Plant Foods Hum Nutr (2009) 64:303–311
DOI 10.1007/s11130-009-0141-0
ORIGINAL PAPER
Antioxidant Activity and Total Phenolic Content of Moringa
oleifera Leaves in Two Stages of Maturity
S. Sreelatha & P. R. Padma
Published online: 11 November 2009
# Springer Science + Business Media, LLC 2009
Abstract Antioxidants play an important role in inhibiting
and scavenging free radicals, thus providing protection to
human against infections and degenerative diseases. Cur-
rent research is now directed towards natural antioxidants
originated from plants due to safe therapeutics. Moringa
oleifera is used in Indian traditional medicine for a wide
range of various ailments. To understand the mechanism of
pharmacological actions, antioxidant properties of the
Moringa oleifera leaf extracts were tested in two stages of
maturity using standard in vitro models. The successive
aqueous extract of Moringa oleifera exhibited strong
scavenging effect on 2, 2-diphenyl-2-picryl hydrazyl
(DPPH) free radical, superoxide, nitric oxide radical and
inhibition of lipid per oxidation. The free radical scaveng-
ing effect of Moringa oleifera leaf extract was comparable
with that of the reference antioxidants. The data obtained in
the present study suggests that the extracts of Moringa
oleifera both mature and tender leaves have potent
antioxidant activity against free radicals, prevent oxidative
damage to major biomolecules and afford significant
protection against oxidative damage.
Keywords Moringa oleifera . Antioxidants . Free radicals .
Scavenging activity
S. Sreelatha (*)
Division of Physical Sciences, NTU,
Singapore, Singapore
e-mail: sree_latha04@yahoo.com
P. R. Padma
Department of Biochemistry & Biotechnology,
Avinashilingam University,
Coimbatore 641043, Tamil Nadu, India
Introduction
The potentially reactive derivatives of oxygen, ascribed as
ROS (reactive oxygen molecules) such as O2−, H2O2 and
OH, are continuously generated inside the human body as a
consequence of exposure to a plethora of exogenous
chemicals in our ambient environment and/or a number of
endogenous metabolic processes involving redox enzymes
and bioenergetic electron transfer [1]. Under normal
circumstances, the ROS generated are detoxified by the
antioxidants present in the body and there is an equilibrium
between the ROS generated and the antioxidants present.
However, owing to ROS overproduction and/or inadequate
antioxidant defense, this equilibrium is hampered favouring
the ROS upsurge that culminates in oxidative stress [2].
The ROS readily attack and induce oxidative damