Effects of Ethanol Leaf and Fruit Extracts of Kigelia africana on Some Oxidative and Biochemical ...

17 Pages·2017·0.31 MB·English

Asian Journal of Research in Biochemistry

1(1): 1-17, 2017; Article no.AJRB.39152

Effects of Ethanol Leaf and Fruit Extracts of Kigelia
africana on Some Oxidative and Biochemical
Parameters of Alloxan -Induced Diabetic Rats

E. N. Uhuo1*, L. U. S. Ezeanyika2 and V. N. Ogugua2

1Department of Chemical Sciences, Godfrey Okoye University, Enugu, Nigeria.
2Department of Biochemistry, University of Nigeria, Nsukka, Nigeria.

Authors’ contributions

This work was carried out in collaboration between all authors. Author ENU designed the study,
performed the statistical analysis, wrote the protocol and first draft of the manuscript. Authors LUSE
and VNO managed the analyses of the study. Authors ENU and LUSE managed the literature
searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/AJRB/2017/39152
Editor(s):
(1) Mohamed Fawzy Ramadan Hassanien, Professor, Biochemistry Department, Faculty of Agriculture, Zagazig University,
Zagazig, Egypt.
Reviewers:
(1) Samuel Ifedioranma Ogenyi, Nnamdi Azikiwe University, Nigeria.
(2) Nina Filip, Grigore T. Popa University of Medicine and Pharmacy, Romania.
(3) Keagile Bati, University of Botswana, Botswana.
(4) Kadiri Oseni, Obafemi Awolowo University, Nigeria.
Complete Peer review History: http://www.sciencedomain.org/review-history/23473

Received 1st November 2017
Accepted 25th January 2018
Original Research Article
Published 6th March 2018

ABSTRACT

Hyperglycaemia, a characteristic feature of diabetics mellitus leads to decreased antioxidant
defense and hence the development of oxidative stress, which is involved in the aetiology of
development of diabetic complications. This work was therefore aimed at evaluating the anti diabetic
and antioxidative potential of the plant. These evidences suggest that good glycemic control and/or
use of antioxidants may play an important role in the prevention of complications associated with
diabetes. Diabetes was induced with single Intra peritoneal injection of alloxan (160 mg/kg b.w)
dissolved in freshly prepared citrate buffer (pH 4.5). Oral administration of Kingelia africana (500
mg/kg b.wt) of methanol leaves and fruits extracts resulted in significant (p>0.05) decrease in the
blood glucose level, MDA, glycosylated haemoglobin, lipid profiles and liver maker enzymes with
corresponding increase in SOD activity, catalase activity, glutathione activity, serum protein
concentration, and Vit.C concentration. In conclusion, K. africana possessed antioxidative properties
evidenced by decrease blood glucose level and its effect on some oxidative parameters of diabetic
rats.
_____________________________________________________________________________________________________

*Corresponding author: E-mail: [email protected];
Uhuo et al.; AJRB, 1(1): 1-17, 2017; Article no.AJRB.39152

Keywords: Diabetes; alloxan; antioxidant; Kingelia africana.

1. INTRODUCTION changes in the structural and functional integrity
of sub-cellular organelles and may produce
Globally, the estimated incidence of diabetes and effects that result in the various complications in
projection for year 2030, as given by diabetic disease [11,12,13,14]. Recently, there
International Diabetes Federation is 350million has been an upsurge of interest in the
[1]. Currently available pharmacotherapies for therapeutic potentials of plants, as antioxidants in
the treatment of diabetes mellitus include oral reducing free radical-induced tissue injury.
hypoglycemic agents and insulin. However these Although several synthetic antioxidants are
drugs do not restore normal glucose commercially available, they are quite unsafe
homeostasis and they are not free from side and their toxicity is a problem of concern.
effects [2]. In view of the adverse effects
A survey of literature revealed that there is no
associated with the synthetic drugs and as plants
experimental evidence of the antidiabetic effects
are safer, affordable and effective, conventional
of Kigelia africana. Therefore, the present work
antidiabetic plants can be explored [3]. Over 400
explores this and will, in addition, study its
traditional plants have been reported for the
potential effect on some oxidative and
treatment of diabetes [4]. Furthermore, following
biochemical parameters of alloxan-induced
World Health Organization recommendations,
diabetic rats.
investigation of hypoglycemic agents from

medicinal plants has become more important [3].
2. MATERIALS AND METHODS
Also, diabetes has been treated orally with

