reducing power of the plant extract(s), gallic acid and ascorbic acid was determined by

assessing the ability to reduce ferric chloride solution (Athukorala et al., 2006). Aliquots of

1ml graded concentration (25-100 µg ml-1

) of the crude extract or standard (gallic acid or

ascorbic acid) was mixed with equal volume of 1% ferriccyanide and 200 mM phosphate

buffer, pH. 6.6. the mixture was incubated at 50oC for 20 minutes. An equal volume of 10 %

trichloroacetic acid was then added and the resulting solution centrifuged at 6000 rpm for 10

minutes. The resulting supernatant, distilled water and 0.1 % ferric chloride were mixed in

the ratio 1:1:2 and the absorbance measured at 700nm. A higher absorbance indicated higher

reducing power

55

3.6.2.3 Total Phenolic Content

The total phenolic content of the plant extract(s) was determined according to the method of

Singleton and colleagues (1999), using the folin-ciocalteau reagent (FCR) which is reduced

by phenols to a mixture of blue oxides with maximal absorbance at around 760 nm. Equal

volumes (100 µl) of plant extract or gallic acid (0-100 µg) were mixed with 500 µl of FCR

and 1.5 ml of 20 % sodium carbonate. The resulting mixture was thoroughly shaken, made up

to 10 ml with distilled water and incubated at room temperature for 2 hours. The absorbance

at 760 nm was determined against a reagent blank. The experiment was performed in

triplicate and the total phenolic content expressed as gallic acid equivalents (GAE)

3.6.2.4 2, 2-Diphenyl-1-picryl-hydrazyl (DPPH) Radical Scavenging Activity

The DPPH radical scavenging activity (DRSA) of the plant extract(s) or standard (ascorbic

acid) was assayed with slight modification of the method of Uddin et al. (2008) which

depends on the production of yellow coloured diphenylhydrazine via reduction of purple

DPPH (maximum absorbance at 517 nm). Graded concentrations (25-100 µl ml-1

) of the

extract or vitamin C were prepared. A 2 ml aliquot of each test solution was then added to 0.5

ml of 1 mM DPPH solution in methanol and the mixture incubated in the dark for 15 minutes

at 37oC, after which the absorbance was measured at 517 nm against a blank containing

methanol and DPPH. This was performed in triplicate. The DPPH radical scavenging activity

was calculated using the equation:

% DRSA = 1- (As /Ab) x100

Where As and Ab are absorbance of the sample and blank respectively. A decrease in

absorbance indicates an increase in DPPH radical scavenging activity.

56

3.6.2.5 DNA Protection Assay

Antioxidant / pro-oxidant activity of crude plant extracts assessed in aqueous system via the

method of hydroxyl radical-induced DNA damage in plasmid pBR322 as described

previously by Russo et al. (2001) with slight modification. To test for DNA damage induced

by hydroxyl radicals, the reaction was conducted in an Eppendorf tube at a total volume of 12

µl containing 0.5 µg pBR322 DNA in 3 µl of 50 mM phosphate buffer (pH 7.4), 3 µl of 2

mM FeSO4 and 2 µl of the hydromethanolic leaf extract of S.alata or S.podocarpa at

concentrations of 10, 100 and 1000 μg ml-1

followed by the addition 4 μl of 30% H2O2. The

resulting mixture was incubated for 60 min at 37 °C. The DNA (super-coiled, linear and open

circular) was analyzed on 1% agarose gels and visualized using ethidium bromide staining.

3.7 In Vivo Studies

3.7.1 Toxicological Studies

3.7.1.1 Organ Toxicity

3.7.1.1.1Treatment of Animals

One hundred and twelve young albino rats (6 – 8 weeks old) were randomly distributed into

seven groups of 16 rats each. Group A which served as control did not receive any dose of

the extract but was administered 1 ml of distilled water daily. Groups B, C, D, received 1 ml

daily dose of 200, 600 and 1000 mg kg-1

body weight extract of Senna alata while groups E,

F, G, received 1 ml daily dose of 200, 600 and 1000 mg kg-1

body weight extract of Senna

podocarpa respectively, for 15, 35 or 63 days. The animals were deprived of food but not

water (8-14 hours) prior to administration of extract. During the period of administration, the

animals were weighed, food and water intake were monitored daily. After 15, 35 or 63 days,

all surviving animals were fasted overnight. Animals were sacrificed after anesthesia

57

(chloroform) by decapitation and blood from each animal was collected separately in labelled

heparinised / EDTA tubes. Selected organs namely the brain, heart, kidney, liver, lungs,

spleen, testes and cauda epididymes were harvested and washed instantly in normal saline.

The washed organs were blotted dry with filter paper, weighed and labelled separately. About

0.2 g of the organs were cut with surgical scissors, kept separately in labelled containers and

frozen (-200C) for further analysis of biochemical parameters. The left over organs were

immediately preserved in 1 % formal saline for histopathological studies.

3.7.1.2 Haematological Parameters

Blood collected in anticoagulant (EDTA) tubes were used to determine populations of white

blood cells (WBC) and red blood cells (RBC) according to Dacie and Lewis, 1991. Packed

cell volume (PCV) was also evaluated.

3.7.1.2.1 Determination of packed cell volume (PCV)/ Haematocrit

PCV was determined by the microhaematocrit method. Haematocirt is the volume of red

blood cells expressed as a percentage of the volume of whole blood in the sample. Lithium

heparinised capillary tube coated with anticoagulant were filled with blood obtained from

EDTA bottles. One end of the capillary tube was sealed with plastercine, and centrifuged for

5 mins at 13000 rpm in a high speed haematocrit centrifuge. The packed cell height of the

sample expressed in percentage (%) was determined using a PCV chart.

3.7.1.2.2 Determination of white blood cell count (WBC)

A white blood cell count estimates the total number of white blood cells in 1 mm3 of blood. A

20 µl aliquot of blood sample was mixed with 380 µl of Turk‟s fluid and the mixture was put

in a Neubauer counting chamber which was subsequently viewed under low power objective

58

(X10) of a light microscope. The four larger corner squares containing uniformly distributed

WBCs were observed and the cells therein counted.

3.7.1.2.3 Determination of differential white blood cell count

A differential white blood cell count provides information on the different types of white

blood cells present in the circulating blood which include neutrophils, lymphocytes,

monocytes, basophils (rarely seen) and eosinophils. Blood from EDTA tube was used to

prepare a thin blood smear on a clean grease free labelled microscope glass slide. The smear

was air dried, fixed with absolute methanol and stained with Leishman stain. The stained

slide was allowed to dry and then examined under the light microscope using the oil

immersion objective.

3.7.1.3 Biochemical Assay

Blood collected into anticoagulant (lithium heparin) tubes were centrifuged at 3000 rpm for

10 minutes at room temperature. The plasma samples were obtained and stored at -40C in

labeled sample bottles until required. The resulting plasma and 10 % liver homogenate was

analyzed to determine the activity of some marker enzymes. Alanine aminotransferase

(ALT), Aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were analyzed by

the method of Obidike et al., 2011 and total protein using the Biuret method. The catalase

and thiobarbituric acid reactives substances (TBARS) levels, (a measure of antioxidant

status) of plasma as well as organ homogenates were also determined according to the

method of Niehaus and Sammuelson, (1968). The 10 % homogenate of selected organs were

also prepared by macerating 1 g of each organ with 9 ml of phosphate buffered saline. The

mixture was centrifuged at 5000 rpm for 5 minutes and the supernatant transferred into

prelabeled Eppendorf tubes.

59

3.7.1.3.1 Determination of biochemical Parameters

3.7.1.3.1.1 Determination of albumin

A 100 µl aliquot of water (blank), plasma or standard (albumin) (100 mg dl-1

) was mixed

with 2500 µl of buffered bromocresol green and incubated for 10 mins at room temperature

after which the absorbance was read against a blank.

Albumin concentration = (OD sample / OD standard) x 100 mg dl-1

3.7.1.3.1.2 Determination of total protein (TP)

Proteins react with cupric ions in alkaline solution to form a purple complex. A 20µl aliquot

of plasma sample, water (blank) or standard (80 g l-1

protein and 0.095 % sodium azide) was

mixed with 1000 µl aliquot of working solution [sodium hydroxide (200 µl), potassium

sodium tartarate (32 mmol l-1

), cupper sulphate and potassium iodide (30 mmol l-1

)],

incubated for 10 mins at room temperature then transferred into cuvettes and the absorbance

of the samples and standard read against the reagent blank within 30mins at 546 nm.

The total protein concentration (g l-1

) = 80 x (OD sample / OD stamdard)

3.7.1.3.2 Determination of enzyme activities

3.7.1.3.2.1 Determination of alanine aminotransferase (ALT)

Alanine aminotransferase catalyse the production of pyruvate from α-ketoglutarate and L-

alanine. Pyruvate, a measure of ALT activity, in the presence of 2,4-dinitrophenylhydrazone

forms pyruvate-hydrazone complex.

A 100 µl aliquot of water/plasma samples was mixed with 500 µl aliquot of working solution

[100 mmol l-1

phosphate buffer (pH 7.4), L-alanine (200 mmol l-1

and α-ketoglutarate (2.0

60

mmol l-1

)] and incubated for exactly 30 mins at 37 oC, then 500 µl of 2,4-

dinitrophenylhydrazine (2 mmol l-1

) was 3added. The resulting solution was further incubated

for 20 mins at room temperature, after which 5000 µl of sodium hydroxide (0.4 mmol l-1

) was

added and the absorbance read against the blank at 546 nm in a spectrophotometer.

