EFFECTS OF XYLOPIA AETHIOPICA EXTRACT AND MELATONIN …

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Udokwu et al. European Journal of Pharmaceutical and Medical Research

151

EFFECTS OF XYLOPIA AETHIOPICA EXTRACT AND MELATONIN ON OSMOTIC

FRAGILITY OF CYCLOPHOSPHAMIDE INTOXICATED WISTAR RATS

Udokwu Euphemia Ifeoma, Okoroiwu I. L., Anonde Andrew Chekwube and Okolie N. J. C. and Obeagu

Emmanuel Ifeanyi*

Department of Medical Laboratory Science, Imo State University, Owerri, Nigeria.

Article Received on 12/08/2020 Article Revised on 01/09/2020 Article Accepted on 22/09/2020

The hormone Melatonin is the main neuroendocrine

secretary product of the pineal gland in animals and an

evolutionary ancient derivative of serotonin with

hormonal properties (slominski et al., 2018). It is also

produced in plants where it functions as a first line of

defence against oxidative stress (Tan et al., 2012).

Xylopia aethiopica, a shrub locally referred to as

Ethiopian pepper, Negro pepper, Guinean pepper,

Senegal pepper, Kili pepper and spice tree in the savanna

zone and coastal regions of Africa is amongst these

plants with great therapeutic potential. It is an

angiosperm belonging to the family Annonaceae (Obodo

et al., 2013), and is among the species that thrive in the

evergreen rain forests of tropical and subtropical Africa

which matures into a slim, tall tree of approximately 60

cm in diameter and up to 30m high with a straight stem

having a slightly stripped or smooth bark.

The red blood cell integrity is largely dependent on its

ability to maintain its membrane constituents that are

mostly polyunsaturated free fatty acid, and if this is

compromised will result to erythrocyte fragility which

becomes more fragile with consequent destruction by the

macrophages. This erythrocyte fragility or red cell

osmotic fragility is the ability of red blood cells to

undergo haemolysis when subjected to stress and the

absolute extent of haemolysis can be measured (Rodak,

2007). When this happens the membranes of the cells

undergo lipid peroxidation leading to oxidative

deterioration of polyunsaturated fatty acid accumulation

of reactive oxygen species (ROS) which are associated

with tissue damage by clearing off the sialic acid from

the cells making them more prone to phagocytosis. Also,

the osmotic fragility of red cells can occur from the

increased phosphorylation of P38 and JNK genes which

promotes increased production of ROS (Robin and

Steven, 2000). Factors such as cell’s size, surface area to

volume ratio, membrane composition and integrity can

equally influence the osmotic fragility of the cells

(Fischbach, et al., 2008). Alteration in the integrity of

blood leads to loss of blood and can be life threatening as

blood is a necessary components of animal body. The

body tends to protect itself from this life threatening

exsanguination by converting the blood from its liquid

state to a solid state in a process known as blood clotting

or coagulation. This formation of a clot is often referred

to as secondary haemostasis and it usually involves two

main pathways namely extrinsic and intrinsic pathways

that make use of clotting factors. Estimation of

coagulation tests like prothrombin time ,activated partial

thromboplastin time etc. are developed to diagnose

disorders of coagulation which can lead to an increased

bleeding (haemorrhage) or obstructive clotting

(thrombosis) (Xiangqun, et al., 2014).

For this study, the cyclophosphamide was chosen

because it is one of the most frequently used antitumor

agents in clinical practice and also its association with

rapidly killing of dividing cells in the body.

Considering the above, this present study was designed

to evaluate the effects of Xylopia aethiopica and

melatonin on osmotic fragility in cyclophosphamide

induced wistar rats, with a view of finding a lasting

solution to the life threatening effects reported about this

cytotoxic drug.

AIM

The research work is aimed at evaluating the effects of

Xylopia aethiopica and melatonin on osmotic fragility in

cyclophosphamide intoxicated adult wistar rats.

SJIF Impact Factor 6.222

Research Article

ISSN 2394-3211

EJPMR

EUROPEAN JOURNAL OF PHARMACEUTICAL

AND MEDICAL RESEARCH

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ejpmr, 2020,7(10), 151-190

INTRODUCTION Cyclophosphamide has been in use clinically to treat a wide range of cancers including malignant lymphomas,

myeloma, leukaemia, mycosis, fungoides, neuroblastoma, adenocarcinoma, retinoblastoma, and breast carcinoma

(Mohammed et al., 2017). Other clinical uses for cyclophosphamide can be seen in immunosuppressive therapy

following organ transplants or as a treatment for autoimmune disorders such as rheumatoid arthritis, Wegener’s

granulomatosis, and nephritic syndrome in children (Chabner et al., 2001).

*Corresponding Author: Obeagu, Emmanuel Ifeanyi

Department of Medical Laboratory Science, Imo State University, Owerri, Nigeria.

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MATERIALS AND METHODS

COLLECTION OF PLANT MATERIALS AND AU

THENTICATION

Pods of Xylopia aethiopica were purchased from Orie-

Ugba vegetable market, Umuahia North Local

Government Area, Abia State, Nigeria. and were taken to

the Department of Forestry and Environmental

Management, Michael Okpara University of Agriculture,

Umudike where they were identified by a botanist/forest

manager. Voucher number MOUAU/VPP/18/012 was

assigned to a specimen sample of the pods which was

deposited in the herbarium of the Department.

PREPARATION OF PLANT EXTRACTS

Extract of the fruit pods was prepared in accordance with

the Soxhlet method described by Jensen, (2007). The

plant materials were subjected to further drying under

shade for 14 days and were pulverized into powder in a

manual blender powered by a Honda petrol engine. One

hundred grams of the powdered sample was introduced

into the extraction chamber of the soxhlet extractor and

extraction was carried out with ethanol as solvent.

Temperature was maintained at 650C throughout the

extraction period of 48 hours. At the end of the period,

the extract in solution was dried in a hot air oven at 40oC

to obtain a dry dark oily extract. The weight of the

extract was taken and percentage yield was calculated

using the formular:

% yield =X x 100

Q 1

Where X = weight of dried extract and Q = weight of

powdered plant material before extraction (100g)

(Bandiola, 2018).

ANIMALS USED FOR STUDY

One hundred and ninety five matured wistar albino rats

were used for the studies. Of the number, 30 were used

for the acute toxicity evaluation of the extract, 35 for

acute toxicity study of cyclophosphamide and 130 were

used for the main study. The rats were kept in aluminum

cages and allowed to acclimatize for two weeks to allow

for proper adaptation to the environment and living

conditions. They were allowed access to feed (Vital feed,

Nigeria) and water ad libitum but were starved for 12

hours prior to commencement of any experiment. All

animal experiments were carried out in compliance with

NIH guidelines for Care and Use of Laboratory Animals

(OECD, 2001). All experiments were carried out in the

Physiology Laboratory of the Department of Physiology

and Pharmacology, College of Veterinary Medicine,

Michael Okpara University of Agriculture, Umudike,

Nigeria.

EXPERIMENTAL DESIGN The rats (130 in number) were assigned to 13 groups of

10 rats each and were treated according to the order

below:

Group I: 10 mg/kg Cyclophosphamide, Food and water

Group II: 10 mg/kg Cyclophosphamide, 400 mg/kg

Extract, Food and water

Group III: 10 mg/kg Cyclophosphamide, 0.5 mg/kg

Melatonin, Food and water

Group IV: 10 mg/kg Cyclophosphamide, 400mg/kg

Extract, 0.5mg/kg Melatonin, Food and water

Group V: 30 mg/kg Cyclophosphamide, Food and water

Group VI: 30 mg/kg Cyclophosphamide, 400 mg/kg


Group VII: 30mg/kg Cyclophosphamide, 0.5mg/kg


Group VIII: 30mg/kg Cyclophosphamide+400mg/kg


Group IX: 50 mg/kg Cyclophosphamide, Food and water

Group X: 50 mg/kg Cyclophosphamide, 400 mg/kg


Group XI: 50mg/kg Cyclophosphamide, 0.5mg/kg


Group XII: 50mg/kg Cyclophosphamide, 400mg/kg


Group XIII: Food and water only

Treatments were done daily via the oral route for twenty

one (21) days. Three animals were sacrificed in each

group for blood collection by cardiac puncture into

EDTA and sodium citrate bottles for haematology and

osmotic fragility studies. Liver and kidney samples were

also collected and preserved in 10% formalin for

histological examination.

DETERMINATION OF RED BLOOD CELLS

OSMOTIC FRAGILITY

The method of Adenkole and Olurenmi, (2014) was

adopted. Sodium chloride solution (200 ml) was

prepared for each sample in concentrations ranging from

0.1-0.85% at pH 7.4. A set of test tubes, each containing

5mls of sodium chloride solution of concentration

ranging from 0.1 to 0.85% were serially arranged in a

test tube rack. One set of test tubes was used to analyze

each sample. A drop of the freshly collected blood was

placed into each of the ten test tubes using a dropper

pipette and each was mixed by gently inverting the test

tubes about three times. The test tubes were then allowed

to stand at room temperature for 30 minutes and then

centrifuged at 3000 rpm for 10 minutes before reading

the absorbance of the supernatant in each test tube on a

spectrophotometer at 540nm. The same procedure was

repeated for each sample collected. Percentage

haemolysis was calculated using the expression:

Percentage Haemolysis = Absorbance of test x100

Absorbance of control

STATISTICAL ANALYSIS

Results were expressed as means ± standard error of

mean (SEM). Statistical analysis was done using one-

way analysis of variance (ANOVA). Significant

differences were assessed at 95% level of significance

between control and treated groups using Duncan and

LSD (Post Hoc) tests. P values less than 0.05 were

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considered significant. Computer software package,

SPSS version 21 was employed.

RESULTS

Result of effects of Xylopia aethiopica extract and

Melatonin on osmotic fragility (0) scores of

Cyclophosphamide intoxicated wistar rats In week one and two and three, there was no significant

difference in all the treatment groups compared with the

control group.

In week one, two and three, treatment with 400mg

Xylopia aethiopica and 0.5mg Melatonin alone and in

combination did not significantly osmotic fragility of rats

exposed to 10, 30 and 50mg Cyclophosphamide.

Table 1: Effects of Xylopia aethiopica extract and Melatonin on osmotic fragility (0) scores of Cyclophosphamide

intoxicated wistar rats.

Treatment 0 (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week 3

(Wk3)

p-

value

p-

value

p-

value

p-

value

F-

value

10m Cyclophosphamide 100±0 a 100.27±0.32

a 100.6±0.31

a 1.000 1.000 .922 .318 1.394

10mg Cyclophosphamide+

400mg Xylopia aethiopia 100±0

a 100.27±0.32

a 100.3±0.35

a 1.000 1.000 1.000 .711 .361

10mg Cyclophosphamide +

0.5mg Melatonin 100±0

a 100.27±0.32

1.000 1.000 .449 .703


400mgXylopia aethiopia +

0.5mg Melatonin

100±0 a 100.27±0.32

a 100.3±0.35

a 1.000 1.000 1.000 .711 .361

30mg Cyclophosphamide 100±0 a 100.27±0.32

a

1.000 1.000 .449 .703


400mg Xylopia aethiopica 100±0

a 100.27±0.32

a

1.000 1.000 .449 .703



a 100.27±0.32

a

1.000 1.000 .449 .703


400mg Xylopia aethiopica+

0.5mgMelatonn

100±0 a 100.27±0.32

a

1.000 1.000 .449 .703


a

1.000 1.000 .449 .703



a 100.27±0.32

a

1.000 1.000 .449 .703

50mg Cyclophoshamide +


a 100.27±0.32

a

1.000 1.000 .449 .703


400mg Xylopia aethiopia+

0.5mg Melatonin

100±0 a 100.27±0.32

a

1.000 1.000 .449 .703

Control 100±0 a 100.27±0.32

a 100.3±0.35

a .711 .361


Melatonin on osmotic fragility (0.1) scores of

Cyclophosphamide intoxicated wistar rats

In week one, two and three, there was significant

increase in osmotic fragility in all the treatment groups

compared with control except group 4 in week one and

three. Osmotic fragility varied significantly from week

one to three in all the treatment except group 13.

In week one and two, there is a dose dependent increase

in osmotic fragility of rats treated with 10, 30 and 50mg

of Cyclophosphamide. Treatment with 400mg Xylopia

aethiopica alone and 400mg Xylopia aethiopica and

0.5mg Melatonin and 0.5mg Melatonin significantly

decreased the osmotic fragility compared with rats

exposed to 10mg Cyclophosphamide. Similarly,

treatment with 400mg Xylopia aethiopica alone and

400mg Xylopia aethiopica and 0.5mg Melatonin

significantly decreased osmotic fragility of rats exposed

to 30mg Cyclophosphamide. Osmotic fragility of rats

treated with 400mg Xylopia aethiopica and 0.5mg

Melatonin was significantly lower than 400mg Xylopia

aethiopica alone. Treatment with 400mg Xylopia

aethiopica and 0.5mg Melatonin significantly decreased

osmotic fragility of rats exposed to 50mg

Cyclophosphamide.

In week three, treatment with 400mg Xylopia aethiopica

alone and in combination 0.5 mg Melatonin significantly

decreased the osmotic fragility of rats exposed to 10mg

Cyclophosphamide.

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Table 2: Effects of Xylopia aethiopica extract and Melatonin on osmotic fragility (0.1) scores of

Cyclophosphamide intoxicated wistar rats.

