University of Nigeria Research Publications

UTOH-NEDOSA, Anastasia U. Aut

hor

PG/M.Sc/03/34657

Title

Evaluation of the Toxic Effects of

Dihydroartemisinin on the Vital Organs of Wister Albino Rats.

Facu

lty Pharmaceutical Sciences

Dep

artm

ent

Pharmacology and Toxicology

Dat

e 2008

Sign

atur

e

EVALUATION OF THE TOXIC EFFECTS OF

DlHYDROARTEMlSlNlN ON THE VITAL ORGANS OF WISTAR

ALBINO RATS.

UTOH-NEDOSA, ANASTASIA UCHECHUKWU

PGlMSCl03134657

A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF

PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARMACEUTICAL

SCIENCES, UNIVERSITY OF NIGERIA NSUKKA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE

(M.SC) DEGREE OF T~:E'UNIVERSITY OF NIGERIA, NSUKKA.

CERTIFf CATICN

This work embodied in this project is an original research work done by

Utoh-Kedosa, Anastasia Uchechukwu (PGIMSCIOS134657) submitted to the

Department of Pharmacology and Toxicology of the University of Nigeria,

Nsukka.

We certify that this work has not been submitted in part or in whole for the

award of a degree or acertificate of this br any other University.

Prof. P.A. Akah Supervisor

Dr. C.O. Okoli Head of Department

Date

Date

DEDICATION

This research work is dedicated to my husband Dr. P.S. Medosa and my

children Kene, Amaka, Ogochi, Ikenna, Chinedu and Ebele.

ACKNOWLEDGEMENT

I wish to express my gratitude to my Supervisor, Professor P.A. Akah of

University Nigeria, Nsukka; Late Dr. Levi Mybojikwe, Dr. O.N. Adeyanju, Mr.

Theophilus Ojemudia, Dr. Chris Chika Ojemudia; Danjuma Daniel Gushi, and

Saleh Mohammed, all 'of National Veterinary Research Institute (N.V.R.1) Vom;

Dr. N.A. Onyekwelu, Mrs. F.N. Ojiegbu, Mr. A. Abubakar, Mrs. B. Illiyasu, Mr.

Bashir, Mr. L. Domfur and Mrs. P.M. Okam, all of the Nigerian Institute of

Trypanosome Research (N.1.T.R) Vom and Dr. P.S. Nedosa, Mrs. J.J. Ndor, Mr.

Goddy Njoku, Pharm. V. Onoja and Mr. Joe Longshak, all of University of Jos

and finally to Amaka and lkenna Nedoss without whom my work would not have

been completed.

TABLE OF CONTENTS

Title page - - -

Certification - -

Dedication - - -

Acknowledgement -

- Table of contents - -

List of tables-- -

List of figures - -

Abstract - -

CHAPTER ONE:

Introduction - - - - - -

Malaria as a world problem - - -

Malaria parasite resistance to chloroquine -

Uncomplicated and complicated malaria -

Dihydroartemisinin -- - - -

Toxicity of artemisinins -.- - - -

The aim of the study - - -

CHAPTER TWO: MATERIALS AND METHODS

2.0 Material and Methods- - - -

2.1 Reagents - - -

2,2 Methods - .- -

- - 2.2.1 brug treatment - - - - - - . - - 15

2.2.2 Effect of DHA On Rat Body Weight - - - - - -1 6

2.2.3 Estimation of the activities of serim enzymes

ALT, AST and ALP.- - - - - - - -1 6

2.2.4 Haematological investigation - - - - - - -1 8

2.2.5 Gross anatomical observations - - - - - - -20

2.2.6 Histopathological investigations - - - - - - -21

2.2.7 Results presentation - - - - - - - -22

CHAPTER THREE: RESULT

3.1 Effect of DHA on body weight of the albino rats - - - -23

3.2 Effect of DHA on serum ALT, AST and ALP enzyme activities.- -23

3.3 Haematological effects of dihydroartemisinin. - - - - -23

3.4 The results of the gross anatomical observations.- - - -24

3.5 The results of the histopathological investigations.- - - -24

CHAPTER FOUR: DISCUSSIONS AND CONCLUSIONS

Discussions and conclusions - - - - - - - -43

Final Conclusions- - - - - - - - - -47

LIST OF TABLES

Table 1:

Table 2:

Table 3:

Table 4:

Table 5:

Table 6:

Table 7:

Table 8:

. Tableg:

Table 10:

The Mean Weights And The Weight Gains Of The Treated And

Effect Of DHA On Serum AST Eazyme Activity In I .U/L - - - - - - -27

Effect Of DHA On Serum ALP Enzyme Activity In K.A. (1.U)- - - - 28

Effect Of DHA Treatment 9 n Packed Cell Volume (PCV) - - - - - 29

Effect Of DHA On Total White Blood Cell (WBC) Count In ~ m ~ .

Effect Of DHA On Percentage Neutrophil Count- - - - - - - - - - - -3 1

Effect Of DHA On Percentage Lymphocyte Count- - - - - - - - - - 32 Effect Of DHA On Percentage Eosinophil Count- - - - - - - - - - - -3 3

Effect Of DHA On Percentage Monocyte Count. - - - - -- - - - - - -34

vii

LIST OF FIGURES

Figure 1 (a) Gross anatomical display of the vital organs of a

control albino rat.- - - - - -

Figure I (b) Gross anatomical display of the vital organs of a

DHA - treated albino rat.- - d -

Figure 2 Photomicrographs of the liver of DHA - treated

and control albino rats.- - - - -

Figure 3: Photomicrographs of the heart of 3HA - treated

and control albino rats.- - - - -

Figure 4: Photomicrographs of the intestine of DHA -

treated and control albino rats.- - - -

Figure 5: Photomicrographs of the lungs uf DHA - treated

and control albino rats.- - - - -

Figure 6: Photomicrographs of the spleen of DHA -

treated and control albino rats.- - - - Figure 7: Photomicrographs of the kidney of DHA -

treated and control albino rats.- - - -,

ABSTRACT

This study is a toxicological evaluation of oral dihydroartemisinin (DHA) in Wistar

albino rats. 2HA was administered for 5 days at a dosage regimen of 2 mglkg

day I, 1 mg/Kg day 2 - 5.

The potential of DHA to produce toxic effects on the liv:.?r, heart, lungs, intestine,

spleen, kidney and blaod cells was investigated. The effect of dihydro-artemisinin

on enzyme activity was also evaluated. The three enzymes used for this

investigation were serum alanine amino transferase, serum aspartate amino

transferase and serum alkaline phosphatase.

In one experiment, DHA was administered by oral inlribation to adult albino rats

once. In a second experiment DHA was adm nistered to weaned baby albino rats

and repeated after an interval of 7 days (1 week).

Dihydroarternisinin treatment significantly elevated the packed cell volume,

(P<0.05); the total white celi count, (P<C.O'I); the percentage neutrophil count

jP<O.OI) and the percentage lymphocyte count, ( P 4 . 0 1 j.

DHA treatment did not affect the serurn level:; of serum alanine amino tranferase,

serum aspartate amino transferase and serurn alkaline phosphatase.

There was no gross an(atomica1 or histopatliological evidence that

dihydroarternisinin treatment produced any t2x1c effec: i;rl the liver, heart, lungs,

intestine, spleen, and kidney on the rats. The study found that oral

dihydroartemisinin had no defiterous effects on the red and white blood cells; did

not alter the values of the serum enzymes ALT, AST and ALP and did not

produce any toxic effects on the vital organs of Wistar albino rats at the dose

tested

CHAPTER ONE

I .O INTRODUCTION

Dihydroarternisinin is a member of the arternisinin group of antimalarial drugs

which are currently employed in the treatment of both complicated and

uncomplicated malaria.