3.7.1.3.2.2 Determination of aspartate aminotransferase (AST)

Aspartate aminotransferase catalyse the production of oxaloacetate from α-oxoglutarate and

L-aspartate. oxaloacetate a measure of AST activity, in the presence of 2,4-

dinitrophenylhydrazone forms pyruvate-hydrazone complex.

A 100 µl aliquot of water (blank) or plasma sample was mixed with 500 µl aliquot of

working solution [100 mmol l-1

phosphate buffer (pH 7.4), L-aspartate (200 mmol l-1

) and α-

oxoglutarate (2.0 mmol l-1

)] and incubated for exactly 30 mins at 37 oC, then 500 µl of 2,4-

dinitrophenylhydrazine (2 mmol l-1

) was added. The resulting solution was further incubated

for 20 mins at room temperature, after which 5000 µl of sodium hydroxide (0.4 mmol l-1

) was

added and the absorbance read against a the blank at 546 nm in a spectrophotometer.

3.7.1.3.2.3 Determination of alkaline phosphatase (ALP)

A 500 µl aliquot of plasma sample was mixed rapidly with 3000 µl aliquot of working

reagent (p-nitrophenylphosphate (10 mmol l-1

), diethanolamine buffer (1 mol l-1

, pH. 9.8) and

0.5 mm l-1

magnesium chloride) in a cuvette and the absorbance read at 1 min interval for 3

mins at 405 nm, in a spectrophotometer.

61

3.7.2 Antioxidant studies

3.7.2.1 Determination of Catalase (CAT) activity

Catalase activity was assayed colorimetrically at 620 nm and expressed a µmoles of hydrogen

peroxide consumed per min or hydrogen peroxide consumed per min per mg of protein as

described by Sinha, 1972. The reaction mixture (1.5 ml) contained 1ml 0.01 M phosphate

buffer (pH 7.0), 100 µl of sample and 400 µl of H2O2.

Catalase enzyme activity = (ΔOD/min/E) x (V/v) x 103

Where : ΔOD/min = change in absorbance/min

E = Molar concentration co-efficient (40)

V = Total volume of reacting sample

v = Volume of sample

3.7.2.2 Determination of lipid peroxidation

Lipid peroxidation resulting in the formation of thiobarbituric acid reactive substances

(TBARS) was measured by the method of Rael et al. (2004). A 100 µl aliquot of plasma was

mixed with 2000 µl working reagent (equal volumes of thiobarbituric acid (0.37 %),

trichloroacetic acid (15 %) and 0.25 M HCl). The mixture was boiled for 15 mins in a water

bath, cooled, centrifuged at 1000 rpm for 10 mins at room temperature and the absorbance of

the clear supernatant was measured against a blank at 535 nm in a spectrophotometer.

The TBARS level expressed as malonyldialdehyde (MDA) level (nmol ml-1

)

= (OD/E) x (V/v) x 106

Where : OD = change in absorbance/min

E = Molar concentration co-efficient (40)

V = Total volume of reacting sample

62

v = Volume of sample

3.7.3 Electrophoresis of Red Blood Cell (RBC) Membrane Proteins

Albino rat erythrocyte plasma membranes (ghosts) were prepared using a modification of the

procedure of Johanning and O'dell (1989). Blood obtained from rats dosed daily for 63 days

with the plants extract was collected into EDTA bottles and centrifuged at 2500 rpm for 15

minutes at 4oC. The plasma and buffy coat were then carefully removed using a Pasteur

pipette. Erythrocytes were then washed 3 times in cold 5 mM sodium phosphate / 0.15 M

sodium chloride and centrifuged as before. Careful aspiration was performed after each wash,

sacrificing packed cells at the interphase to ensure removal of the buffy coat. Packed cells (1

mL) were then lysed in 20 mL of cold 0.01% saponin in 5 mM sodium phosphate, pH 8.0.

The mixture was incubated at 4oC for 10 minutes and centrifuged at 16,000 rpm for 20

minutes at 4oC. The resultant deep red supernatant lysates (enriched cytostolic content) were

discarded, leaving a red pellet of packed ghost over a creamy white layer of leukocyte debris.

The ghost was carefully separated from this protease rich layer, by tilting and rotating the

tube to free the ghost and then aspirating it into a new tube containing ice cold 5 mM sodium

phosphate, pH 8.0 and washed severally by centrifuge until an opaque pellet was obtained.

The protein content of the resulting pellet was assayed using the Biurets method and final

pellet was solubilised with 2 volumes of SDS-PAGE sample loading buffer and boiled for 5

minutes. The membrane protein profile was analysed by SDS-polyacrylamide (10 %) gel

electrophoresis under denaturating conditions, using the discontinuous buffer system of

Laemmli (1970). Equal concentrations of protein were loaded per track on each gel.

63

3.8.1 Genotoxicity Studies

3.8.1.1 Genotoxicological Investigation of Extract Using an Animal (Albino Rat) Model

3.8.1.1.1 Sperm-Head Morphology Assay

Male albino rats (35) were divided into seven experimental groups comprising of 5 animals

per group and one group served as control. The control group received by gavage, 1 ml of

water daily for 63 days, while the experimental groups received also by gavage, 1 ml of sub

chronic doses of 200, 600 and 1000 mg kg-1

body weight, corresponding to low, medium and

high doses of each plant extract. After 63 days from the first exposure, the animals were

sacrificed under anaesthesia, and the cauda epididymis removed and 1mg macerated in

prelabelled tubes containing 5 mls of normal saline. A fraction of each suspension was mixed

with 1% eosin Y solution and a smear made on a clean glass slide. The air dried smear slides

were coded (to avoid bias) and scored for the different sperm head morphologies.

3.8.2 CELL LINE STUDIES

The cytotoxic effect of the extracts on the human erythroid-like K562 cell line was

determined using WST-1 [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-

tetrazolium] cell viability and proliferation assay kit (ScienCell), neutral red uptake assay

(Creppy et al., 2004) and LDH leakage assay (Upur et al., 2008).

3.8.2.1 Cell Culture

Human chronic myelogenous leukemia K562 cells purchased from Cell Line Services

(Germany), were cultured and maintained in a Roswell Park Memorial Institute (RPMI) 1640

medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Sigma

Aldrich). Cells were grown at 37o C in a humidified atmosphere of 5% CO2/air.

64

3.8.2.2 Cell viability Assay (WST-1)

The hydromethanolic leaf extract of Senna alata and Senna podocarpa were tested for in

vitro cytotoxicity, using K562 cells. Cytotoxicity was measured by the reduction of

tetrazolim salt - WST-1 [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-

tetrazolium] - to colored formazan compounds by succinate-tetrazolim reductase which exists

in viable cells. Briefly, 50 μl of media (RMPI 1640) was added to wells in the 96-well plates

from row A to row F (triplicate). Then, 100 μl of diluted plant extract (1mg/ml) was added to

the wells on the first column of rows A - F. Starting from the first column the 100 μl of

solution (50 μl + 50 μl media) was mixed and 50 μl transferred to wells on the second column

by using multichannel micro-pipettor and a serial dilution was done up to the tenth column.

Finally, excessive 50 μl from the tenth column were discarded. The final volume for each

well was 50 μl. The cultured K562 cells were resuspended in RPMI 1640 at 1.39 x 104 cells /

ml after verifying cell viability by a trypan blue dye exclusion assay. Then, 50µl aliquot of

the suspension was pipetted into each well, except the 12th

column wells, in the micro-titer

plate containing medium or extract (final volume 100 μl). Finally, 50 μl of medium was

added to the 12th

column wells as medium control, while column eleven wells served as cell

control. Each sample was replicated 3 times and the cells incubated at 37°C in a humidified

5% CO2 incubator for 24 h. After the incubation period, WST-1 reagent (10 μl of 3.26

mg/ml) was added into each well, the plates carefully shaken and the cells incubated for 3h.

The absorbance values of each well at OD450 – OD630 were read using an ELISA Reader

(Molecular Device, Menlo Park, USA). The IC50 values were determined by plotting the drug

concentration versus the survival ratio of the treated cells. Assays were performed at least

three times, and data shown are representative of those assays.

% Cytotoxicity = 1- [(Absorbance of test / Absorbance of control) x 100]

65

A dose-response curve was plotted to enable the calculation of the concentration that killed

50% (CC50) of the K562 cells.

3.8.2.3 Cell Viability (Neutral Red Uptake Assay)

Cells were seeded in 24-well microplates (x104 cells

ml

-1), routinely cultured in a humidified

incubator for 24 h. Cells were maintained in culture and exposed to Senna alata or Senna

podocarpa aqueous methanolic leaf extract as well as nicosan – a herbal drug, over a range

of concentrations (0.0 – 100.0 µg ml–1

). After 24 h exposure to the test sustance, neutral red

(NR) uptake test was performed according to the procedure described by Upur et al. (2008).

Briefly, at the end of the treatment, the medium with or without test substance was discarded

and 200 µl of freshly prepared neutral red solution (50 µg ml

–1) was added to every well

and

re-incubated for an additional 4 h at 37°C. Thereafter, the cells were carefully washed twice

with 200 µl of PBS to eliminate extracellular neutral red. The incorporated dye was eluted

from the cells by adding 200 µl elution medium (50% ethanol supplemented with 1% acetic

acid, v/v) into each well followed by gentle shaking of microplate for 15 min. The plates were

then read at 540 nm using a microplate reader (Molecular Device, Menlo Park, USA). The

percentage of viable cells was calculated and the IC50 was determined and expressed as

microgram per milliliter.

