Comparative in vitro Assessment of Drumstick (Moringa oleifera) and Neem (Azadiracta indica) Leaf Extracts for Antioxidant and Free Radical Scavenging Activities

Abstract

The current research was aimed at comparing the leaf extracts of two medicinal plants (Moringa oleifera and Azadiracta indica) for antioxidant and free radical scavenging potentials in different extracting solvents (absolute ethanol, 70 and 50% ethanol). Different in vitro assays such as total phenolic and flavonoid content, 2-2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, metal chelating activity, reducing power and total antioxidant capacity were employed in the study. The results revealed that A. indica contained more phenols and flavonoids than M. oleifera with the different extracting solvents. The amount of phenols and flavonoids in A. indica played a pivotal role in scavenging more of the DPPH radical at a lower inhibitory concentration, IC50 of 77.94 µg mL·1 than in M. oleifera at 118.96 µg mL·1 in absolute ethanol. Moringa oleifera was a better scavenger of the DPPH radical in 70 and 50% ethanol. In absolute ethanol, A. indica also chelated 50% of the metal ion at IC50 of 0.22 µg mLG1 which was even better than ascorbic acid (5.95 µg mL·1) and gallic acid (0.503 µg mL·1) standards. The values for A. indica were also comparably better than those of M. oleifera for reducing power and total antioxidant capacity at the respective concentrations. The results are indicative of the antioxidant and free radical scavenging potentials of M. oleifera and A. indica. Comparatively, A. indica was better than M. oleifera in doing the job and absolute ethanol extracts were better than 70 and 50% ethanol extracts in the scavenging potential.

Full text

Research Journal of Medicinal Plant 9 (1): 24-33, 2015
ISSN 1819-3455 / DOI: 10.3923/rjmp.2015.24.33
© 2015 Academic Journals Inc.
24
Comparative in vitro Assessment of Drumstick (Moringa oleifera) and
Neem (Azadiracta indica) Leaf Extracts for Antioxidant and Free
Radical Scavenging Activities
U.B. Ekaluo, E.V. Ikpeme, O.U. Udensi, E.E. Ekerette, S.O. Usen and S.F. Usoroh
Department of Genetics and Biotechnology, University of Calabar, Calabar, Nigeria
Corresponding Author: U.B. Ekaluo, Department of Genetics and Biotechnology, University of Calabar, Calabar,
Nigeria
ABSTRACT
The current research was aimed at comparing the leaf extracts of two medicinal plants
(Moringa oleifera and Azadiracta indica) for antioxidant and free radical scavenging potentials
in different extracting solvents (absolute ethanol, 70 and 50% ethanol). Different in vitro assays
such as total phenolic and flavonoid content, 2-2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging, metal chelating activity, reducing power and total antioxidant capacity were employed
in the study. The results revealed that A. indica contained more phenols and flavonoids than
M. oleifera with the different extracting solvents. The amount of phenols and flavonoids in
A. indica played a pivotal role in scavenging more of the DPPH radical at a lower inhibitory
concentration, IC of 77.94 µg mLG than in M. oleifera at 118.96 µg mLG in absolute ethanol.
50 1 1
Moringa oleifera was a better scavenger of the DPPH radical in 70 and 50% ethanol. In absolute
ethanol, A. indica also chelated 50% of the metal ion at IC of 0.22 µg mLG which was even better
50 1
than ascorbic acid (5.95 µg mLG) and gallic acid (0.503 µg mLG) standards. The values for
1 1
A. indica were also comparably better than those of M. oleifera for reducing power and total
antioxidant capacity at the respective concentrations. The results are indicative of the antioxidant
and free radical scavenging potentials of M. oleifera and A. indica. Comparatively, A. indica was
better than M. oleifera in doing the job and absolute ethanol extracts were better than 70 and 50%
ethanol extracts in the scavenging potential.
Key words: Antioxidants, scavenging, free radical, M. oleifera, A. indica, phenols, flavonoids
INTRODUCTION
Over the years, plants have been the major source of products for prevention and treatment
of ailments in traditional medicine. This is because plants are naturally endowed with inherent
bioactive compounds with medicinal properties capable of preventing or mitigating disease
conditions. In recent time, with the advancement in molecular biology which has provided an
indebt understanding of the molecular structures and actions of these bioactive compounds, much
interest has been paved to medicinal plants occasioned by their invaluable medicinal properties.