several medicinal plants based on folklore 2.1 Plant Materials
medicine since ancient times.
The leave and fruit of Kingelia africana were
Kigelia africana (Lam.) Benth (Family:
collected from Omor, Aghamelu Local
Bignoniaceae) plant has many medicinal
Government Area, Anambra State, Nigeria. The
properties due to the presence of numerous
plant was authenticated by the Department of
secondary metabolites. These compounds
Plant Science and Biotechnology, University of
include iridiods, flavonoids, naphthoquinones,
Nigeria Nsukka.
volatile constituent, etc. [5,6,7 ].
2.2 Chemicals
Experimentally, the plant has shown

antibacterial, antifungal, antineoplastic, These were analytical grade products and
analgesic, anti-inflammatory, antioxidant include: ethanol methanol (BDH), ethylene
properties [8]. Crude extracts of herbs and diamine tetraacetate (EDTA), hydrochloric acid
spices and other materials rich in phenolics are (BDH), sulphuric acid (BDH), Trichloloroacetic
of increasing interest in the food industry acid (TCA), 2-thiobarbituric acid (TBA), Alloxan
because they retard oxidative degradation of Monohydrate (sigma-Aldrich, USA), 1-Chloro-2, 4
lipids and thereby improve the quality and dinitrothiobenzene, glutathione peroxidase kit
nutritional value of food. Flavonoids are groups (Randox Laboratories Limited, UK), Protein kit
of polyphenolic compounds with known (Randox Company, USA), Superoxide
properties, which include free radical scavenging, Dismutase kit (Randox Company, USA),
inhibition of hydrolytic and oxidative enzymes Glycosylated haemoglobin kit (Randox
and anti-inflammatory action [9]. company,USA), Sorbitol kit, Lipid profile kit,
Glucose test (Life Scan Inc, California, USA).
An enhanced oxidative stress has been observed

in diabetic patients as indicated by increased free 2.3 Extraction of the Plant Material
radical production, lipid peroxidation and
diminished antioxidant status [10]. In diabetes The leaves and fruits of K. africana were air-dried
mellitus, alterations in the endogenous free at room temperature for after which it was
radical scavenging defence mechanisms may grounded into powders using Rancilio Rocky
lead to ineffective scavenging of reactive oxygen (Rigtig Kaffe A/S, Skanderborg, Denmark). A
species, resulting in oxidative damage and tissue quantity of 500 mg each of the powdered leaves
injury. Oxidative stress is currently suggested as and fruits of K. fricana macerated in 1.5 litres of
the mechanism underlying diabetes and diabetic Ethanol for 72h. The solution was filtered with
complications. Oxidative stress may cause Whatman no 4 and concentred using rotary
oxidative damage of cellular membranes and evaporator (Model Modulyo 4K, England).

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2.4 Animals At the end of the experiment, rats were
starved for 12h and blood glucose levels
Male Wistar Albino rats between 12 to 14 weeks were determined. Blood samples were
of age, with an average weight of 108±5g, were received into clean dry centrifuge tubes and
obtained from the Department of Zoology, use for the analysis of the parameters.
University of Nigeria Nsukka. They were housed
in the animal facilities of Department of Home 2.7 Estimation of Biochemical parameters
Science and Dietetics, University of Nigeria,
Nsukka for one week before starting the All the chosen biochemical and oxidative
experiment. The animals were allowed free parameters were estimated using bio-diagnostic
excess to a standard diet, water and maintained kits and the procedures were strictly followed as
under optimum conditions of temperature, outlined in the manual guide.
relative humidity and light period. (12h light/12h
dark). 2.8 Statistical Analysis

2.5 Induction of Diabetics
Results were reported as mean± SEM, where
appropriate. Both one-and two-way analysis of
The rats were fasted (12h) prior to injection v a r ia n c e (ANOVA) was used to analyse
of alloxan dissolved in cold citrate buffer (pH 4.5) the experimental data and Ducan multiple test
in a dose of 160 mg/kg intra-peritoneally. The rage was used to compare the group means
base line blood glucose level was determined obtained after each treatment with control
before the induction. On the fourth-day blood measurement. Significant value was taken at p <
samples were taken from the tail vein to measure 0.05.
the blood glucose level using Accu-check
glucose meter (Roche, Germany). Rat with 3 . R E SULTS
blood glucose level of 200 mg/dl and above were

considered diabetic and used for the study [15].
3.1 Qualitative Phytochemical Composi-

tion of Ethanol Leaf and Fruit Extracts
The treatment was for a period of 4 weeks in
of K. africana
which the bloods obtained were used for

parameters analysis.
Table 1 Shows relative trace presence of
2.6 Experiment Begin saponin and terpenoids in all the
extract samples. In the same vein,
Thirty (30) male Wister albino rats with an hydrogen cyanide and steroid were found to
average weight of 108±5 g were classified into 6 be present in trace concentrations. Relative
groups (5 rats per group) and subjected to moderate amount of soluble carbohydrates
treatment as follows. was found in all the extracts. Interestingly,
flavonoid was found in high concentration in the
Group i: Normal control rats. extracts.

Group ii : Diabetics untreated rats. 3.2 Quantitative Phytochemical Composition
of Ethanol Leaf and Fruit Extracts of
Group iii: Diabetic rats treated 2.5 mg/kg bwt K. africana
glibenclamide
Table 2 shows the quantitative composition of
Group iv: Diabetics rats treated with 500 mg/kg bioactive compounds present at various
btw ethanol leaf extracts. concentrations. A significant increase of
flavonoid was recorded in ethanol fruit compared
Group v: Diabetic rats treated with 500 mg/kg with the leaf extracts. Trace concentration of
btw ethanol fruit extracted. hydrogen cyanides was found in the extracts. All
the extracts contained moderate concentration of
Group vi: Diabetic rats treated with an equal alkaloid.
ratio of ethanol leaf and fruit extracts.

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Table 1. Qualitative phytochemical composition of ethanol leaf and fruit extracts of K. africana.

Extract Soluble carbohydrate Tannin Alkaloid Hydrogen cyanide Saponin Flavonoid Reducing Steroid GlycosideT T e penoid
sugar
Ethanol leaf + + + + + ++ + + ++ +
Ethanol fruit + ++ ++ + + +++ + + ++ +
NB: + Present in trace concentration, ++ Present in moderately high concentration, +++ Present in very high concentration

Table 2. Quantitative phytochemical composition leaf and fruit extracts of K. afrcana

Extract Soluble Tannin Alkaloid Hydrogen Saponin Flavonoid Reducing Steroid Glycoside
carbohydrate (mg/100 g) (mg/100 g) cyanide (mg/100 g) (mg/100 g) sugar (mg/100 g) (mg/100 g)
(mg/100 g) (mg/100 g) (mg/100 g)
Ethanol leaf 0.96±0.02 4.11±0.65 2.67±0.12 0.03±0.001 0.52±0.02 2.32±0.04 50.85±3.36 0.53±0.03 2.39±0.15
Ethanol fruit 0.96±0.17 9.08±0.14 3.54±0.11 0.95±0.02 0.56±0.03 3.63±0.02 26.95±5.14 0.57±0.01 2.874±0.14

Table 3. Effect of ethanol extracts of leaves and fruits of K. africana on sugar level of diabetic rats

Treatment Groups Sugar Level (mg/dl)
Before induction 72 Hours after induction After 21 days treatment
Group 1 (Normal Control) 76.20±5.02ab 78.80±2.71ab 75.40±4.22ab
Group 2 (Diabetic Untreated) 67.40±3.50ab 558.40±14.01ac* 405.40±15.96ac*
Group 3 (Standard Control) 66.40±3.91ab 321.00±115.16ab* 241.20±116.79ab*
Group 4 (Diabetic + Ethanol Leaf Extract) 72.20±4.96ab 314.80±159.19ab* 184.40±54.50ab
Group 5 (Diabetic + Ethanol Fruit Extract) 76.40±9.07ab 464.80±159.32ac* 273.20±93.59ab*
Group 6 (Diabetic + Ethanol Leaf and Fruit Extract) 64.60±3.20ab 479.60±142.28ac* 269.60±108.64ab*
Results are expressed in mean ± SD; n = 5
Mean values with different letters as superscripts across the column compared with group 2 (diabetic untreated) are considered significant (p<0.05) while mean values with
asterisk (*) as superscripts across the row compared with the sugar level before the experiment are considered significant (p>0.05)

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3.3 Effects of Ethanol Extracts of Leaves 3.7 Effect of Ethanol Leaf and Fruit
and Fruits of K. africana on Sugar Extract of K. africana on Glycosylated
Level of Diabetic Rats Haemoglobin Concentration in
Alloxin-Induced Diabetic Rats
The sugar levels of rats before the experiment

in all groups were determined. Significant
The mean HbA1c level decreased significantly
(p<0.05) increased sugar level of induced
(p<0.05) in all the test groups compared with the
rats was observed compared with the sugar
HbA1c level of untreated diabetics rats (Group
level the rats before the experiment as shown
2).Change in HbA1c level was observed in
in Table 3. Reversibly, sugar level of rats
Group 6 rats treated with a combination of leaf
after 21 days treatment reduced
and fruit extract in ratio of 1:1 compared with
significantly (p<0.05) compared the level of
Group 2 rats untreated. A significant increase
sugar at 72 hours after induction and before
(p<0.05) HbA1c level was recorded in all the test
induction.
groups against the normal control rats (Fig.3).
Group 6 (diabetic+ ethanol leaf and fruit extracts,
3.4 Body Weight of Diabetic Rats ratio 1:1) demonstrated a non-significant
Treated with Ethanol Extracts of (p>0.05) reduction of HA1c concentration
Leaves and Fruits of K. africana compared with groups 4 and 5 rats orally fed with
before and after Experiment single plant extract (Fig. 3).

3.8 Effects of Ethanol Extracts of Leaf
Significant (p<0.05) increased in the body weight
and Fruit of K. africana on
of test groups compared with diabetic rats after
treatment was observed. Conversely, non- Malondialdehyde(MDA) Concentration
significant (p>0.05) decrease was observed in in Alloxan-Induced Diabetic Rats
the body weights of the animals in other groups 6
after the experiment compared with the body Lipid peroxidation measured as malondialdehyde
weights of the animals before the experiment of (MDA) observed significantly increase (p<0.5) in
the control group. (Table 4) all the test groups compared with untreated
control as shown in Fig.4. A significant decrease
3.5 Effects of Ethanol Leaf and Fruit of MDA concentration was recorded in group 6
Extracts of K. africana on Sorbitol treated with the combination of the plant extract
compared with the groups administered with
Concentration in Alloxan-Induced
single extract (Groups 4 and 5). Similarly, a
Diabetic Rats
significant decrease (p<0.05) of MDA

concentration was observed in the test groups
Sorbitol concentration in all the test groups
compared with MDA concentration of untreated
decreased significantly (p<0.05) compared with
diabetic group.
the untreated diabetic animals (Group 2). A

significant (p<0.05) reduction of sorbitol 3.9 Effects of Ethanol Leaf and Fruit
concentration was recorded in groups 6 treated
Extracts of K. africana on Vitamin C
with a combination of the leaf and fruit extracts of
Concentration in Alloxan-Induced
K.africana compared with the diabetic untreated
Diabetic Rats
rats (Fig. 1).

There was a general decrease in vitamin C
3.6 Effect of Ethanol Leaf and Fruit
concentration in all the test groups and the
Extract of K. africana on Total Protein untreated diabetic group compared with the
Concentration in Alloxan-Induced vitamin concentration of normal control rats
Diabetic Rats (Group 1). There was statistically significant
increase (p<0.05) of vitamin C concentration in
Fig. 2 reveals observable significant increased group 6 rats treated with a combination of
(p>0.05) of total protein in all test groups ethanol leaf and fruit extracts compared with
compared with the positive control rats (Group other test groups. The diabetic rats administered
2). Total protein concentrations in groups 6 fed 2.5 mg/kg b.wt of glibenclamide demonstrated an
with a combination of the leaf and fruit extract increased (p<0.05) vitamin c level compared with
showed significant increase (p<0.05) compared the vitamin C concentration of rats in Group 2
with other test groups. (diabetic untreated rates), see Fig.5

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3.10 Effects of Ethanol Leaf and Fruit (p<0.05) increase in SOD activities of all
Extracts of K. africana on Catalase test groups compared with the untreated
Activity in Alloxan-Induced Diabetic diabetic rats (Group 2) as shown in Fig. 7.
Superoxide dismutase activities of the test
Rats
groups (Group 6) administered with the

combination of the extracts was significantly
Across the test groups was recorded a
increased (p<0.05) compared with other test
statistically significant increase (p<0.05) catalase
groups treated with the single extracts (Groups 4
activity (Fig.6) compared with the untreated
and 5). The same observation was noted in the
diabetic rats (positive control; Group 2). Similarly,
test treated with the standard drug. However, the
a significant increase (p<0.05) of catalase activity
activities of SOD in the diabetic rat administered
was observed in the diabetic rats treated with
with 2.5 mg /kg b.wt of glibenclamide increased
reference drug (glibenclamide) in comparison
significantly (p<0.05) as against all the test
with the catalase activity of all the test groups. In
groups.
the same pattern, Group 6, ethanol leaf + fruit