Malaria is an illness produced by parasitaemia with one of four species of the

genus Plasmodium:- Plasmodium vivax, Plasmodi~irn ovale, Plasmodi~im

malariae and Plasmodium falciparum. Malaria plasmodia are pathogenic to man,

producing intermittent rigors or chills and anaemia with occasional enlargement

c;f the spleen. In malaria, one of the four species of Plasmodium invades human

tissue; freqcrently those of the liver and erythrocytes of the blood.

In humans, malaria has separate systemic and central effects. Davey and Crews

(1972) described the systemic and central (brain) implications of P, falciparum

infection: as "centrilobular congestion and degeneration" in the liver and

congestion / degeneration of brain capillaries which often result in cerebral

complications. Thus, cases of coma ending fatally are not uncommon in

untreated first infections with the parasite. The incubation period of P. falciparum

is 10-1 5 days.

Malaria is z health problem which causes death and debility in most of the

tropical and subtropical areas of the world (WHO 2001). I t is thus one of the most

important health problems which continuou~.ty engage the attention of the World

Health Orgnaisation's centre for Disease Co-i t rd and Prevention (WHO, 2001).

Chloroyuine was the most effective drug for the clinical management of malaria

between the 1950s and the 1970s (Goth, 1976). The malaria parasite, gradually

developed resistance to chloroquine (Bartelloni et al, 1967; Bloum (1967).

Chloroquine resistance became of great concern for clinical malaria management

especially for Plasmodi~irn faleiparurn malaria which is capable of producing very

severe symptoms and even death in complicated or severe malaria (WHO,

2001).

Artemisinin antimalarial drugs derived form the extract of a Chinese herb

qinhaosu used in China for treating fevers for centuries, have in the past three

decade been found to be efficacious in clinical management of chloroquine-

resistant malaria (Klayman, 1985; Meshnick et al, 1996; China Cooperative

Research Group on qinghaosu and its derivatives, 1982; Ekong and Warhurst,

(1990); Mishra et al, 1995).

Malaria areas of Asia and Africa South of the Sahara (including Nigeria), have

accepted and use artemisinin anti-malarials for the clinical management of

especially uncomplicated malaria (WHO Africa Malarial Report, 2003; The

Guardian News, 2005). However, because artemisinin anti-n~alarials originated

from a Chinese herb, and did not undergo the rigors of orthodox drug

develoment, the half of the world which has a strictly scientific culture want

more animal toxicity studies done on arternisinin anti-malaria drugs to enable

them strongly back its use (Public Health Agency of Canada Committee to

Advice on Tropical Medicine and Travel, 2000).

1 . Malaria as a world problem: .

Thirty six percent of the world population live in malaria areas, while 29 percent

live in areas where malaria which was thought to have been brought under

control, has been si.gnificantly re - established (WHO, 2001). The World Health

Organization estimates that "each year 300 to 500 million clinical cases of

malaria occur making it one of?he most conimon infectious diseases worldwide"

(WHO, 2001).

The World health organization estimates that one 'hundred and ten million

Africans are at an epidemic risk of malaria and that malaria epidemics occur in

Africa on the average of every five years (WHO, 2003). Home - based

management of fever was recommended by the World Health Organisation for

improving the coverage of prompt and effective treatment of fever to allow

mothers treat children as soon as fever is detected (WHO, 2003). This program

was launched in Ghana, Nigeria and Uganda in June 2002 (WHO, 2003). Each

year malaria causes more than one rrrillion deaths of African children (Nosten

and Price, 1995). This position highlights the need in Africa for the search for

new anti-malarial drugs especially new forms, which are extremely potent.

1.2 Malarial Parasite Resistance to Chloroquine:

Chloroquine resistance by P. faciparum hat; been increasing over the years in

South Eastern Asia, South America and Africa (Bartelloni et al, 1967; Bloum,

1967; WHO, 2003). Drug resistant strains cf P. falciparurn are now found in all

malaria endemic tropical regions except the Arabian Peninsula, Central America

and the Caribbean regions (Brooks et al., 1995). With the widespread

establishment of chloroquine resistance, '9y malaria parasites especially P.

falcipar-urn; there are only two classes of compounds that are useful for the

management of severe malaria-the chinchcna alkaloids (quinine and qwindine)

and arternisinins (Woodrow, 2005). There i: general acceptance that to combat

anti-malaria drug resistance, combinations of anti-malarial drugs that include an

arternisinin derivative should be used, and if possible, these should be

formulated in a single tablet (Tran et al, 2004). The emergence of resistance to

chloroquine and pyrimethamine - sulfadoxine; in south East Asia led, to the

introduction of artemisinin containing co-nbinations (Nosten et al, 1991).

Combination of artemisinins with mefloquine provided much improved cure rates

in South East Asia (Nosten et al., 1994; L~oareesuwan et al., 1994).

I .3 Uncomplicated and complicated malaria

Malaria is caused by the bite of an infected female anopheles mosquito during

which it releases (injects) Plasmodium spcrozoites (the result of a continuous

replication of the united male and female garnetocytes or reproductive cells of the

Malaria Parasite) into the blood cf an uniiifected human (Nchida, 1993). The

sporozoites circulate in the blood for 15 tc 60 minutes and disappear into the

tissue cells especially the liver cells to b2gin what is known as the exo-

erythrocytic stage of the asexual cycle of the malaria parasite (Ukoli, 1990).

These tissue sites also form the reserve pool of the malaria parasite. The form of

the malaria parasites released by the bursting of the liver cells is called the

merozoite. These rnerozoites re-infect new liver cells or infect the red blood cells

to grow at the expense of the contents of the red blood cells, a stage called the

erythrocytic asexual stage of the cycle. The proliferative activity of the

erythrocytic stage called schizogony releases hosts of merozoites into the blood

stream when the almost empty victimized red blood cells burst (Ukoli, 1990). The

large number of red blood cells destroyed by the malaria parasites result in

anaemia and lowered oxygen carrying powel- of the blood, in the human victim,

while the toxins and parasite bye - producrs -eleased in the blood stream by the

malaria parasites result in malaria fever and intermittent chils or rigors (Ukoli,

1990). Brooks, et al (1995), confi~m that paroxysms of malaria are closely related

to the events in the blood stream. They posit that in the early stages of infection,

the parasite releasing cycles (as invaded erythrocytes burst) are frequently

asynchronous and the fever pattern irregular but later paroxysms may occur at

regular 48 or 72 hour intervals (although. P. 'alcipanlrn pyrexia may last 8 hours

or longer and may exceed 41°c).

Brooks et al (1998) also pointed out that as the malaria progresses,

splenomegally and to a lesser extent hepatomegally appear and a normocytic

anaemia develops particularly in P, falciparm infections.

That malaria affects many widely separated organs in the human body was

supported by Cruikshank (1968). He pointed out that "malaria organisms can

sometimes be detected in films from bone rrarrow aspirated by sternal puncture

and in some cases this methoc may be used for diagnostic purposes".