3.8.2.4 Cell Death (Lactate Dehydrogenase Leakage Assay)

Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme, present in most eukaryotic cells,

which is released into the culture medium upon cell death due to plasma membrane damage.

K562 cells (5 x 104 cells ml

–1well

–1) were pre-incubated

in 24-well plates for 24 hours at 5%

CO2–95% air 37°C. Cell viability was assessed by lactate dehydrogenase (LDH) leakage

through the membrane into the medium. Aliquots (150µl) of the cell culture supernatants

66

from control and serial concentration of extract treated cultures were tested after 6 or 7 days

incubation for the presence of LDH using a LDH assay kit (ScienCell).

In this test, three wells

were used for each concentration of

extract, and three independent experiments were

performed.

3.9 DATA ANALYSIS

Statistical Analysis: Data was analyzed using one-way analysis of variance (ANOVA)

followed by Tukey Post Hoc Test, using SPSS 16.0 computer software package (SPSS Inc;

Chicago, U.S.A). Differences at P<0.05 were considered significant.

67

4.0 RESULTS

4.1 PRELIMINARY SCREENING

Five plants namely Cajanus cajan (beans and leaves), Smilax kraussiana (leaves), Alchornea

cordifolia (leaves), Senna alata (leaves), Senna podocarpa (leaves) and Jana, a herbal

formulation were screened for their membrane stabilizing activity, using the osmotic fragility

test.

4.1.1 MEMBRANE STABILISING EFFECT OF AQUEOUS ETHANOLIC

EXTRACT

Membrane stabilization was conferred on HbSS RBC by the hydroethanolic extract of five

medicinal plants namely Cajanus cajan seed (38.34 – 40.18%)(Fig.2) and leaf extract of

(Fig.3) Smilax kraussiana (18.97 – 45.52%), (Fig.4) Alchornea cordifolia ( -67.63 –

62.85%), (Fig.5) Senna alata (34.63 – 44.17%), (Fig.6) Senna podocarpa (34.25 – 67.12%),

(Fig.7) Cajanus cajan (31.85 - 57.53%), as well as (Fig.8) Jana-herbal formula ( 64.34 -

77.26%). Optimal membrane stabilizing concentration of Senna podocarpa and JANA was

1000 µg ml-1

, while 600 µg ml-1

was recorded for the other extracts. Two plants Senna alata

and Senna podocarpa were thus selected for further investigation, since there is no

documented report on these plants in relation to SCA management.

68

69

70

71

72

73

74

75

4.1.2 EXTRACTION YIELD AND PHYTOCHEMICAL COMPOSITION

For S. alata, aqueous methanolic extract gave the highest yield, followed by water extract and

lastly aqueous ethanolic extract (Table 1). On the other hand, S. podocarpa water extract

gave the highest yield, followed by aqueous methanol and then aqueous ethanol.

Senna alata contained keto-sugar, while alkaloid was detected in S.podocarpa and both S.

alata as well as S. podocarpa contained deoxy-sugar (Table 2a). Presence of tanins, saponins,

flavonoids, steroids, anthraquinones and cardiac glycosides were confirmed (Table 2b) in S.

alata and S. podocarpa, while phlobatanin was found only in S. podocarpa

76

Table 1. Percentage yield of plant material(s) after extraction with different solvents

% Yield

Ethanol (80%) Methanol (80%) Water

Alchornea cordifolia

(Leaves) 26.51 ND ND

Cajanus cajan (seeds) 20.32 ND ND

Cajanus cajan (leaves) 22.01 ND ND

Senna alata (leaves) 23.17 27.67 24.03

Senna podocarpa (leaves) 5.5 9.81 13.95

Smilax kraussiana (leaves) 11.9 ND ND

77

Table 2a Phytochemical screening of ethanolic (80%) extracts

TEST AC CCB CCL SA SK SP

Fehlings soln + - + + + -

Fehlings +H2SO4 + Heat + _ + + + -

Dragendoff - - - - - +

Meyers - - - - - -

Barfoed’s (Monosacchs) + - + - - -

Resorcinol (Keto-sugar) ++ + ++ + + -

Phlorogucinol (Pentose) - - - - - -

Keller-Killanni (Deoxy-sugar) + +++ + + + +

Key: - Negative; + positive; ++ more positive; +++ most positive

AC – Alchornea cordifolia leaves;CCB – Cajanus cajan beans;CCL – Cajanus

cajan leaves; SA – Senna alata leaves; SK – Smilax kraussiana leaves; SP – Senna

podocarpa leaves

Table 2b Phytochemical screening of methanolic (80%) extracts

TEST S. alata S. podocarpa

TANIN + +

SAPONIN + +

FLAVONOID + +

PHLOBATANIN - +

STEROIDS + +

TERPENOID - -

ANTHRAQUINONE + +

CARDIAC GLYCOSIDE + +

Key: - Negative; + positive

78

4.1.3 PROXIMATE COMPOSITION

The chemical compositions (Table 3) of the two plants showed no significant difference in

their ash or moisture content. S. alata had significantly higher lipid, protein and crude fibre

contents while the carbohydrate and energy contents of S. podocarpa were significantly

higher.

79

Table 3 Proximate composition of aqueous-methanolic leaf extract of Senna alata and

Senna podocarpa

Parameter S.alata S.podocarpa

%Moisture 5.67±0.14 6.20±0.15

%Lipids 11.2±0.18ᵃ 3.35±0.10

%Protein 5.28±0.09ᵃ 1.97±0.06

%Crude fibre 33.33±0.18ᵃ 20.72±0.59

%Ash 6.61±0.20 6.78±0.16

%Carbohydrate 43.58±0.81 67.18±0.13ᵃ

Energy (Kcal) 295.94± 0.62 306.19±0.97ᵃ

Values (g/100gwt) expressed as percentage mean ± SEM; n=3; superscript (a) shows

significant difference (P<0.05) between means on the same row.

80

4.2 IN VITRO STUDIES

4.2.1 MEMBRANE STABILISING EFFECT OF AQUEOUS METHANOLIC

EXTRACT

The mean corpuscular fragility values of S.alata and S.podocarpa hydromethanolic leaf

extract treated HbSS RBC were significantly (P < 0.05)lower than the untreated HbSS RBC.

The hydromethanolic leaf extract of S.alata (Fig.9) exhibited a membrane stabilizing effect

(44.44 – 66.01%) with a 200 µg ml-1

optimal concentration, whereas hydromethanolic leaf

extract of S.podocarpa (Fig.10) showed a much lower level of membrane stabilization (5.72 –

17.71%) with 800 µg ml-1

supplementation conferring optimal stabilizing effect.

81

82

83

4.2.2 EFFECT OF EXTRACT ON HEMOGLOBIN POLYMERIZATION

The test substances, S.podocarpa, S.alata, parahydroxybenzoic acid and Nicosan, had

significantly higher antisickling activity when compared to the untreated control at all

sampling times (Fig.11). After 120 minutes of incubation, Nicosan exhibited a significantly

(P < 0.05) higher antisickling activity than S.podocarpa as well as parahydroxybenzoic acid,

while after 150 minutes incubation time the antisickling activity of S.podocarpa was

significantly lower than that of S.alata, parahydroxybenzoic acid and Nicosan in that order

(Fig.12).

Pre incubation of HBSS RBCs with S.alata for up to 90 minutes did not significantly alter the

antisickling profile after 175 minutes. Optimal pre incubation time of 30 minutes prior to

deoxygenation conferred an antisickling effect that lasted for 210 minutes (Fig.13). Pre

incubation of HBSS RBCs for 150 minutes with S.podocarpa prior to deoxygenation,

conferred an antisickling effect that lasted for 210 minutes, while no significant (P > 0.05)

changes in antisickling profile was observed for 30, 60, 120 and 180 minutes pre incubation

time (Fig.14).

84

s.podoca

rpa

S.alata

Nicosa

n

PABA

Buffer

ed saline

-300

-200

-100

0

100

Fig.11 Antisickling efficiency of Senna alata, Senna podocarpa, nicosan,

parahydroxybenzoic acid and and buffered saline. Suspension (10% hematocrit) of HbSS

erythrocytes were incubated with test solution (2.5 mg ml-1

) for three hours at 37oC,

aliquots were then withdrawn, deoxygenated with 2% sodium metabisulphite and the

percentage of sickle cells determined. (PABA- Parahydroxybenzoic acid)

TREATMENT GROUP

An

tisic

kli

ng

acti

vit

y (

%)

85

30 60 90 120

150

180

-300

-200

-100

0

100

s.podocarpa

S.alata

PABA

Nicosan

Buffered saline

Fig.12 Antisickling potencies of Senna alata, Senna podocarpa, nicosan,

parahydroxybenzoic acid and and buffered saline. Suspension (10% hematocrit) of HbSS

erythrocytes were incubated with test solution (2.5 mg ml-1

) at 37oC, then aliquots were

withdrawn at various times, deoxygenated with 2% sodium metabisulphite and the

percentage of sickle cells determined. (PABA- Parahydroxybenzoic acid)

An

tisic

kli

ng

Acti

vit

y(%

)

86

-150

-100

-50

0

50

0mins

30mins

60mins

90mins

150mins

180mins

120mins

Time (mins)

Fig.13 Antisickling potency of Senna alata. Suspension (10% hematocrit) of HbSS

erythrocytes was incubated with S.alata (2.5 mg ml-1

) at 37oC, then aliquots were

withdrawn every thirty minutes, deoxygenated with 2% sodium metabisulphite at 37oC, and

the percentage of sickle cells determined at thirty-five (35) minutes interval .