Treatment 0.1 (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week 3

(Wk3) p-value p-value p-value p-value f-value

10mg Cyclophosphamide 92.2±0.12 h 92.05±0.29

e 97.02±0.69

b .000 .000 .000 .000 41.529


400mg Xylopia aethiopica 90.1±0.23

g 89.39±0.28

d 83.95±0.29

a .000 .011 .172 .000 154.267


0.5mg Melatonin 89.54±0.28

g 89.14±0.28

d .000 .045 .368 1.029


400mg Xylopia aethiopica +

0.5mg Melatonin

88.1±0.06 f 88.39±0.28

cd 85.41±0.3

a .036 .811 1.000 .000 47.286

30mg Cyclophosphamide 98.2±0.06 j 97.77±0.31

g .000 .000 .242 1.887



i 94.97±0.3

f .000 .000 .652 .237



d 85.73±0.27

b .000 .004 .168 2.820



0.5mg Melatonin

85.21±0.01 c 85.34±0.27

b .000 .000 .662 .223


g .000 .000 .798 .075



i 94.5±0.3

f .000 .000 .096 4.683



b 89.26±0.28

d .000 .023 .000 470.987



0.5mg Melatonin

82.12±0.02 a 83.34±0.26

a .000 .000 .010 21.376

Control 87.51±0.01 e 87.64±0.28

c 85.37±0.3

a .091 29.403



Cyclophosphamide intoxicated wistar rats In week one and two, there is a dose dependent increase

in osmotic fragility of rats treated with 10, 30 and 50 mg

of Cyclophosphamide. Osmotic fragility of rats exposed

to 10mg Cyclophosphamide and treated with 400mg

Xylopia aethiopica was significantly higher 10mg

Cyclophosphamide alone. Similarly, treatment with

400mg Xylopia aethiopica alone and 400mg Xylopia



Cyclophosphamide. Treatment with 400mg Xylopia

aethiopica and 0.5mg Melatonin alone and in

combination significantly decreased osmotic fragility of

rats exposed to 50mg Cyclophosphamide.


alone and in combination 0.5mg Melatonin significantly


Cyclophosphamide.




Week 1

(Wk1)

Week 2

(Wk2)

Week 3


10mg Cyclophosphamide 88.1±0.01 ef

89.54±0.28 g 91.27±0.5

c .000 .000 .000 .002 22.699

10mg Cyclophosphamide

+400mg Xylopia aethiopica 89.2±0.64

g 86.53±0.27

f 83.44±0.29

a .000 .000 1.000 .000 44.219



fg 88.14±0.28

g

.000 .000 .098 4.635


+400mg Xylopia aethiopica +

0.5mg Melatonin

89.02±0.14 f 88.36±0.28

g 85.36±0.3

b .000 .000 .019 .000 61.092


g

.000 .000 .024 12.523



e 85.33±0.27

ef

.000 .362 .003 45.415

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d 83.32±0.26

cd

.281 .356 .018 15.076



0.5mg Melatonin

82.2±0.06 c 83.53±0.26

cd

.000 .683 .008 24.037

50mg Cyclophosphamide 92.1±0.01 i 95.4±0.3

i

.000 .000 .000 119.485



c 82.52±0.26

bc

.000 .004 .091 4.909



b 81.56±0.26

b

.000 .000 .005 29.927



0.5mg Melatonin

78.13±0.03 a 78.31±0.25

a

.000 .000 .510 .522

Control 85.2±0.12a 84.33±0.27

de 83.39±0.29

a .005 14.440





in osmotic fragility of rats treated with 10, 30 and 50

mg/kg of Cyclophosphamide. Treatment with 400mg

Xylopia aethiopica alone and 400mg Xylopia aethiopica

and 0.5 mg Melatonin and 0.5 mg Melatonin

significantly decreased the osmotic fragility compared

with rats exposed to 10mg Cyclophosphamide. Similarly,










Cyclophosphamide.




Cyclophosphamide.




Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)

p-

value p-

value p-

value p-

value f-value

10mg Cyclophosphamide 85.23±0.02 g 86.47±0.27

f 89.17±1.46 b .000 .000 .000 .044 5.516

10mg Cyclophosphamide + 400mg Xylopia aethiopica

81.53±0.01 e 82.72±0.26

cd 80.65±0.28 a .000 1.000 .444 .002 21.806

10mg Cyclophosphamide + 0.5mg Melatonin

80.39±0.25 d 81.6±0.26

c

.000 .036

.029 11.140

10mg Cyclophosphamide + 400mg Xylopia aethiopica + 0.5mg Melatonin

79.08±0.57 c 78.3±0.25

b 79.33±0.28 a .000 .000 .982 .227 1.919


h

.000 .000

.008 24.148 30mg Cyclophosphamide + 400mg Xylopia aethiopica

89.11±0.05 h 88.65±0.28

g

.000 .000

.186 2.542


83.02±0.01 f 84.95±0.27

e

1.000 .001

.002 51.160

30mg Cyclophosphamide + 400mg Xylopia Aethiopica + 0.5mg Melatonin

75.23±0.02 b 77.54±0.25

b

.000 .000

.001 87.840


i

.000 .000


80.15±0.01 d 81.36±0.26

c

.000 .008

.009 21.890


79.13±0.02 c 77.4±0.25

b

.000 .000

.002 49.378

50mg Cyclophosphamide + 400mg Xylopia aethiopica+ 0.5mg Melatonin

70.12±0.06 a 75.35±0.24

a

.000 .000

.000 447.667

Control 83.1±0.01 f 83.02±0.26

d 78.94±0.28 a

.000 116.704

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Cyclophosphamideintoxicated wistar rats

In week one and two, there was a dose dependent

increase in osmotic fragility of rats treated with 10, 30

and 50 mg/kg of Cyclophosphamide. Osmotic fragility of

rats exposed to 10mg Cyclophosphamide and treated

with 400mg Xylopia aethiopica and 0.5mg Melatonin

was significantly lower than 10mg Cyclophosphamide

but osmotic fragility of other treatment was significantly

higher compared with 10mg Cyclophosphamide.

Similarly, treatment with Melatoninalone and 400mg

Xylopia aethiopica and 0.5mg Melatonin significantly

decreased osmotic fragility of rats exposed to 30mg








Cyclophosphamide.




Week 1

(Wk1)

Week 2

(Wk2)

Week 3


10mg Cyclophosphamide 72.12±0.58 e 70.47±0.22

ef 79.12±0.51

b .000 .145 .000 .000 98.344



g 71.23±0.23

f 66.54±0.23

a .000 .001 .252 .000 511.581



f 67.42±0.21

c

.000 .000 .000 347.082



0.5mg Melatonin

69.01±0.05 c 66.09±0.21

b 66.11±0.23

a .013 .000 .070 .000 84.995


j

.000 .000 .007 26.773



h 76.42±0.24

i

.000 .000 .002 48.958



ef 72.41±0.23

g

.000 .000 .057 6.989



0.5mg Melatonin

74.92±0.33 g 73.92±0.23

h

.000 .000 .071 5.989


h

.000 .000 .448 .707



c 68.36±0.22

cd

.049 .066 .020 13.889



a 65.37±0.21

b

.000 .000 .000 629.949



0.5mg Melatonin

65.12±0.01 b 63.29±0.2

a

.000 .000 .001 83.202

Control 70.21±0.12 d 69.48±0.22

de 67.47±0.24

a .000 50.546






and 50mg of Cyclophosphamide.

Osmotic fragility of rats exposed to 10mg

Cyclophosphamide and treated with 400mg Xylopia

aethiopica and 0.5 mg Melatonin alone and in

combination was significantly lower than 10mg

Cyclophosphamide. Similarly, treatment with

Melatoninalone significantly decreased osmotic fragility

of rats exposed to 30mg Cyclophosphamide. Treatment


alone and in combination significantly decreased osmotic

fragility of rats exposed to 50mg Cyclophosphamide.

In week three, osmotic fragility of rats exposed to 10mg


aethiopica alone and in combination 0.5 mg Melatonin

was not significantly different from of rats exposed to

10mg Cyclophosphamide.

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Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)

p-

value p-

value p-

value p-

value f-value

10mg Cyclophosphamide 33.21±1.61 d 33.3±0.11

d 35.73±2.36 b 1.000 .000 .000 .513 .747


30.15±0.05 c 25.17±0.08

b 30.9±0.11 b .376 .000 .008 .421 1.004


26.58±0.08 b 23.74±0.08

a

1.000 .105

.000 633.634


31.12±0.05 cd 31.2±0.1

ca 31.21±0.11 b 1.000 .000 .006 .737 .321


f

1.000 .000

.456 .681 30mg Cyclophosphamide + 400mg Xylopia aethiopica

40.13±0.01 e 40.24±0.13

f

1.000 .000

.451 .697


38.12±0.02 e 38.22±0.12

e

1.000 .000

.453 .689


40.12±0.01 e 40.23±0.13

f

1.000 .000

.451 .697


i

1.000 .000


48.13±0.01 g 48.26±0.15

h

1.000 .000

.450 .699


44.15±0.01 f 44.27±0.14

g

1.000 .000

.449 .702


40.15±0.01 e 40.26±0.13

f

1.000 .000

.449 .702

Control 23.1±0.58 a 23.17±0.07

a 23.18±0.08 a

.985 .015



Cyclophosphamideintoxicated wistar rats In week one and two, there was a dose dependent


and 50 mg/kg of Cyclophosphamide.

In week one, osmotic fragility of rats exposed to 10mg



combination was not significantly different compared to

rats exposed to 10mg Cyclophosphamide. Similarly,





rats exposed to 30 mg Cyclophosphamide. Treatment

with 0.5mg Melatonin alone and in combination with

400mg Xylopia aethiopicasignificantly decreased


Cyclophosphamide.

In week 2, treatment with 400 mg Xylopia aethiopica

alone and 400 mg Xylopia aethiopica and 0.5 mg

Melatonin and 0.5mg Melatonin significantly decreased

the osmotic fragility compared with rats exposed to

10mg Cyclophosphamide. Similarly, treatment with 400

mg Xylopia aethiopica and 0.5mg Melatonin


to 30mg Cyclophosphamide. Treatment with 400mg



Cyclophosphamide.




was significantly lower than rats exposed to 10mg

Cyclophosphamide.

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Treatment

0.6

(Wk1) (Wk2) (Wk3) A.Wk

Week

(Wk1)

Week 2

(Wk2)

Week 3


10mg Cyclophosphamide 20.13±0.01 bc

20.18±0.06 d 20.25±0.06

d .000 .000 .000 .323 1.371



b 18.18±0.06

c 19.62±0.07

c .000 .000 .000 .000 113.968



b 18.1±0.06

c

.001 .000 .031 10.733



0.5mg Melatonin

16.1±0.01 b 16.14±0.05

b 16.15±0.06

b .007 .000 .000 .712 .359


20.18±0.06 d

.000 .000 .474 .622



bc 22.21±0.07

f

.000 .000 .470 .635



bc 20.18±0.06

d

.000 .000 .474 .622



0.5mg Melatonin

18.13±0.01 b 18.18±0.06

b

.000 .000 .457 .676


i

.000 .000 .454 .687


400mg Xylopia aethiopicaa 32.13±0.01

d 32.22±0.1

h

.000 .000 .452 .694



cd 26.21±0.08

g

.000 .000 .529 .474



0.5mg Melatonin

21.13±0.01 bc

21.19±0.07 e

.000 .000 .455 .683

Control 8.47±4.36 a 13.24±0.04

a 13.24±0.05

a .365 1.198



Cyclophosphamide intoxicated wistar rats In week one and two, treatment with 400 mg Xylopia





treatment with 400 mg Xylopia aethiopica and 0.5mg

Melatonin significantly decreased osmotic fragility of

rats exposed to 30mg Cyclophosphamide. Treatment



to 50mg Cyclophosphamide.


Cyclophosphamide and treated with 400 mg Xylopia

aethiopica alone and in combination 0.5mg Melatonin


Cyclophosphamide.


Cyclophosphamideintoxicated wistar rat.

Treatment

0.7


Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)

p-value p-value p-value p-value f-value


f 16.28±1 c .000 .000 .000 .395 1.088


12.12±0.05 c 12.15±0.04

c 13.05±0.05 b .000 .000 .018 .000 133.330


12.02±0.04 c 13.22±0.04

d

.000 .000

.000 451.130


11.13±0.01 b 11.16±0.04

b 11.16±0.04 ab .000 .000 .582 .722 .345


h

.000 .000


20.14±0.01 h 20.19±0.06

i

.000 .000

.451 .697

30mg Cyclophosphamide + 16.14±0.01 f 16.18±0.05

g

.000 .000

.459 .669

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0.5mg Melatonin 30mg Cyclophosphamide + 400mg Xylopia aethiopica+ 0.5mg Melatonin

15.14±0.01 e 15.18±0.05

f

.000 .000

.452 .693


j

.000 .000


15.14±0.01 e 15.18±0.05

f

.000 .000

.452 .693


16.14±0.01 f 16.18±0.05

g

.000 .000

.459 .669


14.14±0.02 d 14.18±0.04

e

.000 .000

.497 .556

Control 10.2±0 a 10.23±0.03

a 10.23±0.04 a

.711 .361




In week one and two, treatment with 400mg Xylopia





treatment with 400mg Xylopia aethiopica and 0.5mg

Melatonin and the combination significantly decreased





Cyclophosphamide.





Cyclophosphamide.


Cyclophosphamide induced toxicity rats.

Treatment

0.8


Week 1

(Wk1)

Week 2

(Wk2)

Week 3



e 13.94±0.66

c .000 .000 .000 .630 .499



bc 10.17±0.03

c 10.14±0.04

b .000 .000 .005 .837 .183



b 9.15±0.03

b

.000 .000 .000 332.650



0.5mg Melatonin

9.04±0.02 b 9.06±0.03

b 9.07±0.03

ab .021 .000 .109 .744 .311


f

.000 .000 .731 .136


400mg Xylopia aethiopica 13.13±0

d 13.17±0.04

e

.000 .000 .449 .703



c 11.18±0.04

d

.000 .000 .521 .493



0.5mg Melatonin

11.14±0.01 c 11.17±0.04

d

.000 .000 .454 .685

50mg Cyclophosphamide 20.13±0.01 f 20.18±0.06

g

.000 .000 .456 .681



bc 10.13±0.03

c

.000 .000 .633 .266



b 9.14±0.03

b

.011 .000 .480 .607


400mg Xylopia Aethiopica +

0.5mg Melatonin

10.12±0.02 bc

10.15±0.03 c

.000 .000 .501 .545

Control 7.8±0.17 a 7.83±0.02

a 7.83±0.03

a .973 .027

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Cyclophosphamide intoxicated wistar rats In week one, osmotic fragility of rats at exposed to 10mg


aethiopica and 0.5mg Melatonin and the combination

was significantly higher than rats treated with 10mg







Cyclophosphamide.