Treatment of uncomplicated malaria

Uncomplicated malaria is usually managed with oral anti-malarials and

symptomatic therapy; in contrast to moderate or severe malaria. The particular

combinations of anti-malarials used for management were reviewed by Kremsner

and Kristna (2004). The combination approach to malaria treatment in

uncomplicated malaria has been discussed ~lxtensively by Kremsner and Krishna

(2004); Adjuik et at (2004); Olliaro and Taylor. (2004).

Complicated malaria

Complicated malaria is the malaria resulting rrom congestion and degeneration of

some systemic organs. I t is sometimes called severe malaria or malignant

malaria. Plasomodium falcipanim is the cause of malignant tertian (occurring

every 72 hours) malaria. The tertian malaria caused by P. falciparum is termed

malignant because it produces more deadly malaria or malaria with serious

damaging effects on the human body. The fatal or near fatal nature of falciparum

malaria is related to the fact. that the P. falcipar~lm parasites may be in the

circulation in very large num3ers as they readily invade red blood cells of all

ages. Also, the parasite undergoes its development and asexual multiplication in

the internal organs of the body, hence its damaging effects can affect may

internal organs simultaneously (Brooks et 21, 1998). Anaemia, shock and high

fever thus affect the furictioning of these internal organs. The local pathological

changes resulting from P. falclparum infection according to Davey and Crews

(1972) are: "permeability of the capillary walls permitting the escape of fluid from

the blood into the tissues; concentration of red blood corpc~scles and slowing or

stoppage of the blood flow and reduction and redistribution of the renal

circulat~on. These general and local ch~nges resulting in a reduction of the

amount of oxygen available to the parenchyna cells (a condition aggravated by

the oxygen require men:^ of the parasites themselves), will lead to death of the

trssues if the process is not reversed (Davey and Crews, 1972).

Brooks et al (1998) gave a more vivid picture of how the red and white blood

cells of a human victim is affected by falciparum malaria:- "normocytic anaemia

of variable severity, may be detected during the malaria". They also noted that

during the paroxyms, there may be transient leucocytosis after which leucopenia

develops, with a relative increase in large rr~ononuclear cells. Liver function test

may give abnormal results during attacks but revert to normal with treatment or

spontaneous recovery. They suggested that the presence of prote~n and casts in

the urine of children with Plasrnodil~m falciparum malaria is suggestive of quartan

nephrosis and that in severe P. falciparum infections; renal damage may cause

oligo~lrea and the appearance of casts, protein and red cells in the urine.

Treatment of complicated malaria

Quinine remains the drug of choice for the management of severe malaria in

Europe and Africa though qu~nine has a problem of causing local toxicity and

hypoglycemia when given parenterally (Katzung, 1995, Woodrow, 2005) Quinine

IS also used for complicated malaria in South East Asia although there is

~ncreasing quinine resistance in South East Asia (Pukrittaya-Kamee et al, 1994).

Several studies compared intramuscular arkmeter and quinine for their efficiency

In the treatment of severe malaria infection in Africa (van Hensbroek et al, 1996;

Murphy et al, 1996; Walker et al., f 993; Danis et al, 1996; Taylor et al, 1993; and

in South East Asia (Tran et al., 1996; Karbwang et al, 1995). Despite improved

parasite clearance parameters in most trials, definite evidence for improved

mortality to complicated malaria with artemether in ind~viduals and meta-analys~s

1s lack~ng (Pittler and Ernst, A999; Mclntosh and Olharo, 2000, Artemether -

Quinine Meta - analysis group, 2001). Recent ev~dence shows that perenteral

artesunate (ARS) is the treatment of choice In adults hospital~zed with severe

malaria (Dorndrop et al, 2005). Artesunate (artesunic-acid), a semisynthetic

derivative of arternisinin, is of particular interest because its solubility in water

facilitates its absorption (Barradell and Fitton, 1995). Artesunate is effective

against parasites which have developed r?sistance to conventional antmalarials

in sub-Saharan Africa (Borrrnan et al, 2002).

Parental artesunate has been used in adults and childrep with severe malaria in

South East Asia (Hein et al, 1992; Cao et al, 1997 and Ha et al, 1997). The

- ' ~ ~ - ~ : s ' ; ! ~ l n r . . . 3".saqgte was cornoarable ~n efficiencv and safety to intravenous

administration (Hein et al, 1992, Ha et al, 1997). Intramuscular artesunate has an

acceptable pharmacokinetic profile in African children (Nealon et al, 2002).

Management of uncomplicated and complicated malaria

Artemisinin derivatives are used for treatment of uncomplicated and complicated

malaria. Successful use of artemisinin derivatives with sulfadoxine-

pyrimetharnine has recently been described in Africa (Dorsey et al, 2003).

Artemether-lufantrine is the only fixed dose artemisin~n - containing combination

that is registered for use in Europe and is licensed as a six dose regimen over

sixty (60) hours in patients weighing over 35kg (van Vugt et al, 2000).

Very few dose ranging / frequency studies have been carried out to ensure that

current regimens fcr uncomplicated malaria have been truly optimized (Woodrow

et al, 2005). In adults, different doses of artesunate given under cover of the

slower acting agent mefloquine; suggested to the authors that a dose of 2mglKg

artesunate was sufficient to reduce parasitaemia rapidly (Angus et al, 2002).

Most physicians currently use an oral dose of 4mglKg per day for three days for

patients with uncomplicated malaria when in combination with a second

antimalarial. However, despite the generally rapid elimination kinetics of

artemisinins, daily dosing of oral artesunate results in parasite clearance kinetics

indistinguishable form twice daily dosing (Nosten et al, 1994). White (1 994),

regarded this indistinguishability of once daily dosing from twice daily dosing as a

suggestion that constant drug levels are not necessary for satisfactory parasite

clearance. He posited that the biological effects of artemisinins extend beyond

their presence at therapeutic concentrations in plasma. Woodrow et al (2005)

regarded this occurrence as being analogous to post-antibiotic effect,

Severe or complicated malaria in hospitalized patients is associated with a

mortality of between 15% and 20% despite appropriate anti-malarial and

supportive treatment (Newton and Krishna, 1998).

Themostable 2 rtemisinin depositories

A new pharmaceutical form of artemisinin h ~ s been developed called artemisinin

depositories. 0.w such preparation with artesunate contains 50mg of artesunate.

The absorptior, rate of iis active components is often as rapid as that obtained

with intra-muscular administration (Herrnann, 1995). These artemisinin

depositories have been found to be efficacious in the management of

uncomplicated and moderately severe malaria in persons who are unable to take

oral artemisinin especially in children (Karunajeewa et al, 2004; Simpson et al,

2006).

1.4 Di hydroartemisinin

Dihydroartemisirlin is the active metabolite of all arternisinin compounds

(arternisinin, ar:os~~nate, arteether, artemeter, etc) and is also available as a drug

in itself. Once absorbed, the artemisinin derivatives are converted primarily to

dihydroarternis!nin (DHA) and subsequently to inactive metabolites through

hepatic P -450 and other enzyme systems. Artemisinin itself is not metabolized to

DHA but acts as the primary anti-malarial. Artemether and arteether contribute to

antimalarial activity probably to a similar extent to which they are converted more

slowly. Artesunate is hydrolyzed to dihydroartemisinin within minutes and its

antimalarial activity is largely mediated by dihydroartemisinin (DHA).

DHA is itself a potent antimalarial with elimination half-life of about 45 minutes

(Batly et al, 1993; lllett et al, 2002). DHA is 90% bound to plasma protein (Batty

et al, 2004).