Worthy of note is the fact that some of these medicinal plants have been reported to exhibit
oxidative stress mitigating properties on oxidative stress related diseases such as cancer, asthma,
cardiovascular diseases, diabetes, arthritis, inflammation etc. (Kottaimuthu, 2008;
Gomez-Flores et al., 2008; Koffi et al., 2009; Tripathy et al., 2010; Oluwole et al., 2011). This

Res. J. Med. Plant, 9 (1): 24-33, 2015
25
oxidative stress occur when there is an imbalance between the production of reactive oxygen species
(free radicals) and the mechanism of detoxifying them (antioxidant production) in the body. When
this occurs, the generated free radicals which are unstable atoms with unpaired valence electrons
(Kadam et al., 2010; Aluko et al., 2013) will attack bio-molecules in the body transforming them
into free radicals such as hydrogen peroxides (H O). This subsequently proliferates into the
2 2
aforementioned disease conditions. Infertility has also been highly linked to the production of free
radicals (Agarwal et al., 2008).
Generally, synthetic antioxidants are the commonest ways used in mopping the deteriorating
effects of these free radicals in the body. The growing panics on the use of these synthetic
antioxidants are the reported side effects orchestrated by their consumption (Kukic et al., 2006;
Vijayakumar et al., 2012; Ikpeme et al., 2013). Due to this, there have been shifts from the use of
synthetic antioxidants to natural antioxidants sourced from medicinal plants following reports on
their safety, accessibility and affordability (Calixto, 2000; Ikpeme et al., 2011, 2012). Undoubtedly,
the reported safety, cost effectiveness and accessibility of these medicinal plants on health has
opened up a new field of research allowing research scholars to study different plants for their
antioxidant potency as alternative measure to the synthetic antioxidants.
Moringa oleifera is one of such medicinal plant reported with antioxidant properties
(Siddhuraju and Becker, 2003; Iqbal and Bhanger, 2006). Moringa oleifera is reported to be
effective in the treatment of rheumatism, infections, hiccough, influenza and internal abscess
(Anwar et al., 2007; Mishra et al., 2011). The leaf extract is capable of reducing hyperglycemia
(Mbikay, 2012). Nutritionally, the leaves contain essential amino acids, vitamins, minerals and
$-carotene which have rendered it an invaluable commodity in the food industries (Sabale et al.,
2008; Sharma et al., 2012). Azadirachta indica commonly known as neem is another medicinal
plant of great importance in traditional medicine and fertility studies (Ekaluo et al., 2010). Neem
oil, bark and leaf extracts are therapeutic in folk medicine for the control of leprosy, intestinal
helminthiasis, respiratory disorders, constipation and skin infections (Biswas et al., 2002). Although
antioxidant properties of M. oleifera and A. indica have been reported in recent researches,
however, comparative reports of the antioxidant properties of these two commonly used medicinal
plants are rare. Thus, the aim of the present research was to assess and compare the in vitro
antioxidant activities and free radical scavenging potentials of M. oleifera and A. indica in different
extracting solvents (absolute ethanol, 70 and 50% ethanol) to ascertain a more reliable antioxidant
source between the two plants.
MATERIALS AND METHODS
Collection of plant materials and extraction: Fresh leaves of M. oleifera and A. indica were
obtained from Staff Quarters, University of Calabar, Calabar and authenticated in the
Herbarium Unit of the Department of Botany, University of Calabar. The fresh leaves were freed
from dirts, air dried at room temperature for one week and then finely milled separately using a
blender (Model: 5KSB655CCSO). Ten grams of the milled sample was soaked in 100 mL of the
three different solvents (absolute ethanol, 70 and 50% ethanol) for 72 h at room temperature. The
soaked samples were shaken intermittently during the extraction period and subsequently filtered
using Whatman No. 1 filter paper. The resulting extracts were concentrated under vacuum in a
rotary evaporator at 45°C for complete solvent removal. A stock solution of each crude extract was
prepared and desired working concentrations were made by appropriate dilutions.

