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alhassangarba01@gmail.com, alhassan.garba01@gmail.com;
International Research Journal of Pure & Applied Chemistry
19(1): 1-6, 2019; Article no.IRJPAC.47673
ISSN: 2231-3443, NLM ID: 101647669
Determination of Antioxidant Activity of Leave
Extracts of Albizia chevalieri Using Free Radical
Scavenging Activity Assay
Alhassan M. Garba
1*
, Habiba R. Isa
1
, Sadiq Abubakar
2
and Saudat Ja’afar
1
1
Department of Chemistry, Kano University of Science and Technology Wudil, P.M.B. 3244,
Kano State, Nigeria.
2
Department of Pure and Industrial Chemistry, Bayero University, Kano, P.M.B. 3011, Kano State,
Nigeria.
Authors’ contributions
This work was carried out in collaboration among all authors. Author AMG performed the statistical
analysis, wrote the protocol and wrote the first draft of the manuscript. Author HRI designed the study.
Author SA managed the analyses of the study and author SJ managed the literature searches. All
authors read and approved the final manuscript.
Article Information
DOI: 10.9734/IRJPAC/2019/v19i130099
Editor(s):
(1) Dr. Hao-Yang Wang, Department of Analytical, Shanghai Institute of Organic Chemistry, Shanghai Mass Spectrometry
Center, China.
(2)
Dr. Surendra Reddy Punganuru, Department of Biomedical Sciences, School of Pharmacy, Texas Tech University Health
Sciences Center, Amarillo, USA.
Reviewers:
(1) Eduardo Martinez-Abundis, Juarez Autonomous University of Tabasco, México.
(2)
Veeravan Lekskulchai, Srinakharinwirot University, Thailand.
(3)
Pratibha Kamble, Ohio State University, Columbus.
Complete Peer review History:
http://www.sdiarticle3.com/review-history/47673
Received 01 March 2019
Accepted 15 May 2019
Published 30 May 2019
ABSTRACT
Dried and powdered leaves of Albizia chevalieri were extracted using ethanol. The extract was
fractionated to give methanol, chloroform and pet-ether. The four extracts obtained; ethanol,
chloroform, methanol and pet-ether were evaluated for antioxidant activity using 2,2-diphenyl-1-
(2,4,6-trinitrophenyl)hydrazyl (DPPH) free radical scavenging activity assay. The results of the
DPPH scavenging activity indicated a concentration-dependent antioxidant activity. The DPPH
scavenging activity of the ethanol, chloroform and methanol extracts were found to be promising.
There is no significant difference in the antioxidant activity between the ethanol, chloroform and
methanol extracts with that of standard Ascorbic acid at 10, 25, 250 and 500 μg/ml concentrations.
This showed that the ethanol, chloroform and methanol leave extracts of the plant has the potency
Original Research Article
Garba et al.; IRJPAC, 19(1): 1-6, 2019; Article no.IRJPAC.47673
2
of scavenging free radicals in vitro and may provide leads in the ongoing search for natural
antioxidants from Nigerian medicinal plants to be used in treating diseases related to free radical
reactions.
Keywords: Albizia chevalier; antioxidant; free radical; extract; scavenging activity assay; DPPH.
1. INTRODUCTION
The plant Albizia chevalieri is a tree or a shrub
that grows up to 12m height under harsh
conditions of the dry savannah from Senegal,
Niger and Nigeria. It has an open and rounded or
umbrella-shaped canopy, bark pale-grayish,
twigs pubescent with white lenticels, leaves with
8-12 pairs of pinnate and 20-40 pairs of leaflets
each. The bark was reported to contain alkaloids
and also tennis sufficient for use in tanning in
Nigeria and Senegal. It is used in Borno-North
eastern Nigeria as purgative, taenicide and also
remedy for coughs. A decoction of leaves is used
in Northern Nigeria as a remedy for dysentery
[1]. There are also reports on the local use of the
leaves extract for cancer treatment in Zaria city,
Kaduna state [2].
Previous studies on Albizia chevalieri have
indicated the presence of phenolic compounds
from Albizia amara with significant antioxidant
activity [3] and Albizia inundata was reported for
effective anti candida activity from Brazilian flora
[4]. Liphophlic extracts of Albizia gummifera
revealed very promising antitrypanosomal activity
[5]. The extracts of Albizia ferruginea were also
reported to have significant antimicrobial activity
on selected microorganisms [6] and Albizia
saman was found to have good antiplasmodial
activity [7]. Albizia lebbeck was reported to
contain 3α, 5-dihydroxy-4β, 7-dimethoxy flavones
and N-Benzoyl-L-phenyl alaninol [8]. As the
focus of medicine shifts from the treatment of
manifest disease to prevention, increasing
awareness on herbal remedies as potential
sources of phenolic antioxidants have grown in
recent years, and several plants are being
screened for their antioxidant properties using
different assays [9].