DHA is available as a fixed drug combination with piperaquines; each tablet

containing 40mg DHA and 320mg of pipzraquine manufactured by Holleykin

Pharmaceuticals. The adult dose of DHA is, 1.6 - 12.8 mglKg per dose (rounded

up or down to the nearest half tablet given at Ohr. 8Hr, 24hr, and 48hr.

Alternatively, the same total dose may be given one dally for three (3) days

(Ashley et al, 2005) DHA is also sold in P-frica as Cotexin@ in 60mg tab!ets.. A

cornparism of the safety and efficacy of the formulation of dihydroarternisinin

produced in China. with the one produced in Thailand and a third one produced

in Vietnam found that they were equally safe and effective (Wlilarratana et al.

2998).

I .5 Toxicity of artemisinins

The artemisinins have impressive parasiticidal properties in vitro and in vivo,

rapidly arresting parasite metabolism in concentrations within the lower

nanomolar range and killing paras~tes misre quickly than the other antimalarial

drugs (White, 1994: Hien and White, 1993; Mclntosh and Olliaro. 2000, a & b).

Xu and Zhang (2004) reported that dihydroartemisinin and arksunate, showed

contra gestational effects. The two drugs caused embryo absorptmn in mice and

rabbits whereas in hamsters and guinea pigs, they induced abortion.

Studies by Kamchonwongpaisan et at (1997) with high doses of artemether on

mouse neuroblastorna cell (Neu 2a1, treated with 3H dihydroartemisinin, showed

that the rat uniformly developed neurolqic symptoms; following intmmuscular

administration of SOmg/Kg / day of arteether for 5 to 6 days. The neurological

symptoms were of the nature of acute neuronal necrosis associated with

vacuolization and axonal swelling in the neurophif in specific areas of the brain

especially the vascular nuclei and red nu ;lei. Also "scattered swollen neurons

were evident in the cerebellar nuclei and reticular formation". Although the

therapeutic indices of the artemisinin derivatives appear to be high (Mien et al,

2003; Hien and White, 1992.; Mclntosh and Olliaro, 2000, a 8 b), there are still

concerns that the neurotoxicity found in animal studies (Brewer et al, 1994, a, b &

p . 'a I Li et a!. 2002; and Kamchonwongpaisan et al. 1997); may occur in humans,

especially in ch~ldren (Johann - Liang and Albrecht. 2003) especially in the face

of preclinical evidence of brainstem toxicity in animals (Brewer et al, 1994,

Brewer et al 7994, a).

Other reported side effects of artemisinins

It has been noted that the administration of artemisinins may be associated with

transient gastrointestinal disturbances and rarely with severe allergic reactions

(Leonard1 et al, 2001) or haemolysis (Orj~h, 1996) Also arternether-lufantr~ne was

reported to induce miid but significant hearing loss (Toovey and Jamieson,

2004).

The leading adverse effects among the 66 adverse events reported by 179

patients were gastrointestinal (nausea, vomiting abdommal pain) and

neurological (convulsions, dizziness, impairment of consciousness, a bnorrnal

reflexes and vertigo) (Simpson et al. 2006).

1.6 The aim the study

Some studies found that artemisinin drugs produce some neurotoxic~ty in animals

(Brewer et a[, 1994, a, b & c; Kamchonwongpaisan et a1,1997). Other studies

found that arternisinin drugs produce contra gestational effects in animals (mice,

rabbits, gurnea p~gs, hamsters etc). (WHO, 2003, Xu and Zhang 2004). No

studies have investigated the systemic tox~c~ty of artemis~n~ns on the system~c

vital organs. The aim of the present study was therefore to evaluate the toxicity of

DHA on vital organs and the blood of Wistar Albino rats

CHAPTER TWO

2.0 MATERIALS AND METHODS

Nine adult Wistar albino rats (weighing 106 - 140 grams and 9 (just - weaned)

baby Wistar albino rats weighjng 75 - 90grams.

Mettler Balance (Model AE 166, Delta range Type); OHAUS Triple Beam

Weighing Scale (700/8000 series) made in US; Automatic Shardon Ellich Duplex

Processor Machine, rnade in Western Germany; Rotary Microton Machine, made

in Western Germany: Nicon Micrographic Microscope (Biphot, Lamp 12V, 10A

Line 220/249V, 50/60Hz, 10 inch muzzle) made in United States; RlCOH

Micrographic Camera Film (35mm Konica fvlinolta, vx 100 Colour film); Water

Bath; W.P.A Linton Spectophotometer connected to a Binatone Automatic

Protection Circuitary, made in UK; Leica Model Binocular CME Light Microscope,

rnade in the United states; Gallenhamp Centrifuge, made in England (Timer 5-20

minutes, Speed 1000-5000RPM): 14ml Cenhfuge Test Tubes; Microhaematocrit

Centrifuge( Hawksley Model), rnade in England (Speed 0-1 5 Minutes); Hawksley

Micro-Haematocrit Reader (Scale 0-100); Laboratory DLC counter, made in US

and 1 m1/5ml Syringes.

2.1 REAGENTS

Dihydroartemisinin (CotecxinB) (Beijin COTEC Co, China) alcohol; xylene;

paraffin wax; Haematoxylin and Eosin; Phosphate Buffer (pH 7.4), AST substrate

(200mM); aspartic acid (2mM), x-Ketoglutarate, ALT Substrate (200mM); L -

alanine (2mM); L-ketoglutarate; Workirlg Pyruvate Standard (4mM);

Carbohydrate Buffer (pH 10); Phenyl - (p) Substrate; 0.01 O~sodii~rn Phenyl - (p)

(0.09g x 2 = 2.189); Stock Phenol Standard ( I m g / 30ml); NaOH (0.5N); l o g

NaOH dissolved in 500ml of distilled water; Sodium Bicarbonate (0.5N); 4-amino

antipyrine and Potassium f e r i cyanide.

2.2 METHODS

2.2.1 Drug Treatment

Test and control Wistar albino rats obtained from the small animal breeding

section of the National Veterinary Research Institute (NVRI), Vom, Plateau State,

were acclaniatised for two weeks at an undisturbed area of the Biochemistry

Laboratory of NVRI, before dihydroartemisinin and distilled water were orally

administered to them.

The study was done in two experiments. Nine adult albino rats were used in the

first experiment while nine baby rats were used in the second experiment.

In the first experimenwht, five rats (3 males and 2 females) were given DHA

(2mg I kg) on the first day and Imglkg for the next four days while 4 rats were

given saline and served as control.

In the second experiment, 5 baby rats (3 ma;es and 2 females) were given DHA

(2 mg/ kg) (day 1) , 1 mglkg (day 2 - 5); for 5 days. At the end of the 5 days, the

rats were allowed to rest for 1 week and the 5 days dosage regimen of DHA

repeated.

Four of the baby rats (2males and 2 femaks) were given distilled water and

served as controls.

2.2.2 Effect of DHA On Rat Body Weight

The test and control rats were weighed 10-1 5 mrnutes before the admln~stration

of the first dose of DHA or distilled water and were weighed again 24 hours after

the end of the admin~stration of the last dose of the drug or d~st~l led water. In the

second experiment the test and control rats were weighed 24 hours after the

administration of the first dose of DHA and weighed again 24 hours after the

administration of the last dose of the first and repeated doses of DHA.