sb
c
AA
Scavenging activity(%)=100-100
A
−×
Res. J. Med. Plant, 9 (1): 24-33, 2015
26
Determination of extract yield (%): The percentage yield of each extract was obtained by
dividing the weight of the concentrated crude extract by the initial weight (10 g) of dry milled
starting material and multiplying the ratio by 100.
Determination of Total Phenolic Content (TPC): The total phenolic contents of the extracts
were determined by the Folin-Ciocalteau method according to Duarte-Almeida et al. (2006). One
hundred microliter of Folin-Ciocalteau reagent was added to 500 µL of the different extract
solutions containing 1000 µg mLG +6 mL of distilled water and shaken for one minute. Thereafter,
1
2 mL of 15% sodium carbonate was added to the mixture and shaken again for 30 sec. Finally,
distilled water was added to the solution to make it up to 10 mL, then left to incubate for 1.5 h at
room temperature. Thereafter, the absorbance at 750 nm was evaluated using a spectrophotometer
(LABTECH UV/VIS Spectrophotometer, India-Single beam 295). Gallic acid monohydrate, a
standard phenol, in the range of 5-150 µg mLG was used to prepare standard reference curve. The
1
Total Phenol Contents (TPC) of the extracts were expressed as Gallic Acid Equivalents (GAE) from
the linear regression curve of gallic acid.
Determination of Total Flavonoid Content (TFC): The total flavonoid contents of each extract
concentration were determined using the aluminum chloride colorimetric method, according to
Dewanto et al. (2002). The different extract solutions (1 mL containing 1000 µg mLG) were diluted
1
with 4 mL of distilled water in a 10 mL volumetric flask. Thereafter, 0.3 mL of 5% sodium nitrite
(NaNO ) solution was added to each extract solution. Five minutes later, 0.3 mL of 10%
2
aluminium chloride (AlCl ) was added; 1 min later, 2 mL of 1.0 M sodium hydroxide (NaOH) was
3
added and finally, 2.4 mL of distilled water was added and mixed properly. Absorbance of the
reaction mixture was read at 510 nm. Rutin, a standard flavonoid in the range of 10-150 µg mLG1
was used to prepare the standard reference curve. Total Flavonoid Content (TFC) of the extracts
were expressed as Rutin Equivalents (RE) from the linear regression curve of Rutin.
DPPH radical scavenging activity: The ability of M. oleiferia and A. indica leaf extracts to
scavenge stable DPPH radical was measured using the method of Mensor et al. (2001). Five
different concentrations of each test extracts were prepared in methanol (20, 40, 60, 80,
100 µg mLG). One milliliter of 0.3 mM of freshly prepared DPPH solution in methanol was added
1
to 2.5 mL solution of each extract concentration and allowed to react in the dark at room
temperature for 30 min. Absorbance of the resulting solution was measured at 518 nm. Methanol
(1 mL) was added to 2.5 mL of each extract concentration was used as blank, while 1 mL of 0.3 mM
DPPH solution added to 2.5 mL of methanol served as a negative control. Ascorbic acid and gallic
acid were used as standard reference compounds (positive controls) for comparison. Percentage
DPPH scavenging activities of the extracts and standards were determined using the following
equation:
Where:
A=Absorbance of sample (extracts or reference standard)
s
A=Absorbance of blank
b
A=Absorbance of negative control
c

cs
c
AA
Inhibition of ferrozine(%) =100
A
−×
Res. J. Med. Plant, 9 (1): 24-33, 2015
27
Results were expressed as inhibitory concentration, IC (concentration of extract or standard
50
required to scavenge 50% of DPPH radicals) which were determined from a linear regression curve
of concentration versus scavenging activity (%).