An

tisic

kli

ng

Axti

vit

y(%

)

87

-100

-50

0

50

100

0mins

30mins

60mins

90mins

150mins

180mins

120mins

Fig.14 Antisickling potency of Senna podocarpa. Suspension (10% hematocrit) of

HbSS erythrocytes was incubated with S.podocarpa (2.5 mg ml-1

) at 37oC, then

aliquots were withdrawn every thirty minutes, deoxygenated with 2% sodium

metabisulphite at 37oC, and the percentage of sickle cells determined at thirty-five

(35) minutes interval .

Time (mins)

An

tisic

kli

ng

Acti

vit

y (

%)

88

4.2.3 EFFECT OF EXTRACT ON HEMOGLOBIN DEPOLYMERIZATION

A contact time dependent significant (P < 0.01) increase in reversal activity by Senna

podocarpa (2.5 mg ml-1

) was observed (Fig. 15) after 60 mins, though the level of reversal

activity was extremely significantly (P < 0.001) lower when compared with the positive

control parahydroxybenzoic acid (PABA) (2.5 mg ml-1

) and Senna alata (2.5 mg ml-1

). S.

alata showed a highly significantly (P < 0.001) increased contact time dependent reversal

activity when compared to with normal saline, S. podocarpa and PABA

89

30

60

90

120

-50

0

50

100

Senna alata

PABA

NS

senna podocarpa

Fig. 15 Effect of Senna alata (SA), Senna podocarpa (SP), parahydroxybenzoic acid (PABA) and and buffered saline (NS)

on presickled RBC. Suspension (35% hematocrit) of HbSS erythrocytes was layered with liquid parrafin and

incubated with 2% sodium metabisulphite for 21/2

hours at 37oC. After incubation, the test solution was carefully

introduced into the solution under oil, aliquots were then withdrawn at 30 minute intervals, and the percentage of

sickle cells determined. Data expressed as mean sem; ANOVA and Tukey Post Hoc test; *** significantly different

from control (buffered saline) P 0.001

***

***

***

***

***

***

***

***

***

***

***

Time (mins)

Reversal A

ctivity(%

)

90

4.2.4 ANTIOXIDANT POTENTIAL OF EXTRACTS

Table.4 shows that the total phenolic content of the extracts expressed as mg gallic acid

equivalent per gram dry weight of extract was highest in the aqueous-methanolic extract of

S.podocarpa (154±0.34) followed by aqueous-methanolic extract of S.alata (102.21±0.11),

then aqueous extract of S.podocarpa (79.28±0.06) and lastly aqueous extract of S.alata

(68.81±0.03). There were significant differences (p<0.05) amongst the four extracts in total

phenolic compounds content. Both S.podocarpa and S.alata aqueous-methanolic extract had

higher total phenolic content than their corresponding aqueous extracts.

The aqueous extracts (Fig.16) showed poor reducing power, but gallic acid, ascorbic acid,

aqueous-methanolic extracts of S.podocarpa and S.alata, exhibited EC50 (effective

concentration) values of 28.13, 38.02, 49.47 and 61.45 µg ml-1

respectively.

The DPPH radical scavenging activity of the extracts showed a dose-dependent response. The

aqueous-methanolic extract (Fig.17) of S. podocarpa and S.alata had similar IC50 (inhibitory

concentration) values of 64.56 µg ml-1

and 65.16 µg ml-1

respectively which were about five

times less potent than ascorbic acid (13.59 µg ml-1

).

91

Table 4. Total amount of phenolic compounds, free amino acids and total anti oxidant

capacity of aqueous and methanolic (80%) plant extract

Plant Total phenolic

(mgGAE/gplant extract) Free amino acids (mg/100g)

Total antioxidant capacity

(mgAAE/gplant extract)

Methanol

(80%) Aqueous

Methanol

(80%) Aqueous

Methanol

(80%) Aqueous

S.alata 102.21±0.11 68.81±0.03 192±0.12 542.92±0.63 855.58±0.44 298.78±0.38

S.podocarpa 154.85±0.34a 79.28±0.06a 418.31±0.99a 547.74±0.45a 958.19±0.59a 559.74±0.07a

a- significant difference (p<0.05) exist within column

92

93

94

4.2.5 DNA PROTECTION ASSAY

Aqueous-methanolic leaf extract of S.podocarpa exhibited a dose dependent DNA protection

activity, while aqueous-methanolic leaf extract of S.alata at 10 µg ml-1

and 1000 µg ml-1

exhibited pro-oxidant activity, but 100 µg ml-1

showed DNA protection activity (Fig. 18).

95

Fig.18 Effect of aqueous-methanolic leaf extract of S.alata / S.podocarpa on hydroxyl radical

induced DNA damage.

Lanes 1-pBR322; 2-plasmid + FeSO4; 3-plasmid + H2O2; 4-plasmid + FeSO4 + H2O2; 5-7 plasmid +

FeSO4 + H2O2+ S.alata (1000 µg ml-1, 100 µg ml-1, 10 µg ml-1 respectively); 8-10 plasmid + FeSO4 +

H2O2+ S.podocarpa (1000 µg ml-1, 100 µg ml-1, 10 µg ml-1 respectively)

Open Circular

linear

SuperCoil

ed

Direction

Of

Run

96

4.3 IN VIVO STUDIES

4.3.1 Sub acute toxicological effects of S.alata and S.podocarpa on biochemical

parameters of albino rats

Following the sub acute administration of S.alata for 15 days (Fig.19), the AST activity was

significantly (p < 0.05) reduced in the plasma at doses of 200- and 600 mg ml-1

, but at 1000

mg ml-1

dose, it showed a very significant (p < 0.01) reduction in AST activity. A

concomitant, though non significant (p > 0.05) reduction in ALT activity was also revealed.

A significant (p < 0.05) and very significant (p < 0.01) reduction in plasma urea level by 200-

and 600 mg ml-1

of S.alata respectively was observed with no significant (p > 0.05) changes

in the albumin level (Fig.20). S.podocarpa (Fig.21) on the other hand, did not significantly (p

> 0.05) alter the plasma AST and ALT activity at all doses tested, but reduced the urea level

very significantly (p < 0.01) at 1000 mg ml-1

dose over the control while the albumin level

was not significantly (p > 0.05) altered (Fig.22).

97

AST

ALT

0

100

200

300

400C0NTROL

SAM

SAH

SAL

Fig.19 Effect of hydromethanolic extract of Senna alata leaves on activity of selected enzymes in the

plasma of albino rat administered 1ml daily gavage dose of water (CONTROL), 200mg/kg.bwt(SAL),

600mg/kg.bwt(SAM) or 1000mg/kg.bwt(SAH) for 15 days. Data are mean SEM; n=5; ANOVA (P 0.05);

significantly different from control at *(P 0.05) **(P 0.01)

*

*

**

ENZYME

AC

TIV

IT

Y (U

/L

)

98

ALB

UREA

0

20

40

60

80

CONTROL

SAM

SAH

SAL

Fig.20 Effect of hydromethanolic extract of Senna alata leaves on the level of selected analytes in the

plasma of albino rat administered 1ml daily gavage dose of water (CONTROL), 200mg/kg.bwt(SAL),

600mg/kg.bwt(SAM) or 1000mg/kg.bwt(SAH) for 15 days. Data are mean SEM; n=5; ANOVA (P 0.05);

significantly different from control at *(P 0.05) or **(P 0.01)

**

*

ANALYTE

An

alyte level (g

/l);(m

g/d

l)

99

AST

ALT

0

100

200

300

400CONTROL

SPM

SPH

SPL

Fig.21 Effect of hydromethanolic extract of Senna podocarpaa leaves on activity of selected enzymes in

the plasma of albino rat administered 1ml daily gavage dose of water (CONTROL), 200mg/kg.bwt(SAL),

600mg/kg.bwt(SAM) or 1000mg/kg.bwt(SAH) for 15 days. Data are mean SEM; n=5; ANOVA (P 0.05).

ENZYME

AC

TIV

IT

Y (U

/L

)

100

ALB

UREA

0

20

40

60

80 CONTROL

SPM

SPH

SPL

Fig.22 Effect of hydromethanolic extract of Senna podocarpa leaves on plasma analyte level of albino

rat administered 1ml daily gavage dose of water(CONTROL), 200mg/kg.bwt(SAL),

600mg/kg.bwt(SAM) or 1000mg/kg.bwt(SAH) for 15 days. Data are mean SEM; n=5; ANOVA (P

0.05); Significantly different from control at **(P 0.01).

**

ANALYTE

An

alyte level (g

/l);(m

g/d

l)

101

4.3.2 Sub chronic toxicological effect S.alata and S.podocarpa on body and

organ weights of albino rats

S.alata at 200 mg kg.bwt-1

significantly (p < 0.05) increased weight of the animals (Fig.23).

The weight of liver relative to the body weight (Fig.24) was significantly increased by 600

mg kg.bwt-1

of S.alata, while no significant changes in the relative weights of other organs

was noticed when compared to the control. The high dose (1000 mg kg.bwt-1

) of S.alata

induced a very significant (p < 0.01) increase in the liver weight relative to brain weight

(Fig.25) when compared to the control.