2.2.3. Estimation of the Activities of Serum Enzymes ALT, AST and ALP

Serum alanine amino transferase and serum aspartate amino transferase

enzymes were assayed through the following procedure: - The ALT or AST

substrate was put in four paired test tubes (to replicate the results) termed the

standard (or STD), (0.4ml); the standard blank (STDBL) (0.5ml); the 'test' "T"

(0.5ml) and the test blank (or TB), (0.5ml). These four test tubes were mixed and

kept warm at 70°C for 3 minutes in a thermostatically controlled water bath. This

was succeeded, by the following actions:

1 . Fresh unhaernolysed serum collected (after centrifugation) from the whole

(unheprinized blood )of the DHA treated or control rat was added only to

the 'Test' test tube (0.1 ml).

2. Working Phenol Standard (already prepared for the assay) was added to

the Standard (STD) (0.1 ml) and the Standard Blank (0.1 ml test tubes).

3. Pyruvate Standard (0.1 ml) was added only to the 'STD', test tube.

4. Test tubes 1 - 4 i. e. STD, STD BL, T and T8 were mixed and incubated

at 3 7 ' ~ for 1 hour.

5. 0.5ml dinitrophenyl hydrazine (DNP) was added to each of the test tubes

'STD', STDBL, T and TB and then mixed and stood at room temperature

for 20 minutes.

6. Working Phenol Standard (0. lml) was added only to the test blank, and

mixed.

7. 0.4 Normal NaOH (5ml) was added to each of the four test tubes "STD'

STD BL, T and TB.

8. The brown coloured hydrazone whis:h the pyruvate produced by the

transfer of amino groups from glutamic or aspartic acid to pyruvic acid

when it reacted with 2-4 dinitrophenyl hydrazine (Reitman & Frankel.

1957), was read with a Linton Cambridge (U K) WPA Specto3hotometer at

514nm.

Method of Assay of Serum Alkaline Phosphate

The same procedure described for the method of assay of serum alanine amino

transferase and serum aspartate amino transferase was followed in the assay of

serum alkal~ne phosphatase. The only difference between two assay procedures

was that ALT or AST substrate was replaced by phenyl phosphate substrate;

pyruvate standard was replaced by 4- ammo antipyrine and 2-4 dinitrophenyl

hydrazine was replaced by potassium ferricyanide in the alkaline phosphatase

assay. Potassium ferricyanide was added to the 4 test tubes - the STD, STD BL,

T and TB and the mixture kept for 15 minutes. At the end of the 15 minutes, the

red - pink colour which was produced (Reitman & Frankel, 1957), was read

immediately with the Linton U.K. WPA Spectophatometer at 510nm. Alkaline

phosphate (in alkaline medium) hydrolysed phenyl phosphatase in 15 minutes (at

PH 10) to release the phenol which reacted with 4 -aminoatipyrine (in the

presence of potassium f&rficyanide) td produce the pink colour read at the

Spectophotometer at 51 Onm.

2.2.4 Haematological Investigation

Blood samples, from the test or control rats were collected'24 hours after the

administration of the last dose of DHA or distilled hater. The blood was collected

through expert bleeding of the rat through the subclavian artery which is very

close to the pulmonary blood supply. '

Estimation of the Packed Cell V O I U ~ ~ - ( P C V ) :

The EDTA anticoagulated blood collected from the DHA - treated or distilled

water treated rat (the control), was carefully put in special blood capillary tubes,

the top of which was heat-sealed using a small flame from a pilot flame of the

Bunsen burner. The filled capillary was placed carefully in one of the numbered

slots of the micro-haematocrit rotor with the sealed end against the rim gasket to

prevent breakage. The number of the slot was noted against the identification

number of the rat from which the blood was obtained. The arranged capillaries

were then centrifuged for 3-5 minutes (RCF 120004 5,000 x g), using the shorter

time when the RCF was 15;000 x g.

. Immediately after centrifuging, the PCV was read in a hand held haematocrit

reader by aligning the base of the red cell column above the sealant on the zero

mark (line) and the top of the plasma column on the 100 mark. The PCV was

read off from the scale; the reading point being the top of the red cell column just

below the buffy coat layer (which consisted of white blood cells and platelets).

Determination of the Total white blood cell coun't (WBC)

The blood cells were dilu'ted in a buffered electrolyte solution. A measured

volume of this sample was passed through an aperture tube between two

electrodes. Interuption of the current flowing between the two electrodes by the

non-conducting blood cells altered the electrical charge ,and a pulse was

produced. The amplitude of each pulse was related to the volume of the blood

cells which produced it. There was a threshold circuit, which ensured that only

the pulse which exceeded a preset threshold.leve1 was counted. The total white

blood cell count was determined from the total number of pulses obtained from a

measured volume of blood.

Determination of the Differential Leucocyte Count.

The differential white blood cell or leucocyte count of the DHA treated and control

rats were done on the stained thin films of blood.

The stained thin blood films were allowed to dry completely before they were

examined. The thin blood films were then examined microscopically and the

different white cells counted.

To enable the different white cells to be seen clearly under the microscope, a

drop of immersion oil was put on the lower third of the blood film and covered

vith a clean cover glass; before viewing the blood film under the microscope. 1

T 'iis was then followed by a systematic examination of the blood film and a

co unting of the different white cells seen in each field using an automatic

chanical diffei-ential cell counter. me

2 - 2 3 Gross Anatomical Observations

The DHA- treated and control rats were' sacrificed and dissected 24 hours after

the administration of the last dose of DHA or distilled water (after the collection of

blood for the enzyme assay and haematological investigations). In the second

experiment, the DHA - treated and control rats were sacrificed 24 hours after the

administration of the last repeated dose of DHA or distilled water.

The dissected rat was mounted on a dissecting board to distinctly display' the

lungs, heart, liver, spleen, intestine, and kidney to aid easy gross anatomical

examination of these orga.ns. Any deviations from the normal presentation or

morphological structure of each of these six organs was noted. Any other

deviations from the normal presentation of the viscera was also noted.

2.2.6 Histopathological investigations

Slide Preparation.

Slides of the microsections of the heart, lungs, liver, intestine, spleen and kidney

of the rats for histopathological investigation were prepared as described below;

The severed organs were put in 10% buffered forma'lin for 6 days to fix them.

Then the organs were dehydrated by puttin9 them successively in 709'0, 80%,

90%, 95% and absolute alcohol. They were then cleared with xylene. The

cleared organs were then impregnated in paraffin wax and embedded in

mounting paraffin Wax on a wooden block. Excess paraffin wax was trimried

from the mounted organ to expose the surface for cutting of the microsections of

the organ. A small portion of the vital organ in question, suspected to have bsen

distressed by the drug treatment and an equivalent portion of the same organ

from a control rat were then cut for histopathological investigation. The cutting of

the microsection of the tissue of the heart, lung, liver, intestine, spleen or kidney,

was done using a Rotary Microton machine.

The sliced tissue was then processed in an Automatic Shaidon Ellich Duplex

Processor for 18 to 24 hours. After such processing, the microtissue for

histopathological examiniation, was stained with Coles Haematoxylin and 1%

Eosin (H & e) Stain and the slide was left for a few days to dry.

The slides of the microsections of the heart, liver, lungs, intestine, spleen and

kidney of the rats were photographed with a 35mm konica Minolta vxlOO

photographic film with a RICOH Micrographic Camera attached to a Nicori

Graphic Microscope. The photographed negatives of the micrographs were later

developed and printed it1 a p h d ~ o ~ a p h i ~ COIOU~ I a b ~ ~ K x y .

2.2.7 Results Presentation

The results, where possible, are presented as mean (+ S.E.M8) subjected t3 a

two-tailed T- test of statistical signif~cance. T h e value of P<O.OS was taken as

significant.