extracts demonstrated a non significant increase
(p>0.05) of catalase activity compared with other 3.12 Effects of Ethanol Leaf and Fruit
test groups (Groups 4 and 5) administered with a Extracts of K. africana on
single plant extract. Percentage Inhibition of SOD
Activity in Alloxan-Induced Diabetic
3.11 Effects of Ethanol Leaf and Fruit Rats
Extracts of K. africana on
Superoxide Dismutase (SOD) Fig.8 demonstrates statistically significant
Activity in Alloxan-Induced Diabetic decrease (p<0.05) of percentage inhibition of
Rats SOD activity in the test groups compared with
the normal control group. A significant reduction
The activities of superoxide dismutase (SOD) (p<0.05) of percentage inhibition of SOD activity
reduced significantly (p<0.05) in all the test occurred in the diabetic untreated rats (group 2)
groups compared with the normal control c o m pared with the percentage inhibition of SOD
(Group 1).There were statistically significant activity in normal control. Diabetic 6 treated with

Fig. 1. Effect of ethanol extracts of leaf and fruit of K. africana on sorbitol concentration in
alloxan-induced diabetic rats

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Table 4. Body weights of diabetic rats treated with ethanol extracts of leaves and fruits of K
.africana before and after experiment

Treatment Groups Body Weight (g)
Before Experiment After Experiment
Group 1 (Normal Control) 92.59±5.87a 130.36±17.83b
Group 2 (Diabetic Untreated) 173.66±12.24a 156.16±13.14a
Group 3 (Standard Control) 94.58±5.80a 107.34±18.41a
Group 4 (Diabetic + Ethanol Leaf Extract) 103.94±8.30a 125.98±14.59b
Group 5 (Diabetic + Ethanol Fruit Extract) 119.83±40.91a 127.94±37.44a
Group 6 (Diabetic + Ethanol Leaf and Fruit 86.71±9.77a 95.12±4.09a
Extract)
Results are expressed in mean ± SD; n = 5
Mean values with different letters as superscripts across the row are considered significant (p<0.05)

Fig. 2. Effect of ethanol extracts of leaf and fruit of K. africana on total protein concentration in
alloxan-induced diabetic rats

a combination of leaf and fruit extracts recorded 3.13 Effects of Ethanol Leaf and Fruit
a significantly (p>0.05) increase of percentage Extract of Kigelia africana on
inhibition of SOD activity compared with groups 4 Glutathione Peroxidase Activity in
and 5 administered monotherapically with leaf
Alloxan-Induced Diabetic Rats
and fruit extracts of K. africana. Conversely,

group 6 treated with ethanol leaf extracts showed
Fig. 9 represents activity of glutathione
significant increase (p<0.05) of percentage
peroxidase (GPx) which increased significantly
inhibition of SOD activity as against groups 4, 7
(p<0.05) in all the test groups treated with both
and 8 of the same treatment pattern.
single and combination of the leaf and fruit of K.
Furthermore, non-significant reduction (p>0.05)
africana extract in comparison with the GPx
of percentage inhibition was observed in
activity of the rats in group 1 (normal control
diabetic administered the n-hexane leaf and fruit
rats). The combination therapy in group 6
extracts compared with the diabetic rats treated
demonstrated significant increase (p<0.05) of
with 2.5 mg/kg body weight of glibenclamide
GPx activity compared with groups 4 and 5 test
(Group 3).

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groups treated with a single plant extract increased in GPx activity significantly
(monotherapy).In the same vein test groups 6 (p<0.05) relative to group 3 treated with the
treated with combined leaf and fruit extracts standard drug.

Fig. 3. Effect of ethanol extracts of leaf and fruit of K. africana on glycosylated haemoglobin
concentration in alloxan-induced diabetic rats

Fig. 4. Effects of ethanol extracts of leaf and fruit of K. africana on malondialdehyde
concentration in alloxan-induced diabetic rats

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Fig. 5. Effects of ethanol extracts of leaf and fruit of K. africana on vitamin C concentration in
alloxan-induced diabetic rats

Fig. 6. Effects of ethanol extracts of leaf and fruit of K. africana on catalase activity in
alloxan-induced diabetic rats

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Fig. 7. Effects of ethanol extracts of leaf and fruit of K. africana on superoxide dismutase in
alloxan – induced diabetic rats

Fig. 8. Effects of ethanol extracts of leaf and fruit of K. africana on superoxide dismutase
percentage inhibition in alloxan-induced diabetic rats

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