CHAPTER THREE

3.0 Materials and Methods

3.1 Materials

3.1.1 Plant Materials

Fresh leaves of Kigelia africana were collected from the University of Cross River State

(UNICROSS) environment, Okuku, Cross River State, Nigeria. The leaves were taken to the

University of Calabar, Department of Botany for identification and authentication. The voucher

number of 206 has been deposited for future reference at the department's herbarium.

3.2 Chemicals and Reagents:

All chemicals and reagent (cyclophosphamide, ethanol, sulphuric acid, ferric chloride,

chloroform, hydrochloric acid, Mayer’s reagent and Wagner’s reagent) used were of analytical

grade. Fresh distilled water was used throughout the experimental period. Assay kits used in the

analysis in this study were products of Randox Laboratories (England).

3.3 Experimental animals

Twenty five (25) male Wistar rats were obtained from the animal holding unit of the Department

of Medical Biochemistry, University of Cross River State. The animals was allowed to

acclimatize for a period of 7 days, in a well-ventilated room at room temperature and relative

humidity of 29°C and 70% respectively with 12 hours natural light-dark cycle. They were

allowed food and water ad libitum. Good hygiene was maintained by daily cleaning and removal

of faeces and spills from their cages. All experiments were carried out in compliance with the
recommendations of Faculty of Basic Medical Sciences, University of Cross River State, Ethics

Committee on guiding principles on care and use of animals.

3.4. METHOD

3.4.1 Preparation of extract of Kigelia africana leaf

The leaves of Kigelia africana were collected around UNICROSS and air dried at room

temperature for a period of 21days until constant weight was obtained. The dried leaves were

then pulverized to powdered form by a machine blender and sieved. Thereafter, 400g of the

pulverized plant material (K. africana) was dissolved in 1200ml of 70% petroleum ether for 72

hours. This was followed with vacuum filtration and extracts was concentrated using an

evaporator water bath at 40°C to obtain a solvent free extract, and stored in a refrigerator at 4°C.

3.4.2 Induction of immunosuppression

Immunosuppression in Wistar rats was induced interperitoneal administration of 10 mg/kg b.w.

of cyclophosphamide for 7 days to induce immunosuppression in designated rat groups.

3.4.3 Treatment of animals

The experimental rats were randomly divided into five (5) groups, with five animals per group

and treated for a period of fourteen (14) days.

Group A: NC (normal control)

Group C: immune compromised rats treated with immunal (Standard control)

Group B: immune compromised rats (induced with cyclophosphamide) without treatment

(immune compromised control)

Group D: immune compromised rats treated with 200mg/kg bwt aqueous leaf extract Kigelia

africana extract (ALEKA1)

Group E: immune compromised rats with 400mg/kg bwt aqueous leaf extract Kigelia africana

extract (ALEKA2)

All administrations were done orally using orpharyngeal cannula once per day for 14days (2

week).

3.4.4 Blood sample collection

At the end of the 14-day experimental period, the anesthesia was performed on all experimental

animals. The anesthetized animals were bled by cardiac puncture. The blood samples were

collected into plain sample bottles for enzyme analysis

3.5 Oxidative stress biomarkers

3.5.1 Determination of superoxide dismutase (SOD) activity

The levels of total SOD activity in the tissues were determined by the method of Martin et al.,
(1987).
Principle

The ability of superoxide dismutase to inhibit the auto oxidation of adrenaline at pH 10.2 makes
this reaction a basis for the SOD assay. Superoxide anion (O 2) generated by the xanthine oxidase
reaction is known to cause the oxidation of adrenaline to adrenochrome. The yield of
adrenochrome produced per superoxide anion increased with increasing pH and also with
increasing concentration of adrenaline. These led to the proposal that auto oxidation of
adrenaline proceeds by at least two distinct pathways, one ofwhich is a free radical chain reaction
involving superoxide radical and hence could be inhibited by SOD.

Procedure
An aliquot of 0.2 ml of each of the tissue homogenates was added to 2.5 ml of 0.05 carbonate
buffer (pH 10.2) to equilibrate in the spectrophotometer and the reaction was started by the
addition of 0.3 ml of freshly prepared 0.3 mM epinephrine to the mixture. The absorbance of the
sample was measured at 450 nm using spectrophotometer.
Change in ab/ min = A5-A1/2.5
 Increase∈|of |sample
% Inhibition ¿ x 100%
Increase∈|of |blank

I unit SOD == Amount that cause 50% inhibition

3.5.2 Determination of catalase activity

Catalase activity was determined according to the method of Sinha (1972).
Principle
This method is based on the fact that dichromate in acetic acid is reduced to chromic acetate
when heated in the presence of H2O2 with the formation of perchromic acid as an unstable
intermediate. The chromate acetate then produced is measured colorimetrically at 570-610nm.
Since dichromate has no absorbance in this region, the presence of the compound in the assay
mixture does not interfere at all with the colorimetric determination of chromic acetate. The
catalase preparation is allowed to split H 2O2 for different periods of time. The reaction us
stopped at a particular time by the addition of dichromate acetic acid mixture and the remaining
H2O2 is determined by measuring chromic acetate colorimetrically after heating the reaction
mixture.
Procedure
A known volume, 1 ml. of the supernatant fraction of the tissue homogenate was mixed with
19ml distilled water to give a 1:20 dilution. The assay mixture contained 4ml of H 2O2 solution
(800 µmoles) and 5ml of phosphate buffer, pH 7.0 in a 10ml flat bottom flask. 1ml of properly
diluted sample was rapidly mixed with the reaction mixture by a gentle swirling motion at room
temperature. 1ml portion of the reaction mixture was withdrawn and blown into 2 ml
dichromate/acetic acid reagent at 60 seconds interval. The hydrogen peroxide contents of the
withdrawn sample were determined by the method described above.

Calculation
The monomolecular velocity constant K for the decomposition of H 2O2 by catalase was
determined by using the equation for a first-order reaction.
K=1/t log So/S
Where so=initial concentration of H2O2 and S = concentration of H 2O2 at 1min interval. The
values of K were plotted against time in minutes and the velocity constant of catalase K (O) at 0
minute was determined by extrapolation.

The catalase content of enzyme preparation was expressed in terms of catalase feiahigkeit or
“Kat f” (which is equivalent to micromole H2O2 consumed per min mg protein) according to Von
Euler and Josephson (1927):
Kat f = KO
Mg protein/ml

3.5.3 Assessment of lipid peroxidation

A breakdown product of lipid peroxidation thiobarbituric acid reactive substances (TBARS) was
measured by the method of Buege and Aust (1978).

Principle
Malondialdehyde, formed from the breakdown of polyunsaturated fatty acids, serves as a
convenient index for determining the extent of the peroxidation reaction. Malondialdehyde
(MDA) has been identified as the product of lipid peroxidation that reacts with thiobarbituricacid
to give a red species absorbing at 535nm.

Procedure
One volume of the test sample and two volume of stock reagent were mixed in a cooked test tube
and heated for 15 minutes on a boiling water bath. After cooling at room temperature, the
precipitate was removed by centrifugation at 1000 x g for 10 minutes and the absorbance of the
supernatant was measured at 532nm against blank containing all the reagents except test sample.
Calculation
The malondialdehyde (MDA) concentration of the sample was calculated from the absorbance
using an extinction coefficient of 1.56 x 10 5 M-1 cm-1 according to the method of Adam-Vizi and
Sergi (1982).
 Absorbance x T . V
MDA (moles / g tissue) ¿
E532 x V s x g tissue

Where E532 = Molar extinction coefficient for MDA

T.V = total volume of reaction mixture
Vs = Volume of Sample
g = gram.

Statistical analysis

Statistical analysis was carried out using one-way analysis of variance (ANOVA) to access the
difference between means. Resulting data were represented as Mean ± standard deviation of
triplicate determinationswith the level of significance set at p<0.05; n = 5. GraphPad Prism
9®(Version 9.3.1, GraphPad Software Inc., San Diego, United States of America) was used for
the statistical analysis.

CHAPTER FOUR

4.0 Result

The result below indicates the effect of petroleum ether extract of Kigelia africana on liver
SOD, catalase and MDA in cyclophosphamide induced immunocompromised rats

The petroleum ether extract of Kigelia africana significantly (p<0.05) increased the serum
catalase concentration at both 200 and 400 mg/ kg body weight when compared with the
normal and standard control of Wistar rats (fig 1)
However , the extract of Kigelia africana significantly (p<0.05) lowered both the SOD and
MDA concentration at both 200 and 400 mg/ kg body weight when compared with the normal
and standard control (fig 2-3)

Fig. 1: Effect of petroleum ether extract of Kigelia africana leaf on CAT concentration in
cyclophosphamide induced immune compromised Wistar rats.Results were expressed as Mean ±
SD (n = 5). *** significant at P<0.05 compared with the control. NC: Normal Control, CP +
IMMU: Cyclophosphamide + immunal (20 mg/Kg bwt), CP: Cyclophosphamide control, CP +
PETLEKA1: Cyclophosphamide + Petroleum ether extract of K. africana (200 mg/Kg bwt), CP
+ PETLEKA2: Cyclophosphamide + Petroleum ether extract of K. africana (400 mg/Kg bwt).
Fig. 2: Effect of petroleum ether extract of Kigelia africana leaf on MDA concentration in
cyclophosphamide induced immune compromised Wistar rats.Results were expressed as Mean ±
SD (n = 5). *** significant at P<0.05 compared with the control. NC: Normal Control, CP +
IMMU: Cyclophosphamide + immunal (20 mg/Kg bwt), CP: Cyclophosphamide control, CP +
PETLEKA1: Cyclophosphamide + Petroleum ether extract of K. africana (200 mg/Kg bwt), CP
+ PETLEKA2: Cyclophosphamide + Petroleum ether extract of K. africana (400 mg/Kg bwt).
Fig. 3: Effect of petroleum ether extract of Kigelia africana leaf on SOD concentration in
cyclophosphamide induced immune compromised Wistar rats.Results were expressed as Mean ±
SD (n = 5). *** significant at P<0.05 compared with the control. NC: Normal Control, CP +
IMMU: Cyclophosphamide + immunal (20 mg/Kg bwt), CP: Cyclophosphamide control, CP +
PETLEKA1: Cyclophosphamide + Petroleum ether extract of K. africana (200 mg/Kg bwt), CP
+ PETLEKA2: Cyclophosphamide + Petroleum ether extract of K. africana (400 mg/Kg bwt).