DPPH, known as 2,2-diphenyl-1-picrylhydrazyl,
(I.U.P.A.C name, 2,2-diphenyl-1-(2,4,6-
trinitrophenyl)hydrazyl with molecular formula
C
18
H
12
N
5
O
6
is a stable free radical that is
commonly used to evaluate the ability of a
compound to act as a free radical scavenger or
hydrogen donor and to measure the antioxidant
activity of tissue extract [10]. Free radicals are
fundamental to any biochemical process and
represent an essential part of aerobic life and
metabolism [11]. Antioxidants offer resistance
against oxidative stress by scavenging the free
radical and many other mechanisms thus
preventing disease progression [12]. The
reaction of DPPH with an antioxidant or reducing
compound produces the corresponding
hydrazine DPPH-H, which can be followed by
color change from purple (absorbance at 515-
528nm) to yellow. The DPPH method is widely
used for the measurement of free radical
scavenging ability of antioxidants [13,14]. DPPH
is a rapid, simple, accurate and inexpensive
assay for measuring the ability of different
compounds to act as free radical scavengers or
hydrogen donors and to evaluate the antioxidant
activity of foods and beverages [15].
This work was designed to investigate the
antioxidant properties of ethanol methanol,
chloroform and pet-ether leaves extracts of
Albizia chevalieri with a view to assessing the
potential of the plant as a source for antioxidants.
2. MATERIALS AND METHODS
2.1 General
The Ethanol was obtained from Sigma Aldrich,
the DPPH and Ascorbic acid was obtained from
chemistry laboratory Bayero University Kano.
While other reagents and chemicals were of
analytical grade supplied by Chemistry
Laboratory, Kano University of Science and
Technology, Wudil. All glass wares used were
washed with detergents and oven dried before
use. The leaves of the plant Albizia chevalieri
were rinsed with clean tap water to remove dust
and impurities.
2.2 Collection of Plant Material
The leaves of plant Albizia chevalieri were
collected on 4
th
November 2017 from
Kududdufawa village Ungogo local government
area of Kano State. The plant was authenticated
by Baha'uddeen Said Adam from the Department
of Plant Biology, Bayero University Kano, with
accession number BUKHAN 0378.
Garba et al.; IRJPAC, 19(1): 1-6, 2019; Article no.IRJPAC.47673
3
2.3 Extraction of Plant Material
The extraction and fractionation of air-dried and
ground plant material were carried out according
to the method described by Prakash [15]. 150 g
of the powdered plant sample was weighed and
percolated in 600 ml of 99% Ethanol in 2L
conical flask for 14 days at room temperature. It
was filtered using Whattman No. 2 filter paper
and the solvent (Ethanol) was completely
removed using rotary evaporator. The extract
obtained was dried under room temperature to
get a thick paste form of the plant extract. A dark
green glassy and a gummy thick paste were
obtained and weighed. It was labelled as AC01.
2.4 Fractionation of Crude Extract
The dried Ethanolic extract was partitioned with
chloroform-water in 1:1 ratio of 100ml in a
separating funnel. The mixture was shaken for
15 minutes and allowed to settle for 2 hours in a
separation funnel, the water and chloroform
fractions were separated in glass beakers. The
chloroform fraction was concentrated at 35
o
C
using rotary evaporator and drying was
completed under room temperature. The
chloroform extract obtained was labelled as
AC02. While the water fraction was discarded.
Similarly, the dried chloroform extract was further
partitioned with Methanol and Pet-ether in 1:1
ratio of 100 ml in a separation funnel. The
mixture was shaken for 15 minutes and allowed
to settle. The methanol and pet-ether fractions
were separated in glass beakers and were
concentrated at 45ºC and 35ºC respectively
using rotary evaporator. The extracts were
completely dried under room temperature and
weighed where the methanol and pet ether
extracts were obtained and labelled AC03 and
AC04 respectively.
2.5 DPPH Free Radical Scavenging
Activity Assay
The determination of the radical scavenging
activity of the crude extract of Albizia chevalieri
was carried out using DPPH (1,1-diphenyl-2-
picrylhydrazyl) as described by Fatope et al. [16]
with slight modification. Various concentrations of
500, 250, 100, 50, 25 and 10 μgml
-1
of each of
the sample extract in methanol were prepared.