Metal (ferrous ion) chelating activity: The ferrous ion chelating activity of Moringa oleifera
and A. indica leaf in different extracting solvents (Absolute ethanol, 70 and 50% ethanol)
concentrations (Absolute, 70 and 50% ethanol) were determined by the method of
Ebrahimzadeh et al. (2009). Here, the ability of the extracts to chelate ferrous ion (Fe ) was
2+
estimated. Different concentrations (20-100 µg mLG) of each extract were prepared and 1 mL of
1
each concentration were mixed with 1 mL of FeSO (0.125 M) and 1 mL of ferrozine (0.3125 mM)
4
and shaken vigorously. After incubating for 10 min at room temperature, the mixture solution was
measured using a spectrophotometer at 562 nm against a blank containing the same components
as stated above but the extracts were replaced with distilled water (1 mL of distilled water). The
blank was incubated under the same conditions as the test samples. Sodium EDTA (Na EDTA) was
2
used as control. The percentage inhibitions of ferrozine (Fe ) by the extracts were determined using
2+
the following equation:
Where:
A=Absorbance of control
c
A=Absorbance of sample
s
Results were expressed as IC (concentration of extract or standard required to chelate 50% of
50
ferrous ions) which were determined from a linear regression curve of concentration versus
chelating activity (%).
Total Antioxidant Capacity (TAC) assay: The Total Antioxidant Capacity (TAC) of M. oleifera
and A. indica leaf extract in different extracting solvents (absolute ethanol, 70 and 50% ethanol)
were determined by the phosphomolybdate method according to Jayaprakasha et al. (2002). An
aliquot (30 µL) of different concentrations (20, 40, 60, 80 and 100 µg mLG) of the test extracts were
1
mixed with 3 mL of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, 4 mM
ammonium molybdate) taken in test tubes. The tubes were capped with aluminium foil and
incubated in a boiling water bath at 95°C for 90 min. The reaction mixture was allowed to cool to
room temperature and the absorbance of the solution was measured at 695 nm against a blank
containing 3 mL of reagent solution and the appropriate volume of the dissolving solvents. The
blank was incubated under the same conditions as the test samples. Ascorbic acid and gallic acid
were used as standard reference compounds to compare the activities of the extracts.
Reducing power assay: Antioxidant activity of the leaf extract of Moringa oleifera and A. indica
in different extracting solvents (absolute ethanol, 70 and 50% ethanol) were determined to assess
their ferric ion (Fe ) reducing ability according to the method of Anandjiwala et al. (2007).
3+
Different concentrations (20, 40, 60, 800, 100 µg mLG) of each extract were prepared and 1 mL of
1
each concentration was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.8) and 2.5 mL of

Res. J. Med. Plant, 9 (1): 24-33, 2015
28
potassium ferricyanide. The mixture was incubated in a water bath at 50°C for 20 min. To this
mixture, 2.5 mL of 10% trichloroacetic acid was added and then centrifuged at 3000 rpm for 10 min.
The upper layer of the solution (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of
0.1% ferric chloride was added. Absorbance of the Pert Prussian blue solution formed was measured
at 700 nm. Ascorbic acid and gallic acid were used as standard reference compounds for comparison
and prepared in same concentrations as the extracts.
Statistical analysis: Analysis of variance (ANOVA) was used to analyze absorbance values for
total phenolic content, total flavonoid content, reducing power and total antioxidant capacity of the
two medicinal plants against the reference standards. Mean separation was done using the Least
Significant Difference (LSD) test.
RESULTS
Extract yield, total phenolic content, total flavonoid content and inhibitory
concentration (IC ) of M. oleifera and A. indica: Following the differences in concentration
50
of the extracting solvents (absolute ethanol, 70 and 50% ethanol), there were concomitant
differences in the percentage yield of the extracts. Moringa oleifera had the highest yield in 50%
ethanol (23.12%) followed by 70% ethanol (21.89%) and absolute ethanol (11.79%).
Azadiracta indica yield also increased as the concentration of the extracting solvent reduced;
absolute ethanol (11.34%), 70% ethanol (17.84%), 50% ethanol (20.14%) as shown in Table 1.
Results for total phenolic and flavonoid content revealed a concentration dependent relationship.
Azadiracta indica had significant amount (p<0.05) of phenols than M. oleifera in the different
extracting solvents. Flavonoid content of A. indica was significantly higher (p<0.05) in absolute
ethanol (128.39 µg RE mgG), 70% ethanol (42.83 µg RE mgG), 50% ethanol (30.89 µg RE mgG)
1 1 1
than absolute ethanol (85.06 µg RE mgG), 70% ethanol (25.05 µg RE mgG) and 50%
1 1
ethanol (8.95 µg RE mgG) of M. oleifera. In DPPH radical scavenging, it required 77.94 µg mgG
1 1
absolute concentration of A. indica to scavenge 50% of the radical compared to 118.96 µg mgG of
1
M. oleifera at the same concentration. Azadiracta indica was also a better metal chelator
(p<0.05) than M. oleifera since it required a lesser amount of its extract to chelate 50% of ferrous
ion (Fe ) in the respective extraction solvent concentration compare to M. oleifera (Table 1).
2+
Concentration effect on free radical scavenging properties of M. oleifera, A. indica and
standards: The result revealed significant differences in the free radical scavenging potential of
M. oleifera and A. indica at the different concentration as shown in Table 2. Although
Table 1: Extract yield, phenolic content, flavonoid content, DPPH radical scavenging and metal chelating activities of M. oleifera and
A. indica
M. oleifera A. indica
--------------------------------------------------------------- -----------------------------------------------------------------
Absolute Ethanol Ethanol Absolute Ethanol Ethanol
Parameters ethanol (70%) (50%) ethanol (70%) (50%)
Extract yield (%) 11.97 21.89 23.12 11.34 17.84 20.14
Phenol content (µg GAE mLG)30.94±0.64 29.90±0.51 29.09±0.05 58.31±0.31 51.40±1.32 43.24±1.71
1de e a b c
Flavonoid content (µg RE mLG)85.06±0.76 25.05±0.29 8.95±1.25 128.39±0.73 42.83±0.48 30.89±2.04
1 b efacd
DPPH radical scavenging (µg mLG)* 118.96±2.57 120.77±1.03 152.79±1.76 77.94±1.94 139.51±1.88 193.89±4.82
1edbfca
Metal chelating activity (µg mLG)** 568.20±0.01 3369.00±0.07 3812.50±0.09 0.22±0.01 9.07±0.06 9.97±0.041
1cb a fed
Means with different superscript along the same horizontal array differ significantly (p<0.05) from each other. *IC values for ascorbic
50
acid and gallic acids are 5.95±0.13 and 0.50±0.02 µg mLG, respectively. **IC value for Na EDTA is 0.02±0.001 mg mLG
1 1
50 2

Res. J. Med. Plant, 9 (1): 24-33, 2015
29
Table 2: Concentration effect on reducing power and antioxidant capacity of M. oleifera, A. indica and standards
M. oleifera A. indica
Conc. of -------------------------------------------------------------- -------------------------------------------------------------- Standards
extracts Absolute Ethanol Absolute Ethanol --------------------------------------
(µg mLG)ethanol (70%) Ethanol (50%) ethanol (70%) Ethanol (50%) Ascorbic acid Gallic acid
1
Reducing power
20 0.685±0.002 0.726±0.006 0.706±0.005 0.696±0.006 0.635±0.015 0.702±0.005 1.909±0.074 -
ebcdfca
40 0.711±0.003 0.728±0.007 0.717±0.003 0.729±0.007 0.748±0.018 0.728±0.007 2.149±0.078 -
dcdcbca
60 0.727±0.007 0.734±0.007 0.758±0.001 0.733±0.007 0.757±0.001 0.747±0.021 2.239±0.098 -
ddbdbca
80 0.735±0.007 0.762±0.160 0.773±0.172 0.744±0.151 0.761±0.160 0.750±0.151 2.459±1.020 -
ecbdcda
100 0.757±0.080 0.765±0.140 0.807±1.004 0.767±0.140 0.775±0.170 0.758±0.060 2.464±1.004 -
ede bcd cea
Antioxidant capacity
20 0.027±0.011 0.032±0.011 0.026±0.011 0.054±0.004 0.056±0.003 0.039±0.011 0.651±0.898 0.400±0.040
ddd c c da b
40 0.032±0.001 0.032±0.001 0.028±0.001 0.059±0.005 0.060±0.005 0.050±0.005 0.778±1.024 0.431±0.051
ddd c c c a b
60 0.041±0.002 0.041±0.002 0.030±0.002 0.071±0.006 0.077±0.008 0.057±0.004 0.849±1.004 0.434±0.051
eee cd dca b
80 0.053±0.003 0.042±0.003 0.031±0.002 0.085±0.030 0.085±0.030 0.065±0.009 1.011±1.051 0.459±0.058
eef fc c da b
100 0.053±0.003 0.048±0.004 0.037±0.004 0.088±0.060 0.089±0.060 0.071±0.031 1.050±1.310 0.460±0.061
eef fc c da b
Means with different superscript along the same horizontal array differ significantly (p<0.05) from each other. -: Particular standard was
not used