Senna podocarpa (Fig.26) at 200 mg kg.bwt-1

dose significantly (p < 0.05) increased liver

weight and concomitantly decreased the kidney weight, with a highly significant (p < 0.01)

increase in the weight of the lungs and testes when compared with the control. The 600 mg

kg.bwt-1

dose however, induced a high significant (p < 0.01) increase and decrease in lungs

and testicular weights respectively. The liver and lungs weight was significantly (p < 0.05)

increased and decreased respectively by 1000 mg kg.bwt-1

dose of S.podocarpa when

compared with the control. The liver weight expressed as fraction of the brain weight (Fig.27)

was very highly significantly (p < 0.01) increased over the control in the 1000 mg kg.bwt-1

dose group.

102

CONTROL

SAL

SAM

SAH

SPL

SPM

SPH

0

10

20

30

40

50

*

Fig.23 Effect of Senna alata (SA)/Senna podocarpa (SP) hydro-methanolic leaf extract on

live weight of Albino rats administered 1ml daily gavage dose of 200mg/kg.bwt(L),

600mg/kg.bwt(M) or 1000mg/kg.bwt (H) for 35 days

Dose Group

% B

OD

Y W

EIG

HT

CH

AN

GE

103

BRAIN

HEART

KID

NEY

LIVER

LUNGS

SPLE

EN

TESTE

S

0

1

2

3

4

5CONTROL

SAL

SAM

Fig.24 Effect of Senna alata (SA-)hydro-methanolic leaf extract on live weight of Albino

rats administered 1ml daily gavage dose of 200mg/kg.bwt(L), 600mg/kg.bwt(M) or

1000mg/kg.bwt (H) for 35 days

*SAH

ORGAN

%B

OD

Y W

EIG

HT

104

HEART

KID

NEY

LIVER

LUNGS

SPLE

EN

TESTE

S

0

100

200

300

400

CONTROL

SAL

SAM**

SAH

Fig.25 Effect of Senna alata (SA-) hydro-methanolic leaf extract on live weight of Albino

rats administered 1ml daily gavage dose of 200mg/kg.bwt(L), 600mg/kg.bwt(M) or

1000mg/kg.bwt (H) for 35 days

ORGAN

% B

RA

IN W

EIG

HT

105

Bra

in

Hea

rt

Kid

neys

Liver

Lungs

Sple

en

Teste

s

0

1

2

3

4

5

CONTROLSPLSPMSPH

*

*** *

***

*

*****

Fig.26 Effect of Senna podocarpa (SP-) hydro-methanolic leaf extract on live weight

of Albino rats administered 1ml daily gavage dose of 200mg/kg.bwt(L),

600mg/kg.bwt(M) or 1000mg/kg.bwt (H) for 35 days

ORGAN

%B

OD

Y W

EIG

HT

106

Hea

rt

Kid

neys

Liver

Lungs

Sple

en

Teste

s0

100

200

300

400

500CONTROL

SPL

SPM***

****** SPH

Fig.27 Effect of Senna podocarpa (SP) hydro-methanolic leaf extract on live weight of

Albino rats administered 1ml daily gavage dose of 200mg/kg.bwt(L), 600mg/kg.bwt(M)

or 1000mg/kg.bwt (H) for 35 days

ORGAN

% B

RA

IN W

EIG

HT

107

At nine weeks, daily dosage of S.alata or S.podocarpa (Fig.28) increased with very high

significance (p < 0.001) the weight of the rats at all doses tested, except the 1000 mg kg.bwt-1

dose of S.alata when compared to the control. The liver weight compared to the control was

significantly (p < 0.05) reduced by 600- and 1000 mg kg.bwt-1

dose of S. alata, (Fig.29)

while the liver weight expressed as a fraction of the brain weight (Fig.30) was however

increased very significantly (p < 0.01) and very highly significantly (p < 0.001) at 200- and

1000 mg kg.bwt-1

dose respectively. S. podocarpa (Fig.31) did not significantly (p < 0.05)

induce weight change in the organs at all the tested doses when compared to the control. The

liver weight as a fraction of brain weight (Fig.32) was very significantly (p < 0.01) elevated

over the control by 200- and 600 mg kg.bwt-1

dose of S.podocarpa, while no significant

weight change was noticed in other organs.

108

0

20

40

60

80

100

CONTROLSALSAMSAH

SPMSPH

SPL

Fig.28 Effect of 63 day daily administration 1ml gavage dose of water(Control),

200mg/kg.bwt(-L), 600mg/kg.bwt(M) or 1000mg/kg.bwt(H) of S.alata(SA-) or S.podocarpa(SP-)

hydromethanolic leaf extract on weight change. Values represent mean sem; n=5; ANOVA.

***Significantly different from the control (p0.001)

***

***

***

***

***

Dose Group

% W

eig

ht

Ch

an

ge

109

Bra

in

Hea

rt

Kid

neys

Liver

Lungs

Spleen

Teste

s

0

1

2

3

4

5

CONTROL

SAL

SAM

SAH

***

Fig.29 Effect of 63 day daily administration 1ml gavage dose of water(Control),

200mg/kg.bwt(-L), 600mg/kg.bwt(M) or 1000mg/kg.bwt(H) of S.alata(SA-) hydromethanolic

leaf extract on organ weight. Values represent mean sem; n=5; ANOVA; Significantly different

from the control at *(p0.05) and **(p0.01)

ORGAN

%B

OD

Y W

EIG

HT

110

Hea

rt

Kid

neys

Liver

Lungs

Sple

en

Teste

s

0

100

200

300

400

500CONTROL

SAL

SAM

***

**

Fig.30 Effect of 63 day daily administration 1ml gavage dose of water(Control),

200mg/kg.bwt(-L), 600mg/kg.bwt(M) or 1000mg/kg.bwt(H) of S.alata(SA-) hydromethanolic

leaf extract on organ weight. Values represent mean sem; n=5; ANOVA; Significantly different

from the control at **(p0.01) and ***(p0.001)

SAH

ORGAN

% B

RA

IN W

EIG

HT

111

Bra

in

Hea

rt

Kid

neys

Liver

Lungs

Sple

en

Teste

s

0

1

2

3

4

5

CONTROL

SPL

SPM

SPH

Fig.31 Effect of 63 day daily administration 1ml gavage dose of water(Control),

200mg/kg.bwt(-L), 600mg/kg.bwt(M) or 1000mg/kg.bwt(H) of S.podocarpa(SP-)

hydromethanolic leaf extract on organ weight. Values represent mean sem; n=5; ANOVA (p

0.05)

ORGAN

%B

OD

Y W

EIG

HT

112

Hea

rt

Kid

neys

Liver

Lungs

Spleen

Teste

s0

100

200

300

400

500CONTROL

SPL

SPM

Fig.32 Effect of 63 day daily administration 1ml gavage dose of water(Control),200mg/kg.bwt(-L), 600mg/kg.bwt(-M) or 1000mg/kg.bwt(-H) of S.podocarpa(SP-)hydromethanolic leaf extract on organ weight. Values represent mean sem; n=5; ANOVA

(p 0.05)

***

****** SPH

ORGAN

% B

RA

IN W

EIG

HT

113

4.3.3 Sub chronic toxicological effect S.alata and S.podocarpa on hematologic

parameters of albino rats

There were no significant (p > 0.05) changes in WBC (total and differential) and platelets

populations (Table 5) between the control and the experimental (Senna alata or Senna

podocarpa) groups, and between the experimental groups. The PCV was however

significantly (p < 0.05) increased in the experimental groups when compared with the

control.

114

Table 5. Effect of nine weeks administration of hydromethanolic leaf extract of Senna

alata and Senna podocarpa on hematologic parameters of albino rats.

HEMATOLOGIC PARAMETER

CONTROL (Water)

200 mg kg.bwt-1 600 mg kg.bwt-1 1000 mg kg.bwt-1

S.alata S.podocarpa S.alata S.podocarpa S.alata S.podocarpa

Hematocrit (%)

31.6±0.68 41.00±1.03* 39.40±2.04* 42.17±0.95* 40.00±1.52* 40.40±1.69* 38.40±0.70*

WBC (x10³) (x103/mm3)

6.04±3.31 7.33±6.23 6.64±7.54 6.32±5.18 6.24±5.87 4.44±1.20 5.70±3.42

Platelets (x103/mm3)

40.20±3.10 44.67±1.41 41.40±1.66 42.17±0.91 34.80±3.37 44.00±1.38 38.20±3.41

Leucocytes (x103/mm3)

58.80±3.81 51.00±1.91 56.80±2.13 54.33±0.99 64.80±3.23 51.00±1.18 61.40±3.70

Monophils (x103/mm3)

0.40±0.4 2.00±0.00* 0.80±0.37 2.00±0.0* 0.00±0.00 1.80±0.37* 0.40±0.40

Eosophils (x103/mm3)

0.60±0.6 2.67±0.61 1.00±0.45 1.50±0.22 0.00±0.00 3.20±0.97* 0.00±0.00

Values are expressed mean ± SEM (n=5); P<0.05 level of significant using ANOVA

followed by tukey multiple comparism test. Asterisk (*) shows that the mean is significantly

different (P<0.05) when compared with the control on the same column.

115

4.3.4 Biochemical effects of S.alata and S.podocarpa on ALT, AST and ALP activity in

albino rats

Table 6 Shows that nine weeks orally administered daily gavage dose of aqueous methanolic

leaf extract of S.alata or S.podocarpa had no significant (P > 0.05) effect on the levels of

ALT and AST activities in the liver at all doses tested compared to the control group, but

200- and 600 mg kg.bwt-1

dose of S.alata caused a significant (P < 0.05) and very highly

significant (P < 0.001) reduction in total protein level respectively. Neither extract

significantly (P > 0.05) altered the activity of plasma ALT, but 1000 mg kg.bwt-1

dose of

S.alata decreased significantly (P < 0.05) the AST activity while ALP activity was

significantly (p < 0.05) increased when rats were treated with 600 mg kg.bwt-1

dose of

S.podocarpa versus the control. Also albumin level was increased, very significantly (P <

0.05) when 1000 mg kg.bwt-1

dose of S.podocarpa was given, while all other treatments were

not significantly (P > 0.05) different from the control.