In week two, treatment with 400mg Xylopia aethiopica

alone and 400 mg Xylopia aethiopica and 0.5mg



10mg Cyclophosphamide. Similarly, treatment with

400mg Xylopia aethiopica and 0.5 mg Melatonin and the










Cyclophosphamide.

Table 10: Effects of Xylopia aethiopica extract and melatonin on osmotic fragility (0.85) scores of

cyclophosphamide intoxicated wistar rats.

Treatment 0.85


Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)

p-value p-value p-

value p-value f-value

10mg Cyclophosphamide 5.7±0.58 abc 10.74±0.03

h 11.88±0.12 d .750 .000 .000 .000 93.078


6.8±0.18 def 6.53±0.02

d 5.72±0.02 c .506 .000 .006 .001 27.192


7.05±0.02 ef 5.92±0.02

b

.095 .000

.000 1509.052


5.3±0.14 abc 5.22±0.02

a 4.72±0.02 a .064 .000 .001 .006 13.917


i

.000 .000


8±0.17 g 7.03±0.02

e

.000 .000

.005 31.130


6.01±0.01 bcd 8.03±0.03

g

1.000 .000

.000 6002.250


5.13±0.01 ab 7.15±0.02

e

.014 .000

.000 6307.375


j

.000 .000


7.12±0.01 efg 7.74±0.02

f

.054 .000

.000 608.567


7.13±0.01 fg 5.14±0.02

a

.050 .000

.000 9891.754

50mg Cyclophosphamide + 400mg Xylopia aethiopica+


a 5.91±0.02 b

.004 .000

.000 1659.689

Control 6.2±0.06 cde 6.22±0.02

c 5.31±0.02 b

.000 200.305

DISCUSSION

Results of osmotic fragility scores indicates that CP

significantly reduced RBC percentage haemolysis in the

treated rats when compared with the control in different

salt concentrations. The result suggest that XA may have

increased the integrity of the RBC cell membranes

following treatment and made them resist the haemolytic

effects of the various salt concentrations. It is established

that the performance of normal functions by the

erythrocytes is highly dependent on their membrane

stability and ability to resist lysis (Adenkola and Oluremi

2014). The impact of free radicals on erythrocyte

membrane is a major cause of reduction in its ability to

resist lysis (Devasagayam et al., 2004; Dragan et al.,

2003). The higher membrane stability observed in all

groups treated with XA may be attributed to the

antioxidant potentials of the extract. Antioxidants have

greatly been implicated in the prevention of cellular

damage and generally consolidate the integrity of

erythrocyte membrane by reducing their oxidative

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161

damage due the impact of free radicals (Adenkola and

Oluremi 2014).

CONCLUSION

This study provides evidence that Xylopia aethiopica is a

valuable medicinal food for combating

cyclophosphamide induced systemic toxicity. Xylopia

aethiopica may provide protective effects for toxicants

capable of inducing oxidative stress. Also It can be seen

that despite the high potent immunosuppressive effect of

cyclophosphamide on blood cells, melatonin and Xylopia

aethiopica have shown to exert their ameliorative effects

through their antioxidant and antitumour properties.

Therefore, they may be of value in the prevention of

diseases arising from the oxidative effects of consumed

toxicant substances like cyclophosphamide.

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CHAPTER ONE

1.0 INTRODUCTION The increase in the cases of cancer has been on the

alarming rate in recent years which has given great

concern to health care providers, and there have been

concerted efforts in awareness creation on the menace of

these cancers. Chemotherapy which is a category of

cancer treatment that uses one or more anti–cancer drugs

as part of a standardized regime is one of the major

categories of the medical discipline specifically devoted

to pharmacotherapy for cancer, and as of 1970′s, these

chemotherapeutic agents (cytotoxic agents) have been

recognised as a definitive treatment or as an adjuvant

therapy in asymptomatic patients with the aim of

improving survival (Slater, 2001).

Cyclophosphamide, a chemotherapeutic agent was

approved for medical use in 1959 in the United States

and it is on the world health organisation′s list of

essential medicines, the most effective and safe medicine

needed in health system (WHO, 2015). It has a molecular

formula of C7H15Cl2N2O2P•H2O and a molecular weight

of 279.1. Its chemical name is 2-[bis(2-

chloroethyl)amino]tetrahydro-2H-1, 3, 2-

oxazaphosphorin 2-oxide monohydrate, and it has the

following structural formula:

Fig 1.1: Chemical Structure of Cyclophosphamide

(Wang and Wang, 2012)

In many cancer patients, it is the first oxazaphosphorine

agent that achieved great success in its clinical

application. Although it has been clinically available for

over a decade, it is still seen to be amongst the front-line

choices of chemotherapy for solid tumors (Wang and

Wang, 2012).

It appears as fine white to off-white crystalline powder

that is soluble in water, saline, and ethanol. It is

odourless and an antineoplastic compound that belongs

to the nitrogen mustard a subclass of alkylating agents. It

is a well-known mutagen and clastogen, as well as an

alkylating agent that produces the highly active

carbonium ion, which reacts with the electron-rich area

of nucleic acids and proteins (Shokrzadeh et al., 2014).

Cyclophosphamide is an immune modulatory agent

which entails regulation of immune system by

suppression and stimulation of the cells and organs of

immune system (Oladunmoye, 2006). As an

immunosuppressant, it alkylates DNA, thereby

interfering with its synthesis and function, particularly in

proliferating lymphocytes (B and T cells), although the

toxicity produced is greater on B-cells than the T-cells

(Schmidt and Koelbl, 2012). This drug exerts its greatest

effect by suppressing humoral immunity. It does that

with alkylating properties that result in nucleotide base

mispairs and DNA/DNA or DNA/protein cross-linking

that lead to major disruptions in nucleic acid function

and the inhibition of DNA synthesis (Zhang et al., 2005).

This cyclophosphamide-induced nucleic acid damage

may lead to DNA mutations that result in cytotoxicity,

carcinogenicity, teratogenecity, and reproductive toxicity

following chronic exposure to cyclophosphamide

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(Meirow and Nugent, 2001). On the other hand,

cyclophosphamide, although historically regarded as an

immunosuppressant, has been shown to act as a strong

adjuvant for either adoptive or active immunotherapy

when used with carefully defined dosages and

combination modalities. To further explain this

stimulatory effects of cyclophosphamide, Proietti et

al.(1998), using mouse models, identified type I

interferon (IFN-I) as an important mediator of

cyclophosphamide immunomodulation. Subsequent

studies showed that IFN-I was indeed induced in vivo by

cyclophosphamide and that this cytokine was responsible

for the expansion of memory CD4+ and CD8

+ T cells.

More recent data indicated that cyclophosphamide can

affect dendritic cell homeostasis and can restore an

activated polyfunctional helper phenotype in tumor-

specific adoptively transferred CD4+ T cells through

IFN-I–dependent mechanisms (Moschella etal., 2013).

Nakaharaetal.(2010), demonstrated in their work that

cyclophosphamide can improve immune responses by

preferentially depleting CD8+ lymphoid-resident

dendritic cells (DCs), thereby leading to diminished Treg

suppression and enhanced effector T-cell function in

vivo.

Cyclophosphamide has been in use clinically to treat a

wide range of cancers including malignant lymphomas,

myeloma, leukaemia, mycosis, fungoides,

neuroblastoma, adenocarcinoma, retinoblastoma, and

breast carcinoma (Mohammed et al., 2017). Other

clinical uses for cyclophosphamide can be seen in

immunosuppressive therapy following organ transplants

or as a treatment for autoimmune disorders such as

rheumatoid arthritis, Wegener’s granulomatosis, and

nephritic syndrome in children (Chabner et al., 2001).

Cyclophosphamide metabolism takes place in the liver

and is relatively inert until the binding phosphorus-

nitrogen is broken by means of metabolism catalysed by

hepatic enzymes of cytochrome P450, which are

responsible for the reaction of initial drug activation via

4-hydroxylation and N-dechloroethylation. Cytochrome

P4502B6, cytochrome P4503A4, cytochrome P4502C9,

and cytochrome P4502C19 have been shown to be the

major cytochrome P450s responsible for the metabolism

of cyclophosphamide and among these enzymes,

cytochrome P4502B6 was pointed out as the main

cytochrome P450 isozyme for the formation of 4-

hydroxy-cyclophosphamide (4-OH-CPA) (Afsharian et

al., 2007). When the inactive cyclophosphamide is

metabolized by P450 oxidase enzymes, it is transformed

into 4-hydroxy-cyclophosphamide that originates

the aldophosphamide. The latter, by its time, is carried to

other tissues, where it is converted into mustard

phosphoramide (the effectively cytotoxic molecule) and

acrolein, which is responsible for the adverse effects

(Tatiane et al., 2007).

The use of cyclophosphamide is however, limited by its

toxicity. Some of the adverse effects may include

alopecia, nausea, vomiting, thrombocytopenia, mucosal

ulcerations, transverse striations in the nails, brief spells

of dizziness, increased skin pigmentation, pulmonary

fibrosis, leukopenia, facial abrasion, haematuria,

diarrhoea, haemorrhagic cystitis, and petechial

haemorrhage in lungs and small bowel (Gitanjah et al.,

2017), but negative effects on the haematological system

have been observed especially in leucocyte and platelet

levels (Azevedo et al., 2007). It also affects cells that

respond to erythropoietic agents resulting in progressive

anaemia (Woo et al., 2008), and as a pro-oxidant drug it

can elicit generation of oxidative stress after

administration (Ikumawoyi et al., 2016). The

cyclophosphamide as a chemotherapeutic drug kills

dividing cells rapidly in the body, including cancer cells

and normal cells (Chakraborty et al., 2009). This

negative health effects associated with

cyclophosphamide present a significant health and safety

threat to laboratory staff, animal handlers, and other

personnel who may be subject to accidental exposure. As

a result of this health and safety threat the Institutional

Biosafety Committee (IBC) has classified

cyclophosphamide as a reportable hazardous chemical

that must be registered on the Institutional Animal Care

and Use Committee (IACUC) protocol as Chemical

Hazards. Despite its wide spectrum of clinical uses,

cyclophosphamide has been implicated in some adverse

conditions like hepatotoxicity, nephrotoxicity

haematotoxicity etc. and in attempt to reduce high level

of toxicity and check increase in the resistance of this

drug, various studies have been conducted in the past to

test the efficacy of low dose of cyclophosphamide in

treatment of carcinomas with positive results. Therefore,

cellular sensitivity to cyclophosphamide can be seen as

a function of cellular thiol concentration, metabolism

by aldehyde dehydrogenases to form inactive

metabolites, and the ability of DNA to repair alkylated

nucleotides.

The hormone Melatonin is the main neuroendocrine

secretary product of the pineal gland in animals and an

evolutionary ancient derivative of serotonin with

hormonal properties (slominski et al., 2018). It is also

produced in plants where it functions as a first line of

defence against oxidative stress (Tan et al., 2012). This

Melatonin, also known as N-acetyl-5-methoxy

tryptamine exists in organisms as different as algae, rats

and humans, and it is synthesized by a number of cells

and tissues but in mammals, the pineal gland is the main

source of this indolamine. The synthetic one has a

molecular formula of C13H16N2O2 and molecular weight

of 232.283.

It serves as an endocrine signal contributing to

entrainment/ regulation of organisms’ circadian rhythms

(Anthony et al., 2016), with maximum formation of this

melatonin occurring during the night. It′s nocturnal

synthesis is mainly regulated by the norepinephrine

released from sympathetic nerve endings, triggering the

transcription and translation of arylalkylamine-N-

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acetyltransferase, the most important enzyme involved in

melatonin synthesis (Ferreira et al., 2013). Melatonin is

involved in important physiological functions like

control of seasonal reproduction as well as influences on

the immune system. It also has anti-ageing effect and can

affect body mass, adipocity and energy metabolism

which vary according to species. This pineal indolamine

is a very potent and efficient endogenous radical

scavenger that reacts with the highly toxic hydroxyl

radical providing on‐site protection against oxidative

damage to biomolecules within every cellular

compartment (Burkhard et al., 1993). It quenches the

peroxyl radical, hypochlorous acid, and singlet oxygen,

all of which cause cell damage. Previous studies showed

that melatonin is preferentially localized inside the

nucleus and can protect nuclear DNA from oxidative

damage by interacting with double-stranded DNA

thereby promoting its stability. More so, it′s powerful

antioxidant action acts either directly on free radical

species or by modulating the gene expression of

antioxidant enzymes such as glutathione peroxidase,

catalase and superoxide dismutase. This antioxidant

effect of melatonin involves DNA repair, and can repair

the oxidation induced by the guanosine (G•) radical

(Ferreira et al., 2013). It can protect tissues from the

oxidative damage caused by glutathione depletion and

ischemia-reperfusion injury. Apart from its antioxidant

property, melatonin is a potential antitumor agent.

Therefore, studying of the effects of melatonin in

chemotherapy seems as an interesting area of

investigation.

Since time immemorial, the use of plants and its extracts

for the treatment and management of diseases has been

in existence all over the globe especially in Africa

particularly in the south Sahara (Woode et al., 2011).