ascorbic acid, a standard reference was a better reducing agent than the test medicinal plants,
however, the absorbance values of the medicinal plants indicated their scavenging potentials. At
20 µg mLG, 70% ethanol extract of M. oleifera reduced significant amount (0.726) of the ferric ion
1
(p<0.05) than A. indica but at 40 µg mLG, 70% ethanol extract of A. indica was better than
1
M. oleifera. There was no significant difference (p>0.05) in the reducing power of M. oleifera at
60 µg mLG, absolute ethanol and 70% ethanol extracts (0.727 and 0.734) and 60 µg mLG, absolute
1 1
ethanol extract (0.733) of A. indica. At 100 µg mLG, 50% ethanol extract of M. oleifera was better
1
in reducing free radical while A. indica was better at 70% ethanol extract of the same
concentration. Ascorbic and gallic acid standards showed significant antioxidant capacity than the
test medicinal plant extracts. At 20 µg mLG, 70 and 50% ethanol extracts of A. indica were the
1
same (p>0.05). Absolute ethanol, 70 and 50% ethanol extracts of M. oleifera (0.027, 0.032 and
0.026) and 50% ethanol extract of A. indica (0.039) showed no significant difference at 20 µg mLG.
1
At 40, 60, 80 and 100 µg mLG, A. indica showed high total antioxidant capacity than M. oleifera
1
in the different extracting solvents.
DISCUSSION
Medicinal plants are regularly screened for free radical scavenging properties basically from the
reports on their safety, efficacy and cost effectiveness (Calixto, 2000; Ikpeme et al., 2011, 2012)
over synthetic antioxidants reported with side effects upon their consumption (Vijayakumar et al.,
2012). As a result of this, various medicinal plants reported with antioxidant properties have been
recommended for pharmaceutical industries and traditional medicine for the control and treatment
of different kinds of ailments.
The results of the current research revealed the presence of reasonable amount of phenols and
flavonoids in both medicinal plant extracts. Polyphenolic and flavonoid compounds are very
important secondary metabolites in plants and are reported to be responsible for the variation in
antioxidant activities in plants (Demiray et al., 2009; Basma et al., 2011; Uyoh et al., 2013) and are
capable of fighting against free radicals by inactivating lipid free radicals or preventing
decomposition of hydrogen peroxide into free radicals due to their redox properties, chelate metal

Res. J. Med. Plant, 9 (1): 24-33, 2015
30
ions, quenching singlet and triplet oxygen (Pokorny et al., 2001; Maisuthisakul et al., 2007;
Balasundram et al., 2006; Javanmardi et al., 2003). This may undoubtedly suggest that plants with
high quantity of polyphenols and flavonoids are good antioxidant sources although quantifying
phenols and flavonoids are not the only yardstick for measuring antioxidant capacity of a substrate
as many in vitro antioxidant assays are always required for a more categorical conclusion.
Approximately, Azadiracta indica contained more phenols and flavonoids in absolute ethanol
extract than Moringa oleifera which suggest why it required lesser amount of the extract
(97.94 µg mLG) to scavenge 50% of DPPH radical compare to M. oleifera at the same concentration
1
(Table 1). The DPPH, a synthetic free radical have been used to measure in vitro ability of a test
substance to scavenge free radicals. The effects of phenolic compounds on DPPH radical scavenging
are thought to be due to their hydrogen donating ability (Ghimeray et al., 2009) to the unstable
DPPH free radical that accepts an electron or hydrogen to become a stable diamagnetic molecule
(Siddaraju and Dharmesh, 2007). It is most likely that the decrease in absorbance of DPPH radical
caused by phenolic compound in our result is due to reaction between antioxidant molecules in the
extracts and the radicals. Although the amount of DPPH scavenged by the two plants in the
respective extracting solvents are not equivalent to ascorbic and gallic acid standards, however, this
amount is adequate to suggest the extract potential in scavenging free radicals.