116

Table 6. Effect of 63 days daily administration of hydromethanolic leaf extract of Senna

alata and Senna podocarpa on activity of some plasma enzymes of albino rats

Groups Dosage

(mg

kgˉ¹)

ALT

(U Lˉ¹)

AST

(U Lˉ¹)

ALP

(U Lˉ¹)

Control

24.18±1.05 19.16±0.79 63.29±10.42

Senna alata 200 27.73±1.67 13.61±2.64 73.29±10.00

600 32.24±7.20 16.41±1.56 78.20±16.78

1000 22.86±0.80 10.05±1.79* 76.18±10.79

Senna podocarpa 200 21.88±1.23 15.39±1.20 76.36±12.87

600 40.16±4.23 17.38±0.10 172.00±26.40*

1000 25.34±4.73 17.38±0.97 90.90±19.33

Values are expressed mean ± SEM (n=5); P<0.05 level of significant using ANOVA

followed by tukey multiple comparism test. Asterisk (*) shows that the mean is

significantly different (P<0.05) when compared to the control on the same column.

117

4.3.5 Antioxidant activity of S.alata and S.podocarpa in albino rats

S.alata and S.podocarpa (Table. 7) did not significantly alter plasma catalase activity at all

doses tested when compared to the control, but 200 mg kg.bwt-1

dose of S.alata reduced

significantly (P<0.05) the liver catalase activity. The level of liver lipid peroxidation was

reduced in a very highly significant (P<0.001) manner over the control, by all the tested doses

of S.alata, while S.podocarpa on the other hand had no significant (P < 0.05) effect on the

liver lipid peroxidation level. The level of lipid peroxidation in the plasma was significantly

(P<0.05) reduced over the control by 200 and 1000mg/kg.bwt of dose of S.alata, but

S.podocarpa at 200 and 600mg/kg.bwt caused a very high significant (P<0.001) reduction in

the level of lipid peroxidation in the plasma.

118

Table 7. Antioxidant effect of 63 days daily administration of hydromethanolic leaf

extract of Senna alata and Senna podocarpa on albino rats.

Groups Dosage

(mg kgˉ¹)

CATALASE TBARS

LIVER PLASMA LIVER PLASMA

CONTROL 0.34±0.04 0.084±0.05 11.35±6.26 5.48±4.68

Senna alata 200 0.07±0.10* 0.297±0.10 5.90±3.24* 2.03±1.08*

600 0.08±0.09 0.037±0.40 7.28±3.55* 3.51±1.24

1000 0.05±0.08 0.042±0.25 6.77±7.57* 2.06±1.04*

Senna

podocarpa

200 0.33±0.05 0.024±0.02 10.12±1.31 2.71±4.20*

600 0.40±0.04 0.012±0.00 10.15±0.86 2.25±1.04*

1000 0.17±0.04 0.098±0.04 10.55±1.04 4.52±1.98

Values are expressed mean ± SEM (n=5); P<0.05 level of significant using ANOVA

followed by tukey multiple comparism test. Asterisk (*) shows that the mean is

significantly different (P<0.05) when compared to the control on the same column.

119

4.3.6 Effect of plant extract on albino rat erythrocyte membrane protein profile

The protein profile (Fig. 33) of albino rat RBC of the control and S. alata experimental

groups did not show distinct differences in their banding pattern. No dose related difference

in the albino rat RBC protein profile (Fig. 34) existed between the control and S. podocarpa

experimental groups

120

121

122

4.3.7 Histopathology

Control liver (Plate 1) exhibited dilated central veins, moderate lymphocytes in portal tracts,

while the S. podocarpa group showed (Plate 2) congestion of sinus and hepatic veins with

moderate lymphocytic inflammation. Moderate lymphocytes in the portal tracts with

destruction of limiting plate were noticed (Plate 3) in S.alata group.

123

Plate 1. Control liver with normal parenchyma and dilated central vein (arrowed)

harbouring moderate lymphocytes. Section stained with haematoxylin - eosin (HE)

solution for light microscopy

X 400

124

Plate 2. Senna podocarpa liver Sinusoidal congestion of parenchyma with dialated

central vein (arrowed) and lymphocytic inflammation. Sextion stained with haematoxylin - eosin (HE) solution for light microscopy

X 400

125

Plate 3. senna alata liver moderately congested parenchyma with dialated

central vein (arrowed) habouring lymphocytes. Section stained with

haematoxylin - eosin (HE) solution for light microscopy

X 400

126

4.3.8 Genotoxic potential of plant extract on albino rats

There was no significant (p > 0.05) increase in the average weight of the testis and cauda

epididymis in the hydromethanolic extract fed albino rat groups (Table 8). In the 1000 mg

kg.bwt-1

S.podocarpa extract treated group, increase in testes weight was significant when

expressed as relative weight (organ weight/brain weight).

Figure 35 shows the frequency of occurrence of the different types of spermheads observed in

albino rats exposed to extracts of S.alata and S.podocarpa The mean percentage of normal

sperm ranged from 51% to 97% in all treatment groups, and not different significantly (P <

0.05) among S. alata treated group or between S. alata treated groups compared with control.

However, treatment with S. podocarpa significantly (P < 0.001) reduced the occurrence

(50.8% to 53.33%) of normal sperms in rats, while various morphological (Plate 4) sperm

abnormalities were recorded in the control and the S. alata / S. podocarpa treatment groups.

Significant increase (P < 0.001) in frequency of spermhead abnormalities including pinhead,

wide acrosome and amorphous head were observed in the S. podocarpa treated group, while

the short hook rat spermhead morphology has the highest occurrence (5.21%) in the 1000 mg

kg.bwt-1

S. podocarpa treated group.

127

Table 8 Effects of methanolic extract of Senna alata and Senna podocarpa on testis and

cauda epididymides of albino rats after 9 weeks exposure

TREATMENT

TESTIS CAUDA EPIDIDYMIS A

bso

lute

Weig

ht

%B

od

y w

eigh

t

% B

rain

wei

gh

t

Ab

solu

te W

eig

ht

% B

od

y w

eigh

t

% B

rain

wei

gh

t

CONTROL 0.99±0.07 0.73±0.04 66.78±4.37 0.16±0.02 0.12±0.02 11.09±1.40

Sen

na

ala

ta

200 mg

kg.bwtˉ¹ 1.10±0.04 0.73±0.04 75.91±3.10 0.21±0.02 0.14±0.02 14.35±1.38

600 mg

kg.bwtˉ¹ 1.02±0.02 0.65±0.04 68.89±3.40 0.15±0.01 0.10±0.01 10.43±0.65

1000 mg

kg.bwtˉ¹ 1.15±0.03 0.67±0.04 77.77±2..26 0.18±0.01 0.10±0.01 12.02±0.87

Sen

na

po

do

carp

a

200 mg

kg.bwtˉ¹ 1.07±0.02 0.72±0,02 77.47±5.05 0.17±0.01 0.12±0.01 12.51±1.28

600 mg

kg.bwtˉ¹ 1.02±0.05 0.60±0.03 73.99±2.80 0.20±0.02 0.12±0.00 14.29±1.21

1000 mg

kg.bwtˉ¹ 1.29±0.07 0.67±0.01 79.76

a ±2.24 0.18±0.02 0.09±0.01 11.11±1.12

aSignificantly different from the control (P<0.05)

128

Nor

mal

Sper

m H

ead

Short H

ook

Lon

g H

ook

Pinhea

d

Wid

e ac

roso

me

Am

orphou

s hea

d

0

50

100

150control

sal

sam

sah

spl

spm

sph

Fig.35 Frequency of occurrence of the different types of spermheads induced by 63 days exposure of

albino rats to Senna alata -200/kg.bwt (SAL), -600mg/kg.bwt (SAM), -1000mg/kg.bwt (SAH), Senna

podocarpa – 200mg/kg.bwt (SPL), 600mg/kg.bwt (SPM) or – 1000mg/kg.bwt (SPH). Values represent

mean SEM;ANOVA (P0.05)

SPERMHEAD MORPHOLOGY

FR

EQ

UE

NC

Y (

%)

129

130

4.4 Cytotoxic potential of plant extract on K562 cell line

Figure 36 shows the viability of K562 cells based on WST-1 assay following exposure to

varying concentrations of different extracts. At concentrations below 75 µg ml -1

all the

extracts sustained cell viability. Senna podocarpa, Nicosan and Senna alata had cell

cytotoxicity (CC50) values of 104.5 µg ml -1

, 115.3 µg ml -1

and 131.8 µg ml -1

respectively.

The neutral red uptake assay revealed that the cytotoxic effect of Senna podocarpa on K562

cells (Fig. 37) was not significantly different (p > 0.05) from that of Nicosan at all

concentrations tested, while the cytotoxicity of Senna alata was significantly (P < 0.05) lower

than that of Senna podocarpa and Nicosan at concentrations greater than 64 µg ml -1

. While

the lactate dehydrgenase leakage assay showed (Fig. 38) that, of all the tested concentrations

of the different extracts, only 8.125 µg ml -1

of Senna alata showed significant (P < 0.05)

cytotoxicity.