This was assumed to be due to poverty and illiteracy,

which limited their accessibility to conventional medical

services. However, a large number of these tropical

plants and their extracts have beneficial therapeutic

effects such as contraceptive, aphrodisiac, fertility

enhancing capacities, as well as anti-oxidant, anti-

inflammatory, anti-cancer, and anti-microbial potentials

(Raji et al., 2006). Also, evidence abound that plant

derivatives have therapeutic potentials against vast

human, animal and plant diseases (Ogbonnia et al.,

2008), and thus, plants have become indispensable to

human and animal existence. In fact, according to Chris-

Ozoko et al. (2015), several conventional products

derived from plants exist today such as Aspirin (a

chemical copy of the analgesic chemical in the bark of

willow tree), Digoxin (from fox glove), morphine (from

Opium Poppy) etc. and interestingly, many herbal

products are now been approved by health agencies and

organizations.

Xylopiaaethiopica, a shrub locally referred to as

Ethiopian pepper, Negro pepper, Guinean pepper,

Senegal pepper, Kili pepper and spice tree in the savanna

zone and coastal regions of Africa is amongst these

plants with great therapeutic potential. It is an

angiosperm belonging to the family Annonaceae (Obodo

et al., 2013), and is among the species that thrive in the

evergreen rain forests of tropical and subtropical Africa

which matures into a slim, tall tree of approximately 60

cm in diameter and up to 30m high with a straight stem

having a slightly stripped or smooth bark. The seeds of

Xylopiaaethiopica which have musky flavours are used

as pepper substitutes in Nigeria and the fruits are

popularly used as a condiment in many local dishes by

ethnic groups and communities in Africa. In the Igbo

community, the fruits are used as spices and aqueous

decoction is used after child birth probably for its

antiseptic properties and to arrest bleeding (Nnodim et

al., 2011). Xylopiaaethiopica is used in the treatment of

cough, biliousness, bronchitis, rheumatism, dysentery,

malaria, uterine fibroid, amenorrhea, boils, sores,

wounds and cuts among others (fetse et al., 2016).

According to Obodo et al. (2013), The seeds of

Xylopiaaethiopica have been shown to contain bitter

chemical constituents like alkaloids, glycosides,

saponnis, tannins, sterols, carbohydrate, protein, free

fatty acids, mucilage’s and acidic compounds; some of

which might be responsible for its documented medicinal

and pharmacological properties like anti-inflammatory,

cytotoxic, hypoglycaemic and antioxidant effects.

Since antiquity, blood has been seen as the

essentialcomponent of life (Hart, 2001). It is a tissue that

consists of fluid plasma in which are suspended a

number of formed elements which are red blood cells,

white blood cells and platelets. These blood cells exist at

fairly constant levels, suggesting the existence of

feedback regulatory mechanisms (Ofem, et al., 2012).

These blood cells which are haematological

parameterscan be used as indicators of toxicity and have

a broad potential application in environmental and

occupationalmonitoring (Raquel et al., 2012).

The red blood cell integrity is largely dependent on its

ability to maintain its membrane constituents that are

mostly polyunsaturated free fatty acid, and if this is

compromised will result to erythrocyte fragility which

becomes more fragile with consequent destruction by the

macrophages. This erythrocyte fragility or red cell

osmotic fragility is the ability of red blood cells to

undergo haemolysis when subjected to stress and the

absolute extent of haemolysis can be measured (Rodak,

2007). When this happens the membranes of the cells

undergo lipid peroxidation leading to oxidative

deterioration of polyunsaturated fatty acid accumulation

of reactive oxygen species (ROS) which are associated

with tissue damage by clearing off the sialic acid from

the cells making them more prone to phagocytosis. Also,

the osmotic fragility of red cells can occur from the

increased phosphorylation of P38 and JNK genes which

promotes increased production of ROS (Robin and

Steven, 2000). Factors such as cell’s size, surface area to

volume ratio, membrane composition and integrity can

equally influence the osmotic fragility of the cells

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(Fischbach, et al., 2008). Alteration in the integrity of

blood leads to loss of blood and can be life threatening as

blood is a necessary components of animal body. The

body tends to protect itself from this life threatening

exsanguination by converting the blood from its liquid

state to a solid state in a process known as blood clotting

or coagulation. This formation of a clot is often referred

to as secondary haemostasis and it usually involves two

main pathways namely extrinsic and intrinsic pathways

that make use of clotting factors. Estimation of

coagulation tests like prothrombin time, activated partial

thromboplastin time etc. are developed to diagnose

disorders of coagulation which can lead to an increased

bleeding (haemorrhage) or obstructive clotting

(thrombosis) (Xiangqun, et al., 2014).

Analysis ofblood parameters is relevant to risk

evaluation of alterations of the haematological system in

humans (Olsonet al., 2000). These haematological

parameters are those parameters that are related to the

blood and blood-forming organs. The evaluation of these

parameters does not only serve diagnostic purpose for

routine clinical management of patients but plays

important purpose of risk assessment in animals and

humans exposed to various risk factors (Saliu et al.,

2012). The reason for the use of haematological

parameters for this purpose is that any form of alteration

or disruption of physiological processes, whether such

disruption is induced chemically, physically or

biologically, will correspondingly alter the

haematological parameters both in humans and animals.

It is also an established fact that chronic diseases affect

the blood cells adversely. Damages mediated by free

radicals can result in the disruption of membrane fluidity,

oxidative DNA, alteration of platelet functions, etc. and

have generally been considered to be linked with many

chronic health problems such as cancers, inflammation,

aging and atherosclerosis. Although organisms possess

antioxidant defence and repair systems that have evolved

to protect them against free radicals but these systems are

insufficient to protect them completely against oxidative

damage.

For this study, the cyclophosphamide was chosen

because it is one of the most frequently used antitumor

agents in clinical practice and also its association with

rapidly killing of dividing cells in the body.

Considering the above, this present study was designed

to evaluate the effects of Xylopia aethiopica and

melatonin on some haematological parameters, in

cyclophosphamide induced wistar rats, with a view of

finding a lasting solution to the life threatening effects

reported about this cytotoxic drug.

AIM

The research work is aimed at evaluating the effects of

Xylopia aethiopica and melatonin on some

haematological parameters in cyclophosphamide

intoxicated adult wistar rats.

MATERIALS AND METHODS

COLLECTION OF PLANT MATERIALS AND AU

THENTICATION

Pods of Xylopia aethiopica were purchased from Orie-

Ugba vegetable market, Umuahia North Local

Government Area, Abia State, Nigeria and were taken to

the Department of Forestry and Environmental

Management, Michael Okpara University of Agriculture,

Umudike where they were identified by a botanist/forest

manager. Voucher number MOUAU/VPP/18/012 was

assigned to a specimen sample of the pods which was

deposited in the herbarium of the Department.

PREPARATION OF PLANT EXTRACTS

Extract of the fruit pods was prepared in accordance with

the Soxhlet method described by Jensen, (2007). The

plant materials were subjected to further drying under

shade for 14 days and were pulverized into powder in a

manual blender powered by a Honda petrol engine. One

hundred grams of the powdered sample was introduced

into the extraction chamber of the soxhlet extractor and

extraction was carried out with ethanol as solvent.

Temperature was maintained at 650C throughout the

extraction period of 48 hours. At the end of the period,

the extract in solution was dried in a hot air oven at 40oC

to obtain a dry dark oily extract. The weight of the

extract was taken and percentage yield was calculated

using the formular:

% yield =X x 100

Q 1

Where X = weight of dried extract and Q = weight of

powdered plant material before extraction (100g)

(Bandiola, 2018).

ANIMALS USED FOR STUDY

One hundred and ninety five matured wistar albino rats

were used for the studies. Of the number, 30 were used

for the acute toxicity evaluation of the extract, 35 for

acute toxicity study of cyclophosphamide and 130 were

used for the main study. The rats were kept in aluminum

cages and allowed to acclimatize for two weeks to allow

for proper adaptation to the environment and living

conditions. They were allowed access to feed (Vital feed,

Nigeria) and water ad libitum but were starved for 12

hours prior to commencement of any experiment. All

animal experiments were carried out in compliance with

NIH guidelines for Care and Use of Laboratory Animals

(OECD, 2001). All experiments were carried out in the

Physiology Laboratory of the Department of Physiology

and Pharmacology, College of Veterinary Medicine,

Michael Okpara University of Agriculture, Umudike,

Nigeria.

EXPERIMENTAL DESIGN The rats (130 in number) were assigned to 13 groups of

10 rats each and were treated according to the order

below:

Group I: 10 mg/kg Cyclophosphamide, Food and water

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Group II: 10 mg/kg Cyclophosphamide, 400 mg/kg


Group III: 10 mg/kg Cyclophosphamide, 0.5 mg/kg


Group IV: 10 mg/kg Cyclophosphamide, 400mg/kg


Group V: 30 mg/kg Cyclophosphamide, Food and water

Group VI: 30 mg/kg Cyclophosphamide, 400 mg/kg


Group VII: 30mg/kg Cyclophosphamide, 0.5mg/kg


Group VIII: 30mg/kg Cyclophosphamide+400mg/kg


Group IX: 50 mg/kg Cyclophosphamide, Food and water

Group X: 50 mg/kg Cyclophosphamide, 400 mg/kg


Group XI: 50mg/kg Cyclophosphamide, 0.5mg/kg


Group XII: 50mg/kg Cyclophosphamide, 400mg/kg


Group XIII: Food and water only

Treatments were done daily via the oral route for twenty

one (21) days. Three animals were sacrificed in each

group for blood collection by cardiac puncture into

EDTA and sodium citrate bottles for haematology and

osmotic fragility studies. Liver and kidney samples were

also collected and preserved in 10% formalin for

histological examination.

HEMATOLOGICAL STUDIES Haematological parameters including Red blood cell

count (RBCC), packed cell volume (PCV), haemoglobin

concentration (Hb), mean corpuscular volume (MCV),

mean corpuscular haemoglobin (MCH), mean

corpuscular haemoglobin concentration (MCHC), white

blood cells count (WBCC) and Platelets (PLT) count

were determined. These parameters were obtained at

once for each blood sample in an Automated

Haematology Analyser (Mindray company, China),

following the procedures by the producer.

DETERMINATION OF RED BLOOD CELLS

OSMOTIC FRAGILITY

The method of Adenkule and Olurenmi, (2014) was

adopted. Sodium chloride solution (200 mls) was

prepared for each sample in concentrations ranging from

0.1-0.85% at pH 7.4. A set of test tubes, each containing

5mls of sodium chloride solution of concentration

ranging from 0.1 to 0.85% were serially arranged in a

test tube rack. One set of test tubes was used to analyze

each sample. A drop of the freshly collected blood was

placed into each of the ten test tubes using a dropper

pipette and each was mixed by gently inverting the test

tubes about three times. The test tubes were then allowed

to stand at room temperature for 30 minutes and then

centrifuged at 3000 rpm for 10 minutes before reading

the absorbance of the supernatant in each test tube on a

spectrophotometer at 540nm. The same procedure was

repeated for each sample collected. Percentage

haemolysis was calculated using the expression:

Percentage Haemolysis = Absorbance of test x100

Absorbance of control

DETERMINATION OF ACTIVATED PARTIAL

THROMBOPLASTIN TIME (APTT)

AUTHOMATED KL340 COAGULATION

ANALYZER

Procedure

After gentle swirling of the reagent vials, enough volume

of reagent 1 (CaCl₂) was pre-warmed for immediate use

in a clean and dry plastic tube maintained at 370C. About

100 µl of test plasma was pipette into a test cuvette at

300C. About 100µl of pre-warmed reagent 2 (APTT

Reagent) was added to the cuvette. The mixture was well

mixed and incubated at 370C for 3 minutes before

forcibly pipetting 100µl of pre-warmed reagent 1 into the

test cuvette while starting the stop watch at the same

time to note time in seconds it takes for blood to clot.

This time in seconds is recorded as the APTT.

DETERMINATION OF PROTHROMBIN TIME

(PT) USING AUTHOMATED KL340

COAGULATION ANALYZER

Procedure

Prothrombin reagent was dispensed into a thoroughly

clean and dry plastic tube for immediate use and pre-

warm at 370C for 10 minutes. About 100 µl of test

plasma was introduced into a test cuvette at 370C and

incubated for 3 minutes before. The mixture was well

mixed and incubated at 370C for 3 minutes before adding

forcibly 200µl of the pre-warmed prothrombin reagent

while starting the stop watch at the same time to record

the time in seconds it takes for blood to clot. This time in

seconds is recorded as the PT.

STATISTICAL ANALYSIS

Results were expressed as means ± standard error of

mean (SEM). Statistical analysis was done using one-

way analysis of variance (ANOVA). Significant

differences were assessed at 95% level of significance

between control and treated groups using Duncan and

LSD (Post Hoc) tests. P values less than 0.05 were

considered significant. Computer software package,

SPSS version 21 was employed.

RESULTS

4.1 PERCENTAGE YIELD OF THE EXTRACT

Following extraction and drying of the extract in

solution, 9.23g of the extract was obtained and

represented 9.23% yield (Table 4.1).

Table 4.1: Percentage yield of XA extract.

Weight of plant material Weight of extract Percentage yield

100g 9.23g 9.23%

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Melatonin on RBC of Cyclophosphamide intoxicated

wistar rats

In week one, and two, there was significant decrease in

RBC in all the treatment groups compared with control

except week three. RBC varied significantly from week

one to three in all the treatment groups except group 1, 9

and 13.