According to Ghimeray et al. (2009) transition metals have played a pivotal role in the
generation of oxygen free radicals in living organisms. These transition elements such as iron and
copper are able to kick start free radical generation because they are oxidation reaction catalyst due
to unpaired electrons on their valence shells which makes them structurally unstable. The unstable
nature of these metals often orchestrate the conversion of H Oto OH in the Fenton reaction and
22
in decomposition of alkyl peroxides to heavy reactive alkyl and hydrogen radicals (Hsu et al., 2006).
Interestingly, chelating agents such as antioxidants may inactivate metal ions and potentially
inhibit the metal dependent processes (Finefrock et al., 2003) by donating electrons to these
unstable-electron-deficient metals. From Table 1, with only 0.22 µg mLG, A. indica chelated 50%
1
of ferrous ions in absolute concentration of ethanol showing even more chelating capacity than
ascorbic and gallic acid standards which chelated 50% of the metal ion at higher concentrations
(5.95 and 0.503 µg mLG, respectively). In 70 and 50% ethanol, A. indica extracts also showed good
1
metal chelating capacity comparable to the two standards. This result suggest that rather than
depending completely on ascorbic acid, gallic acid and other synthetic antioxidant sources to
mitigate the deteriorating effect of free radicals in the body, a more readily and cost effective source
such as A. indica leaf extracts could be adopted. Although M. oleifera extracts did not show good
metal chelating potential, its antioxidant properties is worth recommending as reflected from its
DPPH scavenging capacity in absolute ethanol.
Reducing power is the measure of the extract ability to donate electrons in order to facilitate the
reduction of ferric ion (Fe ) to ferrous ion (Fe ). The absorbance values indicate the concentration
3+ 2+
of Fe , thus, the higher the absorbance values the higher the concentration of Fe which indicate
2+ 2+
the ability of the extract to donate electrons as an antioxidant reservoir (Laandrault et al., 2001;
Yen et al., 2000). This may probably suggest that both M. oleifera and A. indica extracts are good
electron donors (antioxidant sources) following their absorbance values at the respective
concentration of the extracting solvents.
It has been reported that damages mediated by free radicals such as superoxide anion (O G),
2
hydroxyl radical (OH) and peroxyl radical (ROOG) result in the disruption of membrane fluidity,
protein denaturation, lipid peroxidation which brings about generation of oxidative stress evidenced

Res. J. Med. Plant, 9 (1): 24-33, 2015
31
in many chronic diseases (Biglari et al., 2008; Ikpeme et al., 2014). Polyphenols from plant origin
have the capacity to quench these radicals due to their ability to stabilize unpaired electrons
(Anokwuru et al., 2011) and may have implication in prevention and/or curing oxidative stress
diseases (Shukla et al., 2009). Although antioxidant activities of M. oleifera and A. indica obtained
from the various in vitro assays in this study may not be completely obtainable in vivo, the results
probably indicate that the extract of these two medicinal plants can scavenge and/or prevent free
radical generation thereby mitigating oxidative stress mediated diseases in the body.
CONCLUSION
Comparatively, the leaf extract of A. indica had more phenols and flavonoids than M. oleifera
in all the extracting solvents. This culminated why A. indica was able to scavenge 50% of the
DPPH radical in absolute ethanol and also chelated 50% of the metal ion at a lower concentration
than M. oleifera. The absorbance values for reducing power and total antioxidant capacity of
A. indica were also comparably better than M. oleifera. Thus, the results indicate that A. indica
leaf extract is a better antioxidant and free radical scavenger over M. oleifera mostly in absolute
ethanol although more comparative antioxidant studies of the two plant extracts are required to
further authenticate this claim.
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