The histopatholog~cal results are presented as photomicrographs of the tissue:

of the heart, lungs, intestine, spleen, liver and kidney of the DHA- treated rats ir

comparison with those of control rats.

CHAPTER THREE

RESULTS

3.1 Effect of DHA on the body weight of the albino rats.

A comparison of the mean weight of t h ~ rats before and after treatment showed

that both the DHA - treated and the control rats gained weight during the

experiments (Table 1).

3.2 Effect of DHA on serum ALT, AST and ALP enzyme activities.

The results of the study show that the serurn alamine amino transfase, serum

aspartate amino transferase and serum alkaline phosphatase enzyme activities

of dihydroartemisinin - treated albino rats we1.e not significantly (p<0.05) different

from those of the control rats (Table 2, 3 and 4). The only exceptions were the 2

mglkg (day j ) , I mglkg (day 2 - 5) DHA treat1nen.t in which the enzyme activity of

the treated group was significantly different from those of the controls (at P<0.05

for ALT and P<0.01 for AST enzymes, Tables 2 & 3). However, all values

obtained for the ALT, AST and ALP enzyme: for all the rats (treated and control)

were within the normal range of ALT, AS-- and ALP enzyme activity valuer;

(Tables 2, 3 & 4).

3.3 Haematological effects of dihydroartemisinin.

Dihydroartemisinin treatment significantl\/ increased the hernatologiczl

parameters (P4 .05 ) . It produced significant (Pc0.05) increase in the packed cell

volume [Table 5) . It also significantly ( ~ ~ 0 . 0 1 ) elevated the total white blood cell

count (Table 6). DHA-treatment also elevated the percentage netrophil cou

significantly at P< 0.01 level (Table 7). Tt-,ere was also a significant elevation

the percentage lymphocyte count (Pc0.05 and P<O:01) (Table 8).

On the other hand, the percentage eosinuphil count of the DHA - treated rats c

not show a significant difference from those of the control rats (Table 9). Like ti

percentage neutrophil and lymphocyte counts the percentage monocyte counts

of DHA - treated rats increased with reference to those of the control rats (Table

No basophils were found in either the blood of the control rats or those of DHP

treated rats.

3.4 The Results of the Gross Anatomical Observations.

Gross Anatomical observations showed that the heart, liver, spleen, intestir

lungs, kidney and blood of the DHA - treated and control rats appeared both

normal and undisturbed.

3.5. The Results o f the Histopathological Investigations.

There was no evidence of DHA toxicity in the liver (figure 2); the he;

(figure 3); the intestine (figure 4); the lu,ngs [figure 5); the spleen (figure 6) a1

the kidney (figure 7) of the rats.

TABLE 1: THE MEAN WEIGHTS AND THE WEIGHT GAINS OF THE

Groups in the experiment

Control for 2 mglKg (day 1),1 mglKg (day 2- 5) DHA

2 m g m (day 11, 1 mglKg (day 2- 5) DHA repeated after 1 week

Control for 2 mglKg (day I), 1 mglKg (day 2- 5) DHA repeated after 1 week

TREATED AND CONTROL RATS.

Wt at day 0 in grams

Mean: 125.96

Std.

Dev:I 3.07528

S.E.M: 5.84744

Mean7 27.32

Std. Dev:4.63001

S.E.M:2.0706

Mean: 83.88

Std. Dev: 4.1 191

S.E.M: 1.84212

Mean: 79.46

Std. Dev:

3.74072

S.E.M: 1.6729

Wt at end of 1'' treatment in grams

Mean: 163.94

Std. Dev: 15.837%

S.E.M:7.08277

Mean:130.7

Std. Dev: 5.1 3907

S.E.M: 2.29826

Mean: 168.2

Std. Dev: 5.10784

S.E.M: 2.28429

Mean: 1 15.26

Std. Dev: 5.4226

S.E.M: 2.42515

Weight gain for 1" treatment in grams

Mean: 38.1 ***

Std. Dev: 2.75409

S.E.M:l.23167

Mean:3.4

Std. Dev: 0.06442

S.E.M: 0.2881

Mean: 82.98

Std. Dev: 2.55088

S.E.M: 1.14079

Mean: 35.8

Std. Dev: 2.4013

S.E.M: 1.07389

Wt at end of repeat treatment in grams

Mean: 179.5

Std. Dev:

4.67707

S.E.M: 2.09165

Meam1 17.36

Std. Dev:5.44878

S.E.M: 1.41683

- Wt gain for repeat treatment in grams

Mcan:95.62***

Std. Dev:3.16812

S.E.M:f A1683

Mean97.8

Std. Dev:2.43336

S.E.M:I .a8823

-

Std. Dev = Standard Deviation

S.E.M = Standard Error Mean

***(P<O.O?)

TABLE 2: EFFECT 'OF DHA ON SERUM ALT ENZYME ACTIVITY IN 1.1

ILITRE.

Groups in the experiment . N Mean Std. Std enzyme Deviation Me activity

2 mglKg (day I ) , 1 mglKg (day 2- 5 * 9.0000 2.97909 1.3---- , 5) DHA

Control for 2 mglKg (day I), I 4 , 5.0000 1.15470 0.577: mglKg (day 2- 5) DHA

2 mglKg (day I ) , 1 mglKg (day 2- 5 ** 7.0000 2.71 570 1.214! 5) DHA repeated after 1 week

Control for 2 mglKg (day I ) , 1 4 6.0625 2.08542 I .042' mglKg (day 2- 5) DHA repeated after 1 week

ALT Normal Values = 3 - 15 I UIL

TABLE 3: EFFECT OF DHA ON SERUM AST ENZYME ACTIVITY IN 1.UIL.

N Mean Std. Std. Errc enzyme Deviation Mean activitv

2 mglKg (day I ) , 1 mglKg (day 2- 5 ** 9.5000 I .43222 0.64051 5) DHA

Control for 2 mglKg (day A ) , I 4 6.5000 0.45644 0.22822 mglKg (day 2- 5) DHA

2 rnglKg (day I), 1 mglKg (day 2- 5 6.5000 . . 1.57361 0.70374 5) DHA repeated after 1 week

Control for 2 mglKg (day I), 1 4 6.2625 0.59774 0.29887 mglKg (day 2- 5) DHA repeated after 1 week

AST Normal Values = 5 - 18UMll

TABLE 4: EFFECT OF DHA ON SERUM ALP ENZYME ACTIVITY IN K.A. (1.U).

Groups in the experiment N Mean Std. Std. Error enzyme Deviation Mean activitv

2 mglKg (day I), I mglKg (day 2- 5 4.0000 0.88388 0.39528 5) DHA

Control for 2 mglKg (day I), 1 4 3.7500 0.79057 0.39528 mglKg (day 2- 5) DHA

2 mgfKg (day I), 1 mglKg (day 2- 5 3.7500 0.85220 0.381 12 5) DHA repeated after I week 1 Control for 2 mglKg (day I), 1 4 4.1250 + . 0.77728 0.38864 mg1Kg (day 2- 5) DHA repeated after I week

ALP Normal values: 3-+I3 K.A

TABLE 5: EFFECT OF DHA TREATMENT ON PACKED CELL VOLUME (PCV)

' ~ r o u p s in the experiment N Mean . Std. Std. Error PCV Deviation Mean

2 mglKg (day I ) , 1 mglKg.(day 2- 5) 3 * 45.0000 1.00000 0.57735 DHA

Control for 2 mglKg (day I ) , 1 3 40.0000 2.00000 1 .I 5470 mglKg (day 2- 5) DHA

2 mglKg (day I), 1 mglKg (day 2- 5) 3 * 46.0000 1.00000 0.57735 DHA repeated after A week

Control for 2 mglKg (day I), 1 3 40.0000 2.00000 1 .I 5470 mglKg (day 2- 5) DHA repeated after I week

G r o u p s in the experiment N Mean WBC Std. Std. Error Deviation Mean 1000.00000 577.35027

2- 5 ) DHA

Control for 2 mg1Kg (day I ) , 1 3 4700.0000 264.57513 152.75252 mglKg (day 2- 5) DHA

2 mglKg (day I ) , I mglKg (day 3 ** lZO25.0000 2036.1 1149 831.23904 2- 5) DHA repeated after I

/Control for 2 m g ~ g (day I), 1 3 JOJO.OOO~ 421.9d046 172,24014 rnglKg (day 2- 5) DHA repeated after 1 week

TABLE 6: EFFECT OF DHA ON TOTAL WHITE BLOOD CELL (WBC) COUNT

I N mm3.

Groups in the experiment N Mean WBC Std. Std. Error Count Deviation Mean

2 mglKg (day I ) , 1 mglKg (day 3 ** 9050.0000 1000.00000 577.35027 2- 5) DHA

Control for 2 mglKg (day I ) , 1 3 4700.0000 264.57513 152.75252 mglKg (day 2- 5) DHA

I r 2 mg1Kg (day I ) , 1 mg1Kg (day 3 ** 12025.0000 2036.11149 831.23904 2- 5) DHA repeated after 1 1 week

Control for 2 mglKg (day I ) , 1 3 5050.0000 421 .go046 172.24014 mglKg (day 2- 5) DHA repeated after 1 week

I

TABLE 7: EFFECT OF DHA ON PERCENTAGE NEUTROPHIL COUNT.

Groups in the experiment N Mean % Std. Std. Error Neutrophil Deviation Mean Count

2 mglKg (day 2 ) , I mglKg (day 3 ** 24.6667 I .I 5470 0.66667 2- 5 ) DHA

Control for 2 mglKg (day I ) , 1 3 10.0000 I .OOOOO 0.57735 mglKg>(day 2- 5) DHA

2 mglKg (day I), 1 mglKg (day 3 ** 26.0000 2.00000 1 .I5470 2- 5) DHA repeated after ? week

Control for 2 mglKg (day I), I 3 11.0000 1.00000 0.57735 mglKg (day 2- 5 ) DHA repeated after 1 week

TABLE 8: EFFECT OF DHA ON PERCENTAGE LYMPHOCYTE COUNT.

Groups in the experiment N Mean O/O Std. Std. Error Lyrnphoctye Deviation Mean Count

2 mgJKg (day I), 1 mglKg 3 * 76.0000 2.00000 1 .I 5470 (day 2- 5) DHA .

Control for 2 mglKg (day I), I 3 70.0900 2.00000 1 .I 5470 mglKg (day 2- 5) DHA

2 mglKg (day A ) , I mglKg 3 ** 82.0000 1.73205 1.00000 (day 2- 5) DHA repeated after I week DHA

Control for 2 mglKg (day I), I 3 75.0000 1.00000 0.57735 rnglKg (day 2- 5) DHA repeated after 1 week DHA

TABLE 9: EFFECT OF DHA ON PERCENTAGE EOSINOPHIL COUNT.

Groups in the experiment N Mean % Std. Std. Error Eosinophil ' Deviation Mean Count

2 mglKg (day I ) , 1 mgIKg (day 3 1.0000 O.OCOOa 0.00000 2- 5) DHA

Control for 2 mglKg (day I), I 3 3.0000 0.0000" @.OOOOO mg/Kg (day 2- 5) DHA

2 mglKg (day I), I mglKg (day 3 1.0000 0.0000" 0.00000 2- 5) DHA repeated after I week

Control for 2 mglKg (day I), 1 3 0.0000 O.OOOOa 0.00000 mglKg (day 2- 5) DHA repeated after I week I----- " t cannot be computed because the Standard Deviations of both groups are 0.

TABLE 10: EFFECT OF DHA ON PERCENTAGE MONOCYTE COUNT.

Groups in the experiment N Mean O h Std. Std. Error Monoctye Deviation Mean Count

2 mglKg (day I), I mglKg (day Oa - - - 2- 5 ) DHA

Control for 2 mglKg (day I), 1 0" - rnglKg (day 2- 5) DHA.

2 mglKg (day I), I mglKg (day 3 1.3333 0.57735 0.33333 2- 5) DHA repeated after 7 week

Control for 2 mglKg (day ?), ? Oa - mglKg (day 2- 5) DHA repeated after 1 week

a t cannot be computed because at least one of the groups is empty.

HISTOPATHOLOGICAL RESULTS

Figure 2-7 show the photomicrographs of the liver, heart, intestine, lungs, spleen and

k~dney of DHA - treated and control rats. In the photomicrographs, there is no

morphological (structural) difference between the DHA - treated and control vital organs

indicating that DHA d ~ d not adversely affect any of the vital organs.

The funct~onal effuency brought about by the s~gnificant elevat~on of the packed cell

volume (P<0.05) and the total white blood cell count (P<O 01) seem to have influenced

the somewhat enlarged organ structures seen in the photomicrographs of the DHA-

treated organs In comparison with the controls

I 1

I Fig 1A: A grossanatomical display of a control albino rat showing the vital organs.

Fig 18: A grossanatomical display of a DHA - treated albino rat showing the vital organs.

While the vital organ, of the control albino rat has pinkish-red colour,

the DHA-treated albino rat's vital organs have a congested dark red appearance (especially the liver, the spleen and the kidney) suggesting that the blood cell

. composition of the DHA-treated albino rat's vital organs was denser than those

of the controls.

FIGURE 2: PHOTOMICROGRAPHS OF THE LIVER - - -

A: uver !Issue OT z r n g ~ g aay I , B: Liver tissue of control of 2mglkg 1 mglkg day 2 - 5 DHA-treated rat day 1, I mglkg day 2 - 5 DHA-treated

w; L I V ~ I IISSUB VI L;UIIIIUI UI ~ e ~ e d l e d C: Liver tissue of 2mg1kg Zmg/kg day I, I rng/kg day 2 - 5 DHA- day 1, I mglkg day 2 - 5 DHA- treated rat

treated rat

FIGURE 3: PHOTOMICROGRAPHS -- OF THE HEART

A: Heart tissue of 2mgkg day I, B: Heart tissue of control of

I mgfkg day 2 - 5 DHA-treated rat- 2mgIkg day 1, I mgkg day 2 - 5 -

C: Heart tissue of repeated 2rng/kg D: Heart tissue of control of

day 1, I mglkg day 2 - 5 DHA- repeated 2mgkg day 1, I mgkg

treated rat day 2 - 5 DHA-treated rat

- FIGURE 4: PHOTOMICROGRAPHS l OF THE INTESTINE

B: Intestme tissue 07 control of A: Inte~tine tissue of 2mg/kg day 2mglkg day 1, I mgkg day 2 - 5 1, I rnglkg day 2 - 5 DHA-treated rat DHA-treated rat

C: lntestine tissue of repeated D: Intestine tissue of control of

2rnglkg day 1, I rnglkg day 2 - 5 repeated 2mglkg day 1, I rnglkg

DHA-treated rat day 2 - 5 DHA-treated rat

FIGURE 5: PHOTOMICROGRAPHS OF THE LUNGS

A: Lung tissue of 2mgIkg day 1, B: Lungs tissue of control of

1 mglkg day 2 - 5 DHA-treated rat 2mgIkg day 1, I mglkg day 2 - 5 DHA-treated rat

C: Lungs tissue of repeated D: Lungs tissue of control of

2mgIkg day 1, I mglkg day 2 - 5 repeated 2mgkg day 1, I mglkg

DHA-treated rat day 2 - 5 DHA-treated rat

FIGURE 6: PHOTOMICROGRAPHS OF THE SPLEEN

A: Spleen tissue of 2mglkg day 1, B: Spleen tissue of control of

1 mglkg day 2 - 5 DHA-treated rat 2mgIkg day 1, I mglkg day 2 - 5 DHA-treated rat

7 - - - - - - - -

C: Spleen tissue of repeated D: Spleen tissue of control of

2mglkg day 1, Imgkg day 2 - 5 repeated 2mglkg day 1, I mglkg

DHA-treated rat day 2 - 5 DHA-treated rat

FIGURE 7: PHOTOMICROGRAPHS OF THE KIDNEY

A: Kidney tissue of 2mglkg day 1, 1 mglkg day 2 - 5 DHA-treated rat

e-y -- =--- . .

C iWn~3.y %we 0"e~eated 2mglkg day 1, Imglkg day 2 - 5 DHA-treated rat

R: Kidney tissue of control of 2mglkg day 1, I mglkg day 2 - 5 DHA-treated rat

D: Kidney tissue of control of repeated 2mglkg day 1, 1 mglkg day 2 - 5 DHA-treated rat

CHAPTER FOUR

DISCUSSIONS AND CONCLUSIONS

The mean body weight gains of the DHA - treated rats were more than those of

the control rats [Table 1). The body weight gains of the DHA - treated and

contro! rats can not be attributed to the effect of feeding alone since there was

statistically [P<0.01) greater body weight gain in the DHA - treated rats in

comparison with those of the control rats. It is thus concluded that DHA -

treatment must have caused the greater weight gain by the DHA - treated rats.

Since gross anatomical obsewatims showed that the heart, lungs, intestine,

her , spleen and kidney of the DHA - treated rats appeared as normal as those

of the control rats; it is concluded that DHA -- treatment did not inflict any injury

or toxic effect on the organs.

Hoe (1961) and Coles (1986) regarded an estimation of the values of Serum GPT

(alanine amino transferase) and G.O.T. (aspartate amino transferase) as specific

for estimation of liver and heart malfunction.

Serum ALT or G.P.T. is increased in liver necrosis (Gray 19681, Very high values

of G.P.T (ALT) is found in toxic liver disease (Gray, 1968). Also after myocardial

infarction in humans, serum AST (GOT.), increases within 6-12 hours from 5-30

Reitmen Frankel units to values as high a 500 - 600 units within 24 to 48 hours

and returns to normal within 4 .- 7 days. The fact that the serum ALT (GPT) and

4 3

serum serum AST (G.0.T) enzyme activity values of the dihydroartemisinin -

treated albino rats did not vary from their .normal values within 24 hours of

cessation of DHA treatment; indicates that DHA treatment did not damage the

liver hepatocytes or the heart muscle celis (myocardium).

The serum phosphatase levels may be above normal in liver cirrhosis or liver

necrosis and may some times reach levels usually encountered in obstructive

jaundice which are usually more than 30 units / 100rnl (while a high percentage

of hepatic jaundice show phosphatase levels of 13 - 30 units / IOOml), (Gray,

7 968).

Since the serum alkaline phosphatase levels of the DHA - treated rats remained

within the normal ALP enzyme activity values of 3-13 K.A. units, this suggests

that DHA -treatment did not affect the liver hepatocytes adversely.

The intactness of the heart and liver of the DHA - treated rats as shown by the

photomicrographs (figures 2 & 3) confirmed that DHA treatment did not adversely

affect or damage them.

The significant increase in the packed cell volume (PCV) of the DHA - treated

albino rats (P<0.05), observed in the study, is an indication that the red blood cell

volume was increased by DHA - treatment. An increase in the packed cell

volume of the blood, suggests an increase in the oxygen carrying capacity of that

blood.

Neutrophils form the polymorphonuclear leucocytes which increase in number

and circulate during microbial (e.g bacterial' )infection. Neutrophils engage in

migration; chemotactic response; ingestion and microbial killing of pathogenic

micro - or ganisims (Brooks et al, 1995)

That DHA - treatment produced a highly significant increase (Pc0.01) in the

percentage nentrophil count of the white blood cell population, is an indication

that DHA increased the microbial fighting capacity of the rat.

The neutrophil exhibiting left -shift is an immature and precursor cell whose

lobes are not yet fully developed (Cheesbrough, 2000). The presence of left

shifted neutrophils in the blood of a rat treated with the repeated uose of DHA, is

a strong indication that DHA stimulated new production of neutropil white blood

cells.

A distinct population of lymphocytes which function as natural Killer (NK) cells

play a role in antibody dependent cellular cytotoxicity to herpes viruses and other

intracel'lular pathogens (Brooks et all 1995). There are also (CD4) T lymphocytes

which recognize the pathogens antigen complexed with class II MHC (major

histocompartibility complex) proteins on the surface of an antigens - presenting

ce\\ (Macrophage or B cell) and produce cytokines that activate B cell expressing

antibodies that specifically match the a~tigens. Since DHA treatment evoked

significant elevations of the percentage lymphocyte count ( P 4 . 0 1 ) of the wrlite

blood cell population, it is likely that DHA enhanced the antibody production

capacity of the rats.

Monocytes circulating in the blood as phagocytic cells are called macrophages.

Macrophages have a longer life span than circulating granulocytic phagocytes

(neutrophils) and continue their activity at a lower pH (Brooks et al, 1895). When

activated by activators like injury, infection, inflammation, antigen - antibody

complexes etc; they engage in intracellular killing of pathogens and in other

cellular defence actions. Since DHA treatment was found in this study to increase

the percentage monocyte counts (Table 10) it may be concluded that DHA

enhanced the production of more monocytes thus enlarging the macrophages

which engage in phagocytic defence af the body.

Basophils are usually present only in organrsrns which have very severe

infection. It was thus not surprising that basophik were absent in the blood of

both the DHA - treated and control rats.

The significant elevation of the total white blood cell count (P<0.01) by DHA -

treatment shows that DHA enhanced the body's defence capability of the white

blood cell system.

The histopathological findings of this study show that in comparison with the vital

organs of the control rats, the vital organs of the DHA - treated rats did not show

any evidence of cellular I tissue damage or necrosis which could indicate

evidence of toxicity of DHA to these vital organs (figure 2-7). It is thus concluded

- that there is no histopathological evidence that oral administration of

dihydroartemisinin regimen once or repeated after one week was toxic to the

heart, liver, spleen, intestine, lungs and kidney

FINAL CONCLUSION

From the gross anatomical, haematologi~al, serum enzyme activity and

histopathological findings of this study; it is concluded that the 5 day oral

administration of dihydroartemisinin whether given once or repeated after an

interval of one week; produced no toxic effects on the vital organs such as the

liver, heart, spleen, intestine, lungs, kidney and blood of wistar albino rats.

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