131

100 200 300 400 500 600

-50

0

50

100Senna podocarpa

Nicosan

Senna alata

FIG. 36 Cytotoxic effect of aqueous methanolic leaf extract of Senna alata, Senna podocarpa and an herbal drug,

Nicosan, on K562 cells. K562 cells grown on 96-well microplates were incubated for 48 hours in RPMI 1640 mediumsupplemented with 1% fetal bovine serum to which was added varying concentrations of extract. Succinatedehydrogenase activity was determined using WST-1 assay kit. Data expressed as mean ± sem.

Concentration (µg ml-1

)

% C

ell C

yto

to

xicity

132

50 100 150

-400

-200

0

200Senna podocarpa

Nicosan

Senna alata

FIG. 37 Cytotoxic effect of aqueous methanolic leaf extract of Senna alata (SA), Sennapodocarpa (SP) and an herbal drug, Nicosan (NIC), on K562 cells. K562 cells grown on 24-wellmicroplates were incubated for 48 hours in RPMI 1640 medium supplemented with 1% fetalbovine serum to which was added varying concentrations of extract. The cells were washed andthen exposed to neutral red for 4 hrs and the quantity of lysosmally accumulated dye wasdetermined. Data expressed as mean ± sem; * significant difference exist between SA and SP ( p< 0.05).

Concentration (µg ml-1

)

Cell

Cyto

to

xic

ity (

%)

133

0

8.125

16.25

32.5 6

5130

-200

-150

-100

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

Senna podocarpa

Nicosan

Senna alata

FIG. 38 Cytotoxic effect of aqueous methanolic leaf extract of Senna alata, Senna podocarpaand an herbal drug, Nicosan, on K562 cells. K562 cells grown on 24-well microplates wereincubated for 48 hours in RPMI 1640medium supplemented with 1% fetal bovine serum towhich was added varying concentrations of extract. Lactate dehydrogenase levels insupernatants were then determined using an analysis kit. Data expressed as mean ± sem; *significant difference exist between SA and SP ( p < 0.05).

*

Concentration (µg ml-1

)

Cell C

yto

to

xicity (%

)

134

5.0 DISCUSSION

The low value of the ash content could be indicative of low mineral value especially the

macro minerals in the leaf of S. alata and S. podocarpa. The crude fat value for S.alata was

moderate, while that for S. podocarpa was high as compared to those of T. triangulare,

Amaranthus hybridus, Calchorus africanum (Akinhunsi and Salawu, 2005).

The increased left shift in osmotic fragilogram of erythrocytes supplemented with the

extracts, suggests conferment of membrane stabilization, with Jana-herbal formula having the

highest, followed by the hydroethanolic leaf extract of Senna podocarpa, Cajanus cajan,

Senna alata, Smilax kraussiana, Alchornea cordifolia, and Cajanus cajan seed in descending

order respectively. Optimal membrane stabilization was achieved by Senna podocarpa and

Jana at highest concentration tested. The fragilogram generated by Alchornea cordifolia was

erratic and showed membrane destabilizing action, while that of S.krussiana at the highest

concentration tested crenated the RBCs. The hydromethanolic leaf extract of S.alata appeared

to be a better membrane stabilizing agent than S.podocarpa, which may be indicative of

higher activity of its extractable secondary compounds composition. Plants are known to

contain compounds which facilitate the stabilization of red blood cell membranes (Smith et

al., 1983) hence the membrane stabilizing effects of these extracts may be due to the presence

of one or more naturally occurring compounds. The attenuating and potentiating effects of the

extracts on membrane stability needs further investigation with a view to understanding the

mechanism involved.

Both plant extracts inhibited the red blood cell sickling process. S.alata appears to be an early

inhibitor while S.podocarpa is a late inhibitor as indicated by the morphological

normalization of HbSS RBCs following treatment with the hydromethanolic extract. This

suggests some influence on the propensity of HbSS RBC to sickle. Although the mechanism

135

of action of has not been elucidated, HbSS erythrocyte membrane stabilization by the plants

extract may play a role in enhancing RBC rehydration rate which would increase the

solubility of deoxy HbS or the extracts may possibly possess protein binding affinity, which

could disrupt formation of tactoid or fibrilary structures due to deoxy HbS polymerization

inside the RBC, thus increasing the concentration of unpolymerised deoxy HbS in solution.

There were significant (P < 0.05) differences amongst the four extracts in total phenolic

compounds content. Both S.podocarpa and S.alata hydromethanolic extract had higher total

phenolic content than their corresponding aqueous extracts. Plant polyphenolic compounds

play an important role in stabilizing lipid oxidation, including radical scavenging properties

and may thus act as primary antioxidant free radical terminators in sickle cell anemia which

can also be viewed as having oxidant etiology. Several studies have described the antioxidant

properties of medicinal plants, foods and beverages which are rich in phenolic compounds

(Krings and Berger, 2001; Wang et. al., 2008).

The aqueous extracts of S.podocarpa and S.alata had a significantly (P < 0.05) higher content

of free amino acids than their hydromethanolic extracts. These plants may serve as a reservoir

of available amino acid when consumed as teas.

The hydromethanolic extracts of S.podocarpa and S.alata showed significantly (P < 0.05)

higher antioxidant capacities than their respective aqueous extracts. The hydromethanolic

extract of S.podocarpa showed highest overall total antioxidant capacity, which may be

attributed to its higher phenolic content. The DPPH radical scavenging activity showed a

dose-dependent response. The hydromethanolic extract of S. podocarpa and S.alata had

similar IC50 values, which were about five times less potent than ascorbic acid. This may be

attributed to its redox properties which allow them act as a reducing agent, hydrogen donors,

136

singlet oxygen quenchers, metal chelating properties. On the other hand aqueous extract of

both plants showed very weak antioxidant activity.

The aqueous leaf extract of S.podocarpa and S.alata showed poor reducing power. Gallic

acid exhibited an EC50 (effective concentration) value < ascorbic acid < hydromethanolic leaf

extract of S.podocarpa. While the hydromethanolic leaf extract of S.alata had the highest

EC50 value. This might be due to the reducing power since a positive correlation existed

between total phenolic content (TPC) and total antioxidant capacity (TAC), which is in

accordance with previous studies (Zheng and Wang, 2001; Unver et al., 2009)

The antioxidative activity of the phenolic compounds may be attributed to their redox

properties which allow them act as reducing agents, hydrogen donors, singlet oxygen

quenchers, metal chelating agents (Huang et al., 2004) or inhibit various oxidizing enzymes

and regenerate endogenous alpha tocopherol in the lipid bilayer (Letelier et al., 2009).

According to Osman et al., (2004) reducing power has a direct positive correlation with

antioxidant properties. Little quantities of different active principles are present in these

extracts which could provoke multiple and synergestic antioxidant effects (Letelier et al.,

2009)

Different values of each antioxidant assay from the same sample are found in various

publications (Surrasmo et al., 2005; Okoro et al., 2010) which could be caused by variation

in geographical location, environmental conditions or the mechanism of action of the

compounds in the natural sample.

The increased DRSA and TAC values of hydromethanolic over aqueous extract could be due

to the solvent of extraction which may have selectively solubilised more phenolic compounds

as shown by the increase in phenolic content. The hydromethanolic extracts contain more

137

apolar compounds than the water extract since increase in the polarity of a compound causes

an increase of solubility of the compound in the apolar phases in which peroxidations occur.

The free radical scavenging property may be one of the mechanisms by which these plants

are effective in traditional medicine. Phenolic compounds may be responsible for antioxidant

properties of many plants (Uddin et al., 2008). Therefore hydromethanolic extract of

S.podocarpa and S. alata can be more effective antioxidants than their water extract.

The results of the DNA protection assay indicate that hydromethanolic leaf extract of

S.podocarpa (at all doses tested) and S.alata (at 100 µg ml-1

) protects DNA against hydroxyl

mediated damage generated by Fenton chemistry. The ability of the extract to protect DNA

can be attributed to its ability to scavenge hydroxyl radicals before

the radicals oxidize DNA,

thus suppressing the formation of linear DNA and induced a partial recovery of super coiled

DNA

Senna alata and Senna podocarpa are medicinal plants widely used in south west Nigeria to

treat various diseases. The application of these herbs in the management of sickle cell anemia

is being sought; hence the toxicity profile of these herbals was investigated. A subchronic (35

day) study of the hydromethanolic leaf extract of these plants at the tested doses did not

appear to retard growth or affect food consumption, indicating that feed intake and utilisation

of protein and other nutrients were not affected by the intake of the herbals.

Analysis of organ weight in toxicology studies is paramount for identification of potentially

harmful effects of chemicals (Sellers, 2007). Anthropometric results revealed a higher body

weights in the group treated for 63 day treatment of rats with S.alata (26.28-68.15% above

controls) and S.podocarpa (59.75-64.62% above controls). Liver weight relative to body

weight was statistically decreased in rats dosed with 600- and 1000 mg kg.bwt-1

of S.alata,

whereas liver weight relative to brain weight was statistically increased in rats dosed with

138

200- and 1000 mg kg.bwt-1

of S.alata. The liver weight effects may be considered test

substance related since according to Bailey et al., (2004) analysis of organ-to-body weight

ratios is predictive for evaluating liver toxicity. Other organ weights relative to body/brain

weight from the different treatment groups did not reveal any statistical differences except the

increase in liver weight relative to brain weight of the 200- and 600 mg kg.bwt-1

S.podocarpa

treated groups

The rats treated with S. alata and S. podocarpa showed no changes in their daily physical and

behavioural activities. The intake of the extract did not affect the functions of the bone

marrow as reflected by the values of WBC (total and differential) and platelets. They were

neither quantitatively nor qualitatively affected. However, the increase in the PCV values

may be taken to be a reflection of an increased RBC volume, indicative of an impairment of

the cellular dehydration pathway, mediated by K+-Cl

- cotransporter (KCC) or Ca

2+- activated

K+ channel (Gardos pathway) (Muzyamba and Gibson, 2003).