The commercial known antioxidant, Ascorbic
acid (Vitamin C) was used for comparison or as a
positive control. The DPPH in the absence of
plant extract was used as control and the plant
extract in the absence of DPPH was used as
blank. DPPH (1 mM, 200 μl) in methanol was
added to 100 μl solution of each of the plant
extract and allowed to stand at room temperature
in a dark chamber for 30 min. The change in
colour from purple to yellow was then measured
at 517 nm on a spectrophotometer. Measure-
ment was performed in triplicate. The percentage
of radical scavenging activity was calculated
using the following equation:
%
= 100 − −
100
Abs sample = Absorbance of plant extract +
DPPH
Abs blank = Absorbance of plant extract
Abs control = Absorbance of DPPH
(DPPH*) + H-A (DPPH-H) + A*
Purple Yellow
3. RESULTS AND DISCUSSION
3.1 Extraction and Fractionation of Plant
Material
The sequence of extraction followed the order
ethanol, chloroform, methanol and pet-ether. The
extraction of the powdered leaves of Albizia
chevalieri was carried out using 99%v/v ethanol
and the resulting extract was partitioned to give
methanol, chloroform and pet-ether fractions
(Fig. 1). 5.11% of the plant material went into the
ethanol which on partitioning between
chloroform/water, about 1.11% of the ethanol
fraction went into chloroform. Further partitioning
of the chloroform extract resulted in about 0.31%
and 0.19% of the extract going into methanol and
pet-ether respectively. This showed that the
leaves extracts of the plant contain a reasonable
amount of medium polar and non-polar
compounds (Table 1).
3.2 Antioxidant Activity of Albizia
chevalieri
Antioxidant properties of leaves extract of Albizia
chevalieri was evaluated to find a new source of
antioxidant. DPPH radical is a commonly used
substrate for fast evaluation of antioxidant activity
because of its stability in the radical form and
simplicity of the assay [17]. This assay is known
to give reliable information concerning the
antioxidant ability of the tested compounds [18].
The principle behind this assay is the colour
change of DPPH solution from purple to yellow
Garba et al.; IRJPAC, 19(1): 1-6, 2019; Article no.IRJPAC.47673
4
as the radical is quenched by the antioxidant
[19].
The leaves extracts of Albizia chevalieri were
screened for DPPH radical scavenging activity
according to the method described [20] with
slight modification and the result of the screening
is shown in (table 2) as compared to Ascorbic
acid, a known antioxidant. Four different extracts
of Albizia chevalieri (AC01, AC02, AC03, and
AC04) showed high radical scavenging activity at
various concentrations of 10, 25, 50, 100, 250
and 500 μg/ml (Fig. 2).
3.3 Inhibitory concentration at 50% (IC
50
)
The radical scavenging activity of each extract
was determined by calculating the inhibitory
concentration at 50% (IC
50
), the IC
50
of various
extract of Albizia chevalieri and Ascorbic acid.
The lower the IC
50
, the more potent the extract,
this showed that methanol extract has the
highest radical scavenging activity (33.08 μg/ml)
more than the standard Ascorbic acid (36.85
μg/ml), followed by ethanol extract (52.92 μg/ml),
Chloroform extract (54.53 μg/ml) and pet-ether
extract (71.59 μg/ml) [Fig. 3].
Fig. 1. Extraction and fractionation procedure of the powdered leave of the plant
Fig. 2. Free radical scavenging activity of various extract of Albizia chevalieri at different
concentrations
Garba et al.; IRJPAC, 19(1): 1-6, 2019; Article no.IRJPAC.47673
5
Table 1. Weights of extracts recovered and their physical properties
Extracts
Colour
Texture
Weight (g)
Weight (%)
AC01
[crude extract] Dark green Gummy like 7.668 5.11
AC02 [CHCl
3
extract] Black Semi-solid 1.661 1.11
AC03 [methanolic extract] Black Semi-solid 0.468 0.31
AC04 [pet-ether extract] Black Semi-solid 0.290 0.19
Fig. 3. Inhibitory concentration at 50% (IC
50
) of various extracts of Albizia chevalieri
4. CONCLUSION
These findings revealed the potential of Albizia
chevalieiri as a source for natural antioxidants. It
indicates that the plant could be a promising
agent in scavenging free radicals and treating
diseases related to free radical reactions. The
leaves extracts of Albizia chevalieri were found to
have high radical scavenging activity as
compared with standard Ascorbic acid. The
results of the DPPH scavenging activity study
indicate a concentration-dependent antioxidant
activity which increases with increase in the
concentration of the extract.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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_________________________________________________________________________________
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