In week one there is a dose dependent decrease in RBC

of rats treated with 10, 30 and 50 mg/kg of

cyclophosphamide. In week one, treatment with 400mg

Xylopia aethiopica and 0.5 mg Melatonin significantly

increased the RBC of rats exposed to 10mg

Cyclophosphamide. RBC of rats treated with 400 mg

Xylopia aethiopica and 0.5mg Melatonin was

significantly higher than 400mg Xylopia aethiopica

alone. Similarly, treatment with 400mg Xylopia


0.5mg Melatonin significantly increased the RBC of rats

exposed to 30mg Cyclophosphamide. RBC of rats


Melatonin was significantly higher than 400mg Xylopia

aethiopica. Treatment with 400mg Xylopia aethiopica

only significantly increased the RBC of rats exposed to



alone and 400mg Xylopia aethiopica and 0.5 mg

Melatonin significantly increased the RBC of rats

exposed to 10mg Cyclophosphamide. Treatment with

400mg Xylopia aethiopica alone significantly increased

the RBC of rats exposed to 30mg Cyclophosphamide.

Treatment with 400mg Xylopia aethiopica, 0.5 mg

Melatoninand the combination had no significant effect

of RBC of rats exposed to 50mg Cyclophosphamide.

In week three, treatment with 400mg Xylopia

aethiopicaalone and in combination with 0.5mg

Melatonin had no significantly effect on the RBC of rats

exposed to 10mg Cyclophosphamide (Table 4.6).

Table 4.6: Effects of Xylopia aethiopica extract and Melatonin on RBC of Cyclophosphamideintoxicated wistar

rats.

Treatment

RBC (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week 3


10mg Cyclophosphamide 6.35±0.05 cd

5.89±0.01 e 9±4.32

a .000 .000 .942 .656 20.737



d 7±0.06 5.96±0.02

a .000 .001 .970 .002 64.225

10mg Cyclophosphamide + 0.5mg

Melatonin 6.5±0.2

d 5.57±0.01

.000 .000 .001 42.551


400mg Xylopia aethiopica + 0.5mg

Melatonin

7.1±0.2 e 6.3±0.12

f 5.89±0.01

a .838 .000 .965 .000 171.564


5.2±0.06 c

.000 .000 .000 816.750

30mg Cyclophosphamide +400mg

Xylopia aethiopica 6.53±0.03

d 5.87±0.01

c

.000 .000 .000 4335.000


Melatonin 5.93±0.02

d 5.08±0.01

bc

.000 .000 .000 5766.000



Melatonin

6.51±0.02 c 5.27±0.01

c

.000 .000 .000 .119

50mg Cyclophosphamide 5.1±0.1 a 5.08±0.01

bc

.000 .000 .748 2457.600



c 5.23±0.01

.000 .000 .000 777.600

50mg Cyclophoshamide + 0.5mg


a 4.75±0.01

a

.000 .000 .000 405.600



Melatonin

5.19±0.01 a 4.93±0.01

ab

.000 .000 .000 .007

Control 7.3±0.2 e 7.29±0.01

h 7.3±0.03

a .993 .453

4.7: Result of effects of Xylopia aethiopica extract and

Melatonin on HB of Cyclophosphamideintoxicated

wistar rats


decrease in HGB in all the treatment groups compared

with control. HGB varied significantly from week one to

three in all the treatment groups except group 9 and 13.

In week one and two, there is a dose dependent decrease

in HGB of rats treated with 10 and 50 mg/kg of

cyclophosphamide. Treatment with 400mg Xylopia

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aethiopica and 0.5 mg Melatonin significantly increased

the HGB of rats exposed to 10mg cyclophosphamide.

Similarly, treatment with 400mg Xylopia aethiopica

alone and 400mg Xylopia aethiopica and 0.5mg

Melatonin significantly increased the HGB of rats

exposed to 30mg cyclophosphamide. Treatment with

400mg Xylopia aethiopica alone significantly increased

the HGB of rats exposed to 50mg Cyclophosphamide.


alone and in combination with 0.5 mg melatonin

significantly increased the HGB of rats exposed to 10mg

Cyclophosphamide (Table 4.7).

Table 4.7: Effects of Xylopia aethiopica extract and Melatonin on HB of Cyclophosphamide intoxicated wistar

rats.

Treatment HB (Wk1) (Wk2) (Wk3) A.WK

Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)

p-

value p-

value p-

value p-

value f-value

10mg Cyclophosphamide 11.8±0.14 de 10.2±0.06

f 8.03±0.07a .000 .000 .000 .000 374.718


12.2±0.12 ef 12.5±0.12

h 11.3±0.12c .000 .082 .000 .001 29.250



ef 10±0.06ef

.000 .000

.000 132.300

10mg Cyclophosphamide+ 400mg Xylopia aethiopica +


f 11.5±0.12f 10.3±0.12

c .020 .000 .000 .000 99.250

30mg Cyclophosphamide 11.3±0.06 cd 9.1±0.12

cd

.000 .000

.000 290.400 30mg Cyclophosphamide+ 400mg Xylopia aethiopica

12.1±0.12 ef 10.2±0.12

e

.000 .000

.000 135.375



c 9±0.06bc

.000 .000

.000 726.000


0.5mg Melatonin 12.1±0.06 9.6±0.12

e

.000 .000

.000 375.000

50mg Cyclophosphamide 9±0.06 a 9±0.17

b

.000 .000

1.000 0.000 50mg Cyclophosphamide+ 400mg Xylopia aethiopica

10.2±0.06 b 9.4±0.06

d

.000 .000

.001 96.000


0.5mg Melatonin 9±0.06

a 8.3±0.12 a

.000 .000

.006 29.400



a 8.5±0.12 a

.000 .000

.010 21.600

Control 13.2±0.17 g 13±0.06

h 13.1±0.06d

.485 .818


Melatonin on HCT of Cyclophosphamideintoxicated

wistar rats


decrease in HCT in all the treatment groups compared

with control. HCT varied significantly from week one to



decrease in HCT in all the treatment groups compared

with control. HCT varied significantly from week one to



in HCT of rats treated with 10, 30 and 50 mg/kg of

cyclophosphamide. Treatment with 400mg Xylopia

aethiopica alone and 400mg Xylopia aethiopica and 0.5

mg Melatonin significantly increased the HCT of rats

exposed to 10mg Cyclophosphamide. HCT of rats

treated with 400 mg Xylopia aethiopica and 0.5mg


aethiopica alone. Similarly, treatment with 400mg


and 0.5mg Melatonin significantly increased the HCT of

rats exposed to 30mg Cyclophosphamide. HCT of rats




aethiopica alone as well as 400mg Xylopia aethiopica

and 0.5mg Melatonin significantly increased the HCT of



aethiopicasignificantly increased the HCT of rats

exposed to 10mg Cyclophosphamide (table 4.8).

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Table 4.8: Effects of Xylopia aethiopica extract and Melatonin on HCT of Cyclophosphamideintoxicated wistar

rats.

Treatment HCT (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week 3


10mg Cyclophosphamide 37±0.29 f 32±0.17

e 25.6±0.12

a .000 .000 .000 .000 773.368



g 39.1±0.11

g 35.8±0.06

c .000 .000 .000 .000 71.195



f 31.9±0.05

e

.000 .000 .000 768.706



0.5mg Melatonin

40.9±0.06 h 36.9±0.05

f 32.7±0.06

b .000 .000 .000 .000 5044.000


c

.000 .000 .000 4873.500



g 32.2±0.05

e

.000 .000 .000 2381.400



c 28.2±0.05

b

.000 .000 .000 4374.000



0.5mg Melatonin

38.3±0.12 g 30.9±0.05

e

.000 .000 .000 3285.600


b

.000 .000 .081 5.400



c 30.8±0.05

d

.000 .000 .000 294.000



a 26.5±0.05

a

.000 .000 .000 600.000



0.5mg Melatonin

30±0.29 b 26.9±0.05

a

.000 .000 .000 110.885

Control 45.6±0.06 i 43.1±0.05

h 44.3±0.23

d .081 78.167


Melatonin on MCV of Cyclophosphamide intoxicated

wistar rats In week one, two and three, there was significant

decrease in MCV in all the treatment groups compared

with control. MCV varied significantly from week one to


MCV of rats exposed to 10mg Cyclophosphamideand

treated with 400mg Xylopia aethiopica and 0.5 mg

Melatonin alone and in combination was not

significantly different from each other. MCV of rats



aethiopica alone. Similarly, treatment with 0.5mg

Melatoninalone and 400mg Xylopia aethiopica and

0.5mg Melatonin significantly increased the MCV of rats

exposed to 30mg Cyclophosphamide. MCV of rats




aethiopica and 0.5mg Melatonin significantly increased

the MCV of rats exposed to 50mg Cyclophosphamide.

By week 2, MCV of rats exposed to 10mg



combination was significantly different from rats treated

with 10 mg Cyclophosphamide alone. MCV of rats

exposed to 30mg Cyclophosphamide and treated with

400mg Xylopia aethiopica and 0.5 mg Melatonin alone

and in combination was significantly different from each

other and rats treated with 30 mg Cyclophosphamide

alone. MCV of rats exposed to 50mg Cyclophosphamide

and treated with 400mg Xylopia aethiopica was

significantly different from rats treated with 50 mg

Cyclophosphamide alone.

In week three, MCV of rats exposed to 10mg

Cyclophosphamide treated with 400mg Xylopia

aethiopicasignificantly higher MCV of rats exposed to

10mg Cyclophosphamide treated with 400mg Xylopia

aethiopica and 0.5mg Melatonin.

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Table 4.9: Effects of Xylopia aethiopica extract and Melatonin on MCV of Cyclophosphamide intoxicated wistar

rats.

Treatment MCV (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week2

(Wk2)

Week 3


10mg Cyclophosphamide 58.3±0.1 def

54.3±0.1 a 54.8±0.1

a .000 .000 .000 .000 475.000



def 55.9±0.1

d 60±0.6

b .000 .000 .399 .001 30.302


0.5mg Melatonin 59.2±0.1 57.3±0.1

e

.000 .000 .000 216.600



0.5mg Melatonin

57.6±02 cd

58.6±0.1 55.5±0.1 a .000 .718 .000 .000 204.818

30mg Cyclophosphamide 60±0.6 57.9±0.1 ef

.000 .005 .022 13.099


400mg Xylopia aethiopica 59.1±0.1 54.9±0.1

abc

.000 .000 .000 2646.000



bc 55.5±0.1

bcd

.000 .000 .010 21.600



0.5mg Melatonin

58.8±0.1 ef

58.6±0.1 fg

.000 .718 .196 2.400

50mg Cyclophosphamide 55.9±0.1 ab

55.5±0.1 bcd

.000 .000 .196 24.000



a 58.9±0.06

g

.000 .999 .000 2400.000



ab 55.8±0.06

cd

.000 .000 1.000 0.000



0.5mg Melatonin

57.8±0.1 de

54.6±0.06 f

.000 .000 .000 1536.000

Control 62.5±0.1 g 59.13±0.6

g 60.7±0.1

b .272 21.586

4.10: Result of effects of Xylopia aethiopica extract

and Melatonin on MCH of Cyclophosphamide

intoxicated wistar rats


MCH in all the treatment groups compared with control

except week three. MCH varied significantly from week

one to three in all the treatment groups except group 4, 9,

11 and 13.

In week 1, MCH of rats exposed to 10 mg


aethiopica and 0.5 mg Melatonin alone was significantly

different from rats exposed to 10 mg Cyclophosphamide.

MCV of rats treated with 400 mg Xylopia aethiopica and

0.5mg Melatonin was significantly higher than 400mg

Xylopia aethiopica alone. MCH of rats treated with

400mg Xylopia aethiopica and 0.5mg Melatonin singly

and in combination was not significantly different from

the MCH of rats exposed to 30mg Cyclophosphamide.

Similarly, MCH of rats treated with 400mg Xylopia

aethiopica and 0.5mg Melatonin singly and in

combination was not significantly different from the

MCH of rats exposed to 50mg Cyclophosphamide.

By week 2, MCH of rats exposed to 10mg




with 10 mg Cyclophosphamide alone. MCH of rats

exposed to 30mg Cyclophosphamide and treated with

400mg Xylopia aethiopica and 0.5mg Melatonin was

significantly higher than rats treated with 30 mg

Cyclophosphamide alone. MCH of rats exposed to 50 mg


aethiopica was significantly different from rats treated

with 50mg Cyclophosphamide alone.

In week three, MCH of rats exposed to 10mg

Cyclophosphamide treated with 400 mg Xylopia


combination was not significantly different.

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Table 4.10: Effects of Xylopia aethiopica extract and Melatonin on MCH of Cyclophosphamide intoxicated

wistar rats.

Treatment MCH Wk1 Wk2 Wk3 A.Wk

Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)

P-

value p-

value p-

value p-

value F-value

10mg Cyclophosphamide 18.6±0.1 de 17.3±0.1

ab 17.6±0.1 a .008 .091 .999 .000 69.500

10mg Cyclophosphamide+ 400mg Xylopia aethiopica

18.5±0.1 cd 17.9±0.1

cde 19±0.3 a .062 1.000 .989 .015 9.100

10mg Cyclophosphamide + 0.5mg Melatonin 18.6±0.1 de 18±0.1

de

.008 .974

.021 13.500 10mg Cyclophosphamide+ 400mg Xylopia aethiopica + 0.5mg Melatonin

17.7±0.1 ab 18.23±0.1

e 22.27±4.6 a .062 .216 .570 .450 .915

30mg Cyclophosphamide 19±0.1 e 17.5±0.01

abcd

.000 .717

.000 337.500 30mg Cyclophosphamide+ 400mg Xylopia aethiopica

18.6±0.1 de 17.4±0.1

abc

.008 .315

.001 86.400

30mg Cyclophosphamide + 0.5mg Melatonin 18.9±0.1 de 17.7±0.2

abcde

.000 1.000

.007 25.412 30mg Cyclophosphamide+ 400mg Xylopia aethiopica + 0.5mg Melatonin

18.6±0.1 de 18.2±0.1

e

.008 .315

.008 24.000

50mg Cyclophosphamide 17.7±0.1 ab 17.7±0.1

abcde

.062 1.000

1.000 0.000 50mg Cyclophosphamide+ 400mg Xylopia aethiopica

17.4±0.06 a 18±0.06

de

.000 .974

.002 54.000

50mg Cyclophosphamide + 0.5mg Melatonin 17.6±0.06 a 17.5±0.12

abcd

.008 .717

.482 .600 50mg Cyclophosphamide+ 400mg Xylopia aethiopica + 0.5mg Melatonin

17.5±0.06 a 17.2±0.06

a

.001 .020

.021 13.500

Control 18.1±0.06 bc 17.8±0.06

bcde 18±0.06 .008 .091 .999 .276 7.000


and Melatonin on MCHC of Cyclophosphamide



decrease in MCHC in all the treatment groups compared

with control. MCHC varied significantly from week one

to three in all the treatment groups except group 9 and

13.

In week 1, MCHC of rats exposed to 10 mg


aethiopica and0.5 mg Melatonin alone was significantly


MCHC of rats treated with 400 mg Xylopia aethiopica

and 0.5mg Melatonin was significantly higher than

400mg Xylopia aethiopica alone. MCHC of rats treated


singly and in combination was not significantly different

from the MCHC of rats exposed to 30mg

Cyclophosphamide. Similarly, MCHC of rats treated

with 400mg Xylopia aethiopica and 0.5 mg Melatonin

was significantly different from the MCHC of rats

exposed to 50mg Cyclophosphamide.

By week 2, MCHC of rats exposed to 10mg




with 10 mg Cyclophosphamide alone. MCHC of rats

exposed to 30 mg Cyclophosphamide and treated with

400mg Xylopia aethiopica and 0.5 mg melatonin was

significantly higher than rats treated with 30 mg

Cyclophosphamide alone. MCHC of rats exposed to 50

mg Cyclophosphamide and treated with 400 mg Xylopia

aethiopica was significantly different from rats treated

with 50 mg Cyclophosphamide alone.

In week three, MCHC of rats exposed to 10mg


aethiopica and 0.5 mg melatonin alone and in

combination was not significantly different.

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Table 4.11: Effects of Xylopia aethiopica extract and Melatonin on MCHC of Cyclophosphamide intoxicated

wistar rats.

Treatment MCHC (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week3

(Wk3) p-value p-value p-value p-value F-value


d 32±0.6

a .000 .000 .999 .972 .029



de 32±0.1

d 31.6±0.1

a .000 .000 .999 .002 21.000



d 31.5±0.15

bcd

.000 .000 .573 .375



0.5mg Melatonin

30.8±0.1 c 31.2±0.1

bc 56.07±2.4

a .000 .000 .465 .406 1.050

30mg Cyclophosphamide 31.6±0.1 de

30.2±0.1 cd

.000 1.000 .001 73.500



d 31.7±0.1

a

.000 .000 .140 3.375



f 31.9±0.1

d

.000 .000 .000 117.600



0.5mg Melatonin

31.6±0.1 de

31.1±0.1 b

.000 .000 .018 15.000

50mg Cyclophosphamide 31.6±0.1de

31.9±0.1d

.000 .000 .081 5.400



d 30.5±0.11547

a

.000 .739 .002 48.600



de 31.3±0.1

ab

.000 .000 .223 2.077



0.5mg Melatonin

30.3±0.1 b 31.6±0.6

bcd

.000 .000 .000 253.500

Control 28.9±0.1 a 30.2±0.06

a 29.6±0.17

a .226 34.636


and Melatonin on WBC of Cyclophosphamide



decrease in WBC in all the treatment groups compared

with control. WBC varied significantly from week one to

three in all the treatment.

In week one and two, there is a dose dependent decrease

in WBC of rats treated with 10, 30 and 50 mg/kg of


aethiopica alone and 400mg Xylopia aethiopica and 0.5

mg Melatonin significantly increased the WBC of rats

exposed to 10mg Cyclophosphamide. WBC of rats



aethiopica alone. Similarly, treatment with 400mg


and 0.5mg Melatonin significantly increased the WBC of

rats exposed to 30mg Cyclophosphamide. WBC of rats




aethiopica and 0.5mg Melatonin significantly increased

the WBC of rats exposed to 50mg Cyclophosphamide.


aethiopicasignificantly increased the WBC of rats

exposed to 10mg Cyclophosphamide.

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Table 4.12: Effects of Xylopia aethiopica extract and Melatonin on WBC of Cyclophosphamide intoxicated

wistar rats.

WBC (Wk1) (Wk2) (Wk3) A.Wk

Treatment Week 1

(Wk1)

Week 2

(Wk2)

Week 3

(Wk3)

p-

value

p-

value p-value p-value f-value

10mg cyclophosphamide 1.8±0.06 cde

1±0.06 d 0.3±0.06

a .000 .000 .000 .000 148.000


400mg Xylopia aethiopica 2.7±0.12 1.4±0.06

e 0.4±0.06

a .000 .000 .000 .000 64.500


0.5mg Melatonin 2±0.12

de 1±0.06

d

.000 .000 .001 60.000



0.5mg Melatonin

3.5±0.12 g 2.3±0.06

f 1.9±0.06

b .000 .000 .000 .000 392.000

30mg Cyclophosphamide 1.2±0.06 b 0.6±0.06

bc

.000 .000 .002 54.000



cde 1.1±0.12

de

.000 .000 .003 38.400



bc 0.6±0.06

bc

.000 .000 .001 60.000



0.5mg Melatonin

2.3±0.12 ef

1±0.06 a .000 .000 .000 363.000


0.3±0.06a

.000 .000 .081 5.400



a 0.5±0.12

b .000 .000 .482 .600



a 0.1±0

a

.000 .000 .004 37.500



0.5mg Melatonin

1.4±0.06 bc

0.9±0.06 b

.000 .000 .000 507.000

Control 8.4±0.23 h 9.6±0.12

g

.008 12.103

Values are mean± SEM

4.13: Result of effects of Xylopia aethiopicaextract and

Melatonin on Platelet of



platelet in all the treatment groups compared with control

except week three. Platelet varied significantly from

week one to three in all the treatment groups except

group 2 and 13.


decrease in platelet count of rats treated with 10, 30 and

50 mg/kg of Cyclophosphamide. Treatment with 400mg


and 0.5mg Melatonin significantly increased the platelet

count of rats exposed to 10mg Cyclophosphamide.

Platelet count of rats treated with 400 mg Xylopia

aethiopica and 0.5mg Melatonin was significantly higher

than 400mg Xylopia aethiopica alone. Similarly,



significantly increased the platelet of rats exposed to

30mg Cyclophosphamide. Platelet count of rats treated


was significantly higher than 400mg Xylopia aethiopica

alone. Treatment with 400mg Xylopia aethiopica and

0.5mg Melatonin significantly increased the platelet of



and 0.5 mg Melatonin alone and in combination did not

significantly increase the platelet of rats exposed to


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177

Table 4.13: Effects of Xylopia aethiopica extract and Melatonin on Platelet of Cyclophosphamideintoxicated

wistar rats.

Treatment

Platelet (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week 3

(Wk3) p-value

p-

value

p-

value p-value F-value

10mgCyclophosphamide 37±0.29 k 32±0.17

k 25.6±0.11

a .000 .000 .001 .000 20247.621



c 39.1±0.11

d 35.8±0.06

a .000 .000 .002 .999 .001



j 31.9±0.05

h

.000 .000 .000 145.200



0.5mg Melatonin

40.9±0.06 i 36.9±0.05

g 32.7±0.06

a .000 .000 .001 .000 5683.568


e

.000 .000 .000 3307.500



d 32.2±0.05

bc

.000 .000 .000 672.923



g 28.2±0.05

d

.000 .000 .000 1559.294



0.5mg Melatonin

38.3±0.12 f 30.9±0.05

c

.000 .000 .000 4455.375


e

.000 .000 .001 86.400



a 30.8±0.74

a

.000 .000 .000 240.000



c 26.5±0.06

bc

.000 .000 .008 24.870



0.5mg Melatonin

30±0.29 b 26.9±0.06

b

.000 .000 .000 3.594

Control 45.6±0.06 l 43.1±0.06

j 44.3±0.23

b .131 90.067

Table 4.14 Result of effects of Xylopia aethiopica

extract and Melatonin on PT (s) of


In week one and two and three, there was significant

increase in average PT in all the treatment groups

compared with control except group 1 and 3 in week one.

Average PT varied significantly from week one to three

in all the treatment except group 13.

In week 1, average PT of rats exposed to 10 mg


aethiopica and 0.5 mg Melatonin alone was significantly


Average PT of rats treated with 400mg Xylopia

aethiopica and 0.5mg Melatonin singly and in

combination was not significantly different from the

average PT of rats exposed to 30mg Cyclophosphamide.

Similarly, average PT of rats treated with 400mg Xylopia

aethiopica and 0.5 mg Melatonin was not significantly

different from the average PT of rats exposed to 50mg

Cyclophosphamide.

By week 2, average PT of rats exposed to 10mg


aethiopica alone was significantly different from rats

treated with 10 mg Cyclophosphamide alone. Average

PT of rats exposed to 30 mg Cyclophosphamide and


Melatonin singly and in combination was significantly

higher than rats treated with 30mg Cyclophosphamide

alone. Similarly, average PT of rats exposed to 50 mg


aethiopica and0.5mg melatonin singly and in


with 50mg Cyclophosphamide alone.

In week three, average PT of rats exposed to 10mg




with 10mg Cyclophosphamide.

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Table 4.14: Result of effects of Xylopia aethiopica extract and Melatonin on PT (s) of Cyclophosphamide

intoxicated wistar rats.

Treatment PT (s) (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week 3


10mg Cyclophosphamide 13.9±0.12 ab

18.9±0.06 b 20±0.06

b .434 .000 .000 .000 1585.500


400mg Xylopia aethiopica 17±0.06

c 20.1±0.12

de 21±0.17

c .000 .000 .000 .000 283.071



ab 19.2±0.17

bc

.059 .000 .000 160.067

10mgCyclophosphamide+


0.5mg Melatonin

15.1±0.12 b 19±0.12

b 20.9±0.12

c .000 .000 .000 .000 655.750

30mg Cyclophosphamide 17.4±0.23 cd

19.7±0.06 cd

.000 .000 .001 93.353

30mgCyclophosphamide +


cde 21±0.17

f

.000 .000 .000 160.167



c 20.5±0.06

ef

.000 .000 .000 693.600

30mg Cyclophosphamide+ 400mg

Xylopia aethiopica + 0.5mg

Melatonin

17.3±0.12 cd

20.7±0.06 ef

.000 .000 .000 693.600


f

.000 .000 .021 13.669


400mg Xylopia aethiopica

18.9±0.12

cde

21.9±0.12 h

.000 .000 .000 337.500



de 21.1±0.12

fg

.000 .000 .000 156.000

50mgCyclophosphamide+


0.5mg Melatonin

18±0.17 cde

21.7±0.12 gh

.000 .000 .000 315.923

Control 13±0.23 a 13.8±0.06

a 13.7±0.12

a .090 8.143

4-15: Result of effects of Xylopia aethiopica extract

and Melatonin on APTT (s) of



increase in APTT in all the treatment groups compared

with control except group 1 and 3 in week one. APTT

varied significantly from week one to three in all the

treatment except group 13.


increase in APTT of rats treated with 10, 30 and 50

mg/kg of Cyclophosphamide.

In week 1 and 2, treatment with 400mg Xylopia

aethiopica alone, 0.5mg Melatonin alone and 400mg


increased the APTT of rats exposed to 10mg

Cyclophosphamide.

Treatment with 0.5mg of Melatonin and 0.5mg of

Melatonin combined with 400mg Xylopia aethiopica

decreases APTT of exposed rats compared with rats

treated with 30mg Cyclophosphamide. 400mg Xylopia

aethiopica alone significantly increased the APTT of rats

exposed to 30mg Cyclophosphamide. APTT of rats

treated with 400mg Xylopia aethiopica alone and 0.5mg

Melatonin alone was significantly higher compared to

exposed to 50mgCcyclophophamide.


and 0.5mg Melatonin significantly increased average

APTT of rats exposed to 10mg Cyclophosphamide.

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Table 4.15: Effects of Xylopia aethiopica extract and Melatonin on APTT (s) of Cyclophosphamide intoxicated

wistar rats.

treatment AVERAGE APTT (s) (Wk1) (Wk2) (Wk3) A.W k

Week 1

(Wk1)

Week 2

(Wk2)

Week 3

(Wk3) p-value p-value p-value p-value F-value


b 39.2±0.17

b .489 .000 .000 .000 1625.727



cd 35±0.06

c 40.93±0.09

c .000 .000 .000 .000 815.216


Melatonin 32±0.58

b 35.9±0.17

d .000 .000 .003 41.862

10mg Cyclophosphamide+400mg


Melatonin

34.5±0.12 cd

34.4±0.06 b 39.3±0.06

b .000 .000 .000 .000 1176.500

30mg Cyclophosphamide 37.5±0.23f 38.3±0.06

e .000 .000 .028 11.294



g 40.6±0.06

g .000 .000 .003 39.706



c 38.4±0.06

e .000 .000 .000 960.000

30mgCyclophoshamide+400mg


Melatonin

35.9±0.06 cde

39.1±0.12 f .000 .000 .000 614.400

50mg Cyclophosphamide 37±0.06 ef

51.3±0.12 a .000 .000 .000 12269.400



i 51.6±0.17

hi .000 .000 .000 691.200



h 51±0.06

h .000 .000 .000 190.426



Melatonin

36.4±0.35 def

51.4±0.17 hi

.000 .000 .000 1500.000

Control 29.3±0.35 a 29±0.12

a 29.07±0.03

a .602 .554


and Melatonin on osmotic fragility (0) scores of

Cyclophosphamide intoxicated wistar rats In week one and two and three, there was no significant

difference in all the treatment groups compared with the

control group.

In week one, two and three, treatment with 400mg

Xylopia aethiopica and 0.5mg Melatonin alone and in

combination did not significantly osmotic fragility of rats

exposed to 10, 30 and 50mg Cyclophosphamide.

Table 4.16: Effects of Xylopia aethiopica extract and Melatonin on osmotic fragility (0) scores of


Treatment 0 (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week 3

(Wk3) p-value p-value

p-

value

p-

value F-value

10m Cyclophosphamide 100±0 a 100.27±0.32

a 100.6±0.31

a 1.000 1.000 .922 .318 1.394


400mg Xylopia aethiopia 100±0

a 100.27±0.32

a 100.3±0.35

a 1.000 1.000 1.000 .711 .361



a 100.27±0.32

1.000 1.000 .449 .703


400mgXylopia aethiopia +

0.5mg Melatonin

100±0 a 100.27±0.32

a 100.3±0.35

a 1.000 1.000 1.000 .711 .361


a

1.000 1.000 .449 .703



a 100.27±0.32

a

1.000 1.000 .449 .703



a 100.27±0.32

a

1.000 1.000 .449 .703



0.5mgMelatonn

100±0 a 100.27±0.32

a

1.000 1.000 .449 .703

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180


a

1.000 1.000 .449 .703



a 100.27±0.32

a

1.000 1.000 .449 .703



a 100.27±0.32

a

1.000 1.000 .449 .703


400mg Xylopia aethiopia+

0.5mg Melatonin

100±0 a 100.27±0.32

a

1.000 1.000 .449 .703

Control 100±0 a 100.27±0.32

a 100.3±0.35

a .711 .361


and Melatonin on osmotic fragility (0.1) scores of



increase in osmotic fragility in all the treatment groups

compared with control except group 4 in week one and

three. Osmotic fragility varied significantly from week

one to three in all the treatment except group 13.


in osmotic fragility of rats treated with 10, 30 and 50mg

of Cyclophosphamide. Treatment with 400mg Xylopia














Cyclophosphamide.




Cyclophosphamide.

Table 4.17: Effects of Xylopia aethiopica extract and Melatonin on osmotic fragility (0.1) scores of



Week 1

(Wk1)

Week 2

(Wk2)

Week 3



e 97.02±0.69

b .000 .000 .000 .000 41.529



g 89.39±0.28

d 83.95±0.29

a .000 .011 .172 .000 154.267



g 89.14±0.28

d .000 .045 .368 1.029



0.5mg Melatonin

88.1±0.06 f 88.39±0.28

cd 85.41±0.3

a .036 .811 1.000 .000 47.286


g .000 .000 .242 1.887



i 94.97±0.3

f .000 .000 .652 .237



d 85.73±0.27

b .000 .004 .168 2.820



0.5mg Melatonin

85.21±0.01 c 85.34±0.27

b .000 .000 .662 .223


g .000 .000 .798 .075



i 94.5±0.3

f .000 .000 .096 4.683



b 89.26±0.28

d .000 .023 .000 470.987



0.5mg Melatonin

82.12±0.02 a 83.34±0.26

a .000 .000 .010 21.376

Control 87.51±0.01 e 87.64±0.28

c 85.37±0.3

a .091 29.403

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Cyclophosphamide intoxicated wistar rats In week one and two, there is a dose dependent increase

in osmotic fragility of rats treated with 10, 30 and 50 mg

of Cyclophosphamide. Osmotic fragility of rats exposed

to 10mg Cyclophosphamide and treated with 400mg

Xylopia aethiopica was significantly higher 10mg

Cyclophosphamide alone. Similarly, treatment with

400mg Xylopia aethiopica alone and 400mg Xylopia










Cyclophosphamide.




Week 1

(Wk1)

Week 2

(Wk2)

Week 3


10mg Cyclophosphamide 88.1±0.01 ef

89.54±0.28 g 91.27±0.5

c .000 .000 .000 .002 22.699



g 86.53±0.27

f 83.44±0.29

a .000 .000 1.000 .000 44.219



fg 88.14±0.28

g

.000 .000 .098 4.635


+400mg Xylopia aethiopica +

0.5mg Melatonin

89.02±0.14 f 88.36±0.28

g 85.36±0.3

b .000 .000 .019 .000 61.092


g

.000 .000 .024 12.523



e 85.33±0.27

ef

.000 .362 .003 45.415



d 83.32±0.26

cd

.281 .356 .018 15.076



0.5mg Melatonin

82.2±0.06 c 83.53±0.26

cd

.000 .683 .008 24.037


i

.000 .000 .000 119.485



c 82.52±0.26

bc

.000 .004 .091 4.909



b 81.56±0.26

b

.000 .000 .005 29.927



0.5mg Melatonin

78.13±0.03 a 78.31±0.25

a

.000 .000 .510 .522

Control 85.2±0.12a 84.33±0.27

de 83.39±0.29

a .005 14.440





in osmotic fragility of rats treated with 10, 30 and 50

mg/kg of Cyclophosphamide. Treatment with 400mg


and 0.5 mg Melatonin and 0.5 mg Melatonin

significantly decreased the osmotic fragility compared

with rats exposed to 10mg Cyclophosphamide. Similarly,










Cyclophosphamide.




Cyclophosphamide.

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182




Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)

p-

value p-

value p-

value p-

value f-value


f 89.17±1.46 b .000 .000 .000 .044 5.516


81.53±0.01 e 82.72±0.26

cd 80.65±0.28 a .000 1.000 .444 .002 21.806


80.39±0.25 d 81.6±0.26

c

.000 .036

.029 11.140


79.08±0.57 c 78.3±0.25

b 79.33±0.28 a .000 .000 .982 .227 1.919


h

.000 .000


89.11±0.05 h 88.65±0.28

g

.000 .000

.186 2.542


83.02±0.01 f 84.95±0.27

e

1.000 .001

.002 51.160

30mg Cyclophosphamide + 400mg Xylopia Aethiopica + 0.5mg Melatonin

75.23±0.02 b 77.54±0.25

b

.000 .000

.001 87.840


i

.000 .000


80.15±0.01 d 81.36±0.26

c

.000 .008

.009 21.890


79.13±0.02 c 77.4±0.25

b

.000 .000

.002 49.378



a 75.35±0.24 a

.000 .000

.000 447.667

Control 83.1±0.01 f 83.02±0.26

d 78.94±0.28 a

.000 116.704






and 50 mg/kg of Cyclophosphamide. Osmotic fragility of

rats exposed to 10mg Cyclophosphamide and treated


was significantly lower than 10mg Cyclophosphamide

but osmotic fragility of other treatment was significantly

higher compared with 10mg Cyclophosphamide.

Similarly, treatment with Melatoninalone and 400mg










Cyclophosphamide.



treatment 0.4 (Wk1) (Wk2) (Wk3) A.Wk

Week 1

(Wk1)

Week 2

(Wk2)

Week 3



ef 79.12±0.51

b .000 .145 .000 .000 98.344



g 71.23±0.23

f 66.54±0.23

a .000 .001 .252 .000 511.581



f 67.42±0.21

c

.000 .000 .000 347.082



0.5mg Melatonin

69.01±0.05 c 66.09±0.21

b 66.11±0.23

a .013 .000 .070 .000 84.995


j

.000 .000 .007 26.773

30mg Cyclophosphamide + 78.12±0.01 h 76.42±0.24

i

.000 .000 .002 48.958

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183

400mg Xylopia aethiopica



ef 72.41±0.23

g

.000 .000 .057 6.989



0.5mg Melatonin

74.92±0.33 g 73.92±0.23

h

.000 .000 .071 5.989


h

.000 .000 .448 .707



c 68.36±0.22

cd

.049 .066 .020 13.889



a 65.37±0.21

b

.000 .000 .000 629.949



0.5mg Melatonin

65.12±0.01 b 63.29±0.2

a

.000 .000 .001 83.202

Control 70.21±0.12 d 69.48±0.22

de 67.47±0.24

a .000 50.546






and 50mg of Cyclophosphamide.

Osmotic fragility of rats exposed to 10mg



combination was significantly lower than 10mg

Cyclophosphamide. Similarly, treatment with

Melatoninalone significantly decreased osmotic fragility

of rats exposed to 30mg Cyclophosphamide. Treatment


alone and in combination significantly decreased osmotic

fragility of rats exposed to 50mg Cyclophosphamide.




was not significantly different from of rats exposed to





Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)

p-

value p-

value p-

value p-

value f-value


d 35.73±2.36 b 1.000 .000 .000 .513 .747


30.15±0.05 c 25.17±0.08

b 30.9±0.11 b .376 .000 .008 .421 1.004


26.58±0.08 b 23.74±0.08

a

1.000 .105

.000 633.634


31.12±0.05 cd 31.2±0.1

ca 31.21±0.11 b 1.000 .000 .006 .737 .321


f

1.000 .000


40.13±0.01 e 40.24±0.13

f

1.000 .000

.451 .697


38.12±0.02 e 38.22±0.12

e

1.000 .000

.453 .689


40.12±0.01 e 40.23±0.13

f

1.000 .000

.451 .697


i

1.000 .000


48.13±0.01 g 48.26±0.15

h

1.000 .000

.450 .699


44.15±0.01 f 44.27±0.14

g

1.000 .000

.449 .702

50mg Cyclophosphamide + 400mg Xylopia aethiopica +


e 40.26±0.13 f

1.000 .000

.449 .702

Control 23.1±0.58 a 23.17±0.07

a 23.18±0.08 a

.985 .015

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184



Cyclophosphamideintoxicated wistar rats In week one and two, there was a dose dependent


and 50 mg/kg of Cyclophosphamide.

In week one, osmotic fragility of rats exposed to 10mg




rats exposed to 10mg Cyclophosphamide. Similarly,





rats exposed to 30 mg Cyclophosphamide. Treatment

with 0.5mg Melatonin alone and in combination with

400mg Xylopia aethiopicasignificantly decreased


Cyclophosphamide.

In week 2, treatment with 400 mg Xylopia aethiopica

alone and 400 mg Xylopia aethiopica and 0.5 mg



10mg Cyclophosphamide. Similarly, treatment with 400

mg Xylopia aethiopica and 0.5mg Melatonin





Cyclophosphamide.





Cyclophosphamide.



Treatment

0.6


Week (Wk1) Week 2 (Wk2) Week 3 (Wk3) p-value p-value p-value p-value f-value


20.18±0.06 d 20.25±0.06

d .000 .000 .000 .323 1.371



b 18.18±0.06

c 19.62±0.07

c .000 .000 .000 .000 113.968



b 18.1±0.06

c

.001 .000 .031 10.733



0.5mg Melatonin

16.1±0.01 b 16.14±0.05

b 16.15±0.06

b .007 .000 .000 .712 .359


20.18±0.06 d

.000 .000 .474 .622



bc 22.21±0.07

f

.000 .000 .470 .635



bc 20.18±0.06

d

.000 .000 .474 .622



0.5mg Melatonin

18.13±0.01 b 18.18±0.06

b

.000 .000 .457 .676


i

.000 .000 .454 .687


400mg Xylopia aethiopicaa 32.13±0.01

d 32.22±0.1

h

.000 .000 .452 .694



cd 26.21±0.08

g

.000 .000 .529 .474



0.5mg Melatonin

21.13±0.01 bc

21.19±0.07 e

.000 .000 .455 .683

Control 8.47±4.36 a 13.24±0.04

a 13.24±0.05

a .365 1.198



Cyclophosphamide intoxicated wistar rats In week one and two, treatment with 400 mg Xylopia





treatment with 400 mg Xylopia aethiopica and 0.5mg

Melatonin significantly decreased osmotic fragility of





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185





Cyclophosphamide.


Cyclophosphamideintoxicated wistar rat.

Treatment

0.7


Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)



f 16.28±1 c .000 .000 .000 .395 1.088


12.12±0.05 c 12.15±0.04

c 13.05±0.05 b .000 .000 .018 .000 133.330


12.02±0.04 c 13.22±0.04

d

.000 .000

.000 451.130


11.13±0.01 b 11.16±0.04

b 11.16±0.04 ab .000 .000 .582 .722 .345


h

.000 .000


20.14±0.01 h 20.19±0.06

i

.000 .000

.451 .697


16.14±0.01 f 16.18±0.05

g

.000 .000

.459 .669


15.14±0.01 e 15.18±0.05

f

.000 .000

.452 .693


j

.000 .000


15.14±0.01 e 15.18±0.05

f

.000 .000

.452 .693


16.14±0.01 f 16.18±0.05

g

.000 .000

.459 .669


14.14±0.02 d 14.18±0.04

e

.000 .000

.497 .556

Control 10.2±0 a 10.23±0.03

a 10.23±0.04 a

.711 .361




In week one and two, treatment with 400mg Xylopia





treatment with 400mg Xylopia aethiopica and 0.5mg

Melatonin and the combination significantly decreased





Cyclophosphamide.





Cyclophosphamide.

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186


Cyclophosphamide induced toxicity rats.

Treatment

0.8


Week 1

(Wk1)

Week 2

(Wk2)

Week 3



e 13.94±0.66

c .000 .000 .000 .630 .499



bc 10.17±0.03

c 10.14±0.04

b .000 .000 .005 .837 .183



b 9.15±0.03

b

.000 .000 .000 332.650



0.5mg Melatonin

9.04±0.02 b 9.06±0.03

b 9.07±0.03

ab .021 .000 .109 .744 .311


f

.000 .000 .731 .136


400mg Xylopia aethiopica 13.13±0

d 13.17±0.04

e

.000 .000 .449 .703



c 11.18±0.04

d

.000 .000 .521 .493



0.5mg Melatonin

11.14±0.01 c 11.17±0.04

d

.000 .000 .454 .685

50mg Cyclophosphamide 20.13±0.01 f 20.18±0.06

g

.000 .000 .456 .681



bc 10.13±0.03

c

.000 .000 .633 .266



b 9.14±0.03

b

.011 .000 .480 .607


400mg Xylopia Aethiopica +

0.5mg Melatonin

10.12±0.02 bc

10.15±0.03 c

.000 .000 .501 .545

Control 7.8±0.17 a 7.83±0.02

a 7.83±0.03

a .973 .027



Cyclophosphamide intoxicated wistar rats In week one, osmotic fragility of rats at exposed to 10mg



was significantly higher than rats treated with 10mg







Cyclophosphamide.


alone and 400 mg Xylopia aethiopica and 0.5mg



10mg Cyclophosphamide. Similarly, treatment with

400mg Xylopia aethiopica and 0.5 mg Melatonin and the










Cyclophosphamide.

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187

Table 4.25: Effects of Xylopia aethiopica extract and melatonin on osmotic fragility (0.85) scores of

cyclophosphamide intoxicated wistar rats.

Treatment 0.85


Week 1 (Wk1)

Week 2 (Wk2)

Week 3 (Wk3)


10mg Cyclophosphamide 5.7±0.58 abc 10.74±0.03

h 11.88±0.12 d .750 .000 .000 .000 93.078


6.8±0.18 def 6.53±0.02

d 5.72±0.02 c .506 .000 .006 .001 27.192


7.05±0.02 ef 5.92±0.02

b

.095 .000

.000 1509.052


5.3±0.14 abc 5.22±0.02

a 4.72±0.02 a .064 .000 .001 .006 13.917


i

.000 .000


8±0.17 g 7.03±0.02

e

.000 .000

.005 31.130


6.01±0.01 bcd 8.03±0.03

g

1.000 .000

.000 6002.250


5.13±0.01 ab 7.15±0.02

e

.014 .000

.000 6307.375


j

.000 .000


7.12±0.01 efg 7.74±0.02

f

.054 .000

.000 608.567


7.13±0.01 fg 5.14±0.02

a

.050 .000

.000 9891.754



a 5.91±0.02 b

.004 .000

.000 1659.689

Control 6.2±0.06 cde 6.22±0.02

c 5.31±0.02 b

.000 200.305

CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.0 Discussion

In this study, the effect of Xylopia aethiopica extract on

haematological parameters in cyclophosphamide induced

toxicity rats has been evaluated with results obtained

showing significant extract yield and phytochemical

agents’ presence in the extract. Acute toxicity study for

the extract was found to be above 6000 mg/kg body

weight while that of cyclophosphamide was 555.50

mg/kg body weight. Haematological values were also

altered following induction with significant reductions in

WBC count which was not improved following co-

administration with the extract and melatonin. Falls in

RBC, PCV and Hb values due to treatment with CP was

slightly improved when co-treated with extract. The

increase in osmotic fragility scores due to CP was also

significantly ameliorated following treatment with the

extract and melatonin.

The high extract yield obtained following extraction of

Xylopia aethiopica fruits suggests that the plant is rich in

phytocomponents which may be responsible for the

healing properties of the plants. The amount of extract

obtained may directly be due to the method of extraction.

Soxhlet extraction has been reported to be preferred over

cold maceration due to high extract yields (Jensen,

2007). Extract yield obtained following several other

works involving similar techniques also produced high

yields (Akomas et al., 2014; Oshilonyah et al., 2015;

Ijioma et al., 2017). Phytochemical agents present in

plants have been implicated in the pharmacological

effects of medicinal plants, hence the presence of

flavonoids, saponins, alkaloids, phenolic compounds,

terpenes, tannins and cardiac glycosides in the extract

only attests to the medicinal potentials of the plant. Most

green leafy vegetables are known to contain significant

amounts of these healing agents (Saliu et al 2012).

Flavonoids are known to be strong antioxidant agents

and scavenge free radicals (including ROS) and by that

improve the quality of body defense, preventing diseases

like ulcer, cancer and others associated with oxidative

stress. The antidiabetic effects of flavonoids have also

been reported (Oshilonyah et al., 2015). Flavonoids have

also been implicated in wound healing, cellular

regeneration and cytoprotection (Lewis et al., 1999;

Kumar et al., 2013) and may be a useful and readily

available agent for the management of ulcer. It is also

established that the anti-malarial effect of flavonoids

may by the inhibition of fatty acid biosynthesis in

plasmodium parasite and consequent influx of L-

glutamine and myoinositol into infected red blood cells

(Ntie-kang et al., 2014). Alkaloids and tannins are

widely used in the chemotherapeutic treatment of cancer

(Jin-Jan et al., 2012), a characteristic attributable to their

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antioxidant effects and ability to prevent cellular

oxidative damage and lipid peroxidation. Tannins

specifically inhibit the generation of superoxide radicals

(Chung et al., 1998) and have shown some degree of

inflammatory and anti-ulcer effects (Parekh et al., 2005).

Steroids and terpenes increase protein synthesis, promote

growth of muscles and bones (Huang, 2007). Results

obtained following the evaluation of healing effects of

saponins and glycosides show that these can alleviate

cardiaovascular problems due to their anti-

hypercholesterol effects associated (Ijioma et al., 2018).

Phenolic compounds are known to possess numerous

pharmacological activities including increasing bile

secretion, reducing blood cholesterol and other lipids

levels and antimicrobial activity against some bacteria

(Leung, 1980). Phenolics have also been implicated in

antiulcer effects (Matsuda et al., 2003), anti-

inflammatory activity (Araujo and Leon, 2001),

antioxidant potentials (Ghasemzadeh et al., 2011),

cytotoxic and antitumor effects (Murakami et al., 2004),

and antispasmodic activity (Ammon and Wahl, 1991).

The zero toxicity recorded following the administration

of graded doses of the extract to the experimental rats,

even at a high dose of 6000 mg/kg body weight suggests

that the extract have high margin of safety and as such

may be free of any form of acute oral toxicity. This

conclusion agrees with the OECD guidelines for acute

toxicity studies (OECD. 2001) which stipulates that the

expected outcome for any toxic substance is mortality

and that where no mortality is observed at the end of the

study period, then the subject is adjudged as been safe

for oral administration. Similar conclusions were reached

in other studies involving the acute toxicity study of

other medicinal plants (Akomas et al., 2014; Madubunyi

et al., 2012). Toxicity effects following the consumption

of plants usually are as a result of consuming toxic

amounts of phytochemicals (Oshilonyah, 2016). In this

study however, acute toxicity result of Xylopia

aethiopica suggest that the plant may contain only

moderate amounts of these phytoagents sufficient

enough to produce desired pharmacological effects.

An acute toxicity value of 555.0 mg/kg body weight

obtained for cyclosphamide suggest that at this dose the

use of the agent may be toxic. Result of a similar acute

toxicity study carried on cyclophosphamide reveal that

the later can cause severe acute toxicity effects including

mortality of animals to which the agent was administered

(Haubitz, 2007). Other toxicity effects such as

hepatotoxicity and nephrotoxicity (Ma et al., 2002),

hyperammonaemia, enterotoxicity and neurotoxicity

(Jury et al., 2011) and pulmonary toxicity (Malik et al.,

1996) have been reported following the administration of

cyclophosphamide. Therefore the rats that died during

acute toxicity evaluation of cyclophosphamide may have

died due to multiple systemic toxicity effects of the

agent.

Changes in haematological parameters such as WBC,

RBC, PCV and haemoglobin concentration have been

used to assess the degree of toxicity of substances on

experimental animals. Bone marrow is the site of

continued proliferation and turnover of blood cells and

also a source of cells involved in immune activity. White

blood cells form the first of body’s defense against

infections and are major players in immune responses

(Sembulingam and Prema, 2010). Fall in the values of

white blood cells of rats treated with cyclophosmide is

indicative of leukopenia. Thus cyclophosphamide is a

potential immunosuppressant lowers immune strength of

the body. The immunosuppressive effects of

cyclophosphamide may be linked to its cytotoxic effect

which is fully established (Haubitz, 2007, Ma et al.,

2002; Jury et al., 2011, Malik et al., 1996). Leukopenia

observed in this study is in agreement with the results of

a similar study in which the effects of cyclophosphamide

on haematological values was evaluated showed that the

administration of cyclophosphamide to experimental rats

caused severe leucopenia in the treated rats (Frinken and

Barnes, 1988). Co-treatment with Xylopia aethiopica

extract significantly ameliorated leukopenia, an effect

attributable to the phytochemicals present in the extract.

Phytochemicals possess antioxidant properties which

protect body cells from oxidative stress and dangers due

to attack on the cells by oxidative agents (Ijioma et al.,

2017). Results of a similar study have shown that

antioxidant agents can improve the body’s immune

strength by increasing the WBC count of the

experimental animals (Bendich 1993).

Adverse changes in red blood cells parameters may be

indicators of toxicities affecting the liver and bone

marrow as such can be used to assess anemic conditions

(Chernecky, 2003). In this study, treatment with

cyclophomide significantly lowered red blood cells

parameters including RBC, PCV and haemoglobin

concentrations in the treated rats. These results are in

agreement with previous reports on the haematotoxicity

of cyclophosphamide (Frinken and Barnes, 1988; Ukpo

et al., 2017). Irma et al., 2010 had reported that agents

with cytotoxic effects may cause haemolysis of red blood

cells, causing anaemia due to fall in the number of

circulating red blood cells. Thus anaemic effect of

cyclophosmamide may be linked to its cytotoxic

properties of the drug molecules on the cell with

hemolysis as the possible outcome. Co-treatment with

Xylopia aethiopica extract significantly ameliorated this

fall in RBC parameters, suggesting that the extract may

contain substances with blood building property. Idowu

et al., 2017 reported that Xylopia aethiopica improved

RBC count in an experiment designed to study the effect

of the extract on haemtological values. Xylopia

aethiopica may not be nutritionally different from other

green leafy vegetables/herbs people consume for

nutritional purposes. Green leafy vegetables have being

implicated in the elevation of RBC counts because of

their high iron content (Saliu et al., 2012) and potentials

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189

for improving bone marrow functions (Orhue et al.,

2008) and potentials for improving bone marrow.

The decrease seen in RBC values observed with

melatonin agrees with the work in disagreed. However,

Skwarlo-Sonta (2002) concluded that effects exerted by

melatonin on these parameters are different and can

depend on several factors, such as the dose and the way

of melatonin application, species, sex, age of animal, its

immune system maturation, way of immune system

activation and parameter examined as well as the season,

circadian rhyme of both immunity and pineal gland

function, stressful conditions and accompanying

experimental procedure. Hence, a modulator role can be

attributed to melatonin in the haemapoesis and its

functions from this study.

The fall in platelets counts following CP administration

suggests thrombocytopenia may have occurred due to the

cytotoxic effects of CP (Friken and Barnes, 1988; Ukpo

et al., 2017). CP-induced thrombocytopenia and

leucopenia (Glaser and Kiecolt-Glaser 2005) can

increase the PLT destruction and/or reduce the PLTs

production in bone marrow Hackett 2003. Bone marrow

is the organ most affected by immunosuppressant. Loss

of stem cells and inability of bone marrow to regenerate

new blood cells will result in thrombocytopenia and

leucopoenia. Our results also show a significant increase

mean platelet rats treated with XA and melatonin and a

possible synergistic effect. This in effect agrees with the

countering effect of XA on the toxicity of

cyclophosphamide on the bone marrow. The increase in

platelets observed with melatonin can be as a result of

melatonin being acetylated product of serotonin since

previous studies have demonstrated involvement of

serotonin in megakaryocytopoiesis. Yang et al. (2008)

also hypothesized that therapeutic effects of melatonin

may be involved in directly stimulating

megakaryocytopoiesis and having anti-apoptric effect in

megakaryocytopoiesis via activation of Akt/Erk

signaling. The fact that the extract was able to lower

platelets counts may be why it further increased both

prothrombin time (PT) and activated partial

thromboplastin time (APTT) in the test animals,

suggesting that the extract may have anti-platelets

aggregation and possible fibrinolytic activity. Falls in

platelets counts have directly being linked to increased

bleeding and clotting times and has a little advantage of

reducing the risk of blood clots being developed within

the blood vessels and its consequent cardiovascular

problems (Inyang et al., 2011; Torres-Uruttia et al.,

2011; Akomas and Ijioma, 2014).

Results of osmotic fragility scores indicates that CP

significantly reduced RBC percentage haemolysis in the

treated rats when compared with the control in different

salt concentrations. The result suggest that XA may have

increased the integrity of the RBC cell membranes

following treatment and made them resist the haemolytic

effects of the various salt concentrations. It is established

that the performance of normal functions by the

erythrocytes is highly dependent on their membrane

stability and ability to resist lysis (Adenkola and Oluremi

2014). The impact of free radicals on erythrocyte

membrane is a major cause of reduction in its ability to

resist lysis (Devasagayam et al., 2004; Dragan et al.,

2003). The higher membrane stability observed in all

groups treated with XA may be attributed to the

antioxidant potentials of the extract. Antioxidants have

greatly been implicated in the prevention of cellular

damage and generally consolidate the integrity of

erythrocyte membrane by reducing their oxidative

damage due the impact of free radicals (Adenkola and

Oluremi 2014).

CONCLUSION

This study provides evidence that Xylopia aethiopica is a

valuable medicinal food for combating

cyclophosphamide induced systemic toxicity. The

ameliorative effects of Xylopia aethiopica may be

mediated at least through scavenging reactions of free

radicals. Thus, Xylopia aethiopica may provide

protective effects for toxicants capable of inducing

oxidative stress. Also It can be seen that despite the high

potent immunosuppressive effect of cyclophosphamide

on blood cells, melatonin and Xylopia aethiopica have

shown to exert their ameliorative effects through their

antioxidant and antitumour properties. Therefore, they

may be of value in the prevention of diseases arising

from the oxidative effects of consumed toxicant

substances like cyclophosphamide.

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