It is established that the liver plays a significant role in various metabolic processes, and its

xenobiotic function is vital. Therefore, emphasis was laid on the effect these extracts might

have on the function of the organ, 63 days consumption of either S.alata or S.podocarpa did

not alter its total protein level.

S.alata at 200- and 1000 mg kg.bwt-1

significantly and very significantly reduced plasma

AST activity respectively while 600 mg kg.bwt-1

of S.podocarpa increased plasma ALT

activity. The changes in aspartate (AST) and alanine (ALT) aminotransferase enzyme levels

found in the rats correlate with the relative organ weight changes, though the values were not

double that of control, which is one indicator of biological significance (Rhomberg et al.,

2007). Both enzymes are not liver specific but are also found in skeletal and cardiac muscle

(Thrall, 2004). These findings are therefore not considered toxicologically significant. There

139

were no significant changes in the WBC (total and differential) and platelets counts between

the control and the experimental groups. A significant increase in the RBC counts of the

experimental groups over the control suggests that the hydromethanolic leaf extracts of

S.alata and S.podocarpa may not be toxic as they do not affect leucopoiesis but affected the

circulating erythrocytes and possibly hematopoiesis as reflected by changes in packed cell

volumes (PCV) and eosinophils. The increased PCV may also be a reflection of the hydration

state of the RBCs.

Assessment of TBARS was used to measure plasma and tissue concentrations of

malondialdehyde (MDA), a decomposition product of oxidized lipids, thus serving as an

index of plasma and tissue lipid peroxidation. The increased lipid peroxidation level of the

brain of rats administered 35 day daily dosage of S.alata leaf extract, may be due to either

artifactual ex vivo oxidation (Janero, 1990) or that the extract or its product of metabolism

crossed the blood brain barrier, whereas the reduction in hepatic, renal as well as plasma

TBARS supports the former

Thiobarbituric acid reactive substances (TBARS), as a marker of lipid peroxidation, and

catalase a cellular antioxidant were also estimated in the plasma and liver, of rats after 63 day

administration of plant extract. There was a significant reduction in hepatic (S.alata dose

group) and plasma (both plant extracts) TBARS. Overall result of the TBARS indicated

potential of crude extracts to inhibit oxidation in lipid system. The findings of this study

revealed that the leaf extract of S.alata and S.podocarpa increased the antioxidant capacity of

blood and had an inhibitory effect on the basal level of liver lipid peroxidation, a major

consequence of free radical cell damage, which may alter intrinsic membrane properties.

Natural antioxidants, e.g. plants extract, could also protect erythrocyte membrane from lipid

peroxidation induced by oxo-heme oxidants, by terminating the chain reaction or reacting

140

with free radicals, particularly peroxy radicals, which are major propagators of the auto-

oxidation chain of fat. This lends scientific support to the therapeutic use of the plant leaves

in traditional medicine.

Senna alata and Senna podocarpa leaves are employed traditionally in the treatment of a

variety of disease (either singly or in conjunction with other plants) in southwestern Nigeria.

The sperm head morphological evaluation is considered to be essential for the assessment of

male germ cell toxicity by several regulatory bodies like the Organization for Economic

Cooperation and Development (Trivedi, 2010).

The mechanism by which Senna podocarpa leaf extract caused increased a significant

increase in sperm head abnormalities is not quite clear, but generally damage to the sperm

cell may have occurred via physiological, cytotoxic or genetic means.

Testicular germ cells carrying minor gene mutations are not eliminated but are manifested as

morphologically deformed sperm. It is also documented that certain substances are germ cell

mutagens, affecting gene loci in spermatogonial cells thereby increasing the percentage of

sperm abnormality (Letz, 1990). In mice, a rodent, sperm cell morphology is genetically

controlled by numerous autosomal and sex-linked genes (Krazanowska, 1986), thus, the

abnormal sperm population observed in the Senna podocarpa treated group in this study

maybe due to mutagenic effects of some constituents of these plants extract on specific gene

loci of germ cell chromosomes involved in the maintenance of normal sperm structure. And

since no significant testicular weight loss was recorded in this study, testicular germ cells

may not have been destroyed either due to membrane damage or macromolecular

degradation, although the increased incidence of abnormal sperms is of great concern.

141

Toxicological disturbance of male reproductive function can occur at many sites producing a

range of effects, some primary, and some secondary to the initial insult. Target cells include

testis, epididymis, mature sperm and hormonal regulatory system (Creasy, 2001). Senna

podocarpa exposure could have produced pituitary-hypothalamic or sex hormonal effects

which may have affected spermatogenesis (Bruce and Heddle, 1979; Letz, 1990).

Spermatogonia located within the basal compartment of the testis, is outside the blood-tubule

barrier, hence it is exposed to any toxicant entering the interstitial fluid. Genotoxicants that

gain access to the lumenal compartment (containing spermatocytes and spermatids) may

result in irreversible DNA damage which may manifest as head abnormalities.

The sensitivity of the assays used in determining cell cytotoxicity depends on their endpoints.

The results from the WST-1 assay, indicate that both Senna alata and Senna podocarpa

possessed some active principles which increased cell viability at concentrations lower than

100 µg ml¹, these constituents might have increased the activity of mitochondrial

dehydrogenase, induced the expression of growth stimulation or excerted mitogenic effect.

But since some plant extracts have been found to reduce MTT in the absence of cells

(Shoemaker et al., 2004), which could also have been the case with WST-1. The dose-

dependent cytotoxicity observed with higher concentrations of both plant extracts maybe due

to biologically active alkaloids (Ganapaty et al., 2010) present in Senna podocarpa or

antioxidants which can be oxidised in culture medium with deleterious effect on cells in vitro

(Long et al., 2000; Morita et al., 2003; Halliwell, 2008). The lysosome is regarded as the

garbage disposal unit of the cell and its functionality connotes cell survival. Lysosomal

activity was most severely affected by Senna podocarpa (8.13 µg mlˉ¹) followed by Senna

alata (16.25 µg mlˉ¹) and nicosan (65 µg mlˉ¹). The results from the neutral red uptake assay

revealed that Senna alata at concentrations greater than (16.25 µg mlˉ¹) increased lysosomal

142

activity, while the converse was the case for Senna podocarpa as concentrations greater than

(8.13 µg mlˉ¹) impaired lysosomal activity. K562 cell membrane perturbation leads to

increased extracellular lactate dehydrogenase activity which implies cell cytotoxicity. Senna

alata appeared to cause greater membrane perturbation at lower concentrations than senna

podocarpa probably due to the presence of higher saponin content. The observation in this

study maybe of therapeutic significance, since these plants are used in complementary and

alternative medicine (CAM).

CONCLUSION

The hydromethanolic leaf extracts of Senna alata and Senna podocarpa exhibited appreciable

anti-sickling as well as de-sickling effect. Both plants provide exogenous sources of

antioxidants (possibly in form of phenolic phytochemicals) with remarkable activity, which

may be beneficial if incorporated into supplements formulated for SCA patients, to lower

DNA, membrane and possibly protein oxidative damage. The use of these extracts may help

reduce anemia, with indications that the prolonged use at high concentrations may also

induce infertility due to reduced spermatozoa. Thus the extensive prolonged utilization of

these plants orally in traditional medicine is cautioned when fertility is an issue. The results

of the present research have paved the way to envisaging in vivo studies of these extracts as a

therapeutic agent in patients with SCD and may provide a rational explanation for their use in

managing SCD by Nigerian traditional practitioners.

143

SUMMARY OF FINDINGS

OBJECTIVES

FINDINGS

To evaluate the HbSS RBC membrane stabilising effect of S. alata and S. podocarpa and their infleunce on poly merisation/depolymerisation of mutant HbS

The result indicated a significant reduction in lysis of HbSS RBC under osmotic stress and inhibition/reversal of the sickling process by S. alata and S. podocarpa extracts

To determine the antioxi dant activity of S. alata and S. podocarpa

Hydromethanolic leaf extract of S. alata and S. podocarpa significantly increased the blood antioxidant capacity and had an inhibitory effect on the basal liver lipid peroxidation level

To evaluate the long term toxicological effect(s) of S. alata and S. podocarpa on albino rats

Long term oral administratiion of hydromethanolic leaf extract of S. alata and S. podocarpa appeared toxic

To determine the effect of S. alata and S. podocarpa on RBC membrane protein Profile of albino rats

Both extract did not alter albino rat erythrocyte membrane profile

To evaluate the cytotoxic effect of S. alata and S. Podocarpa on K562 cells

Both extract reduced K562 cell viabilty

144

CONTRIBUTION(S) TO KNOWLEDGE

1. This study has established the membrane stabilizing potential of hydromethanolic leaf

extract of Senna alata and senna podocarpa, via reduction in the median corpuscular fragility

2. Hydromethanolic leaf extract of Senna alata possess greater antisickling activity than that

of Senna podocarpa

3. Hydromethanolic leaf extract of S.alata and S.podocarpa increases the antioxidant capacity

of blood and also suppress the basal level of lipid peroxidation of liver.

4. Hydromethanolic leaf extract of Senna alata and Senna podocarpa functions as an

antianemic agent by increasing the hematocrit level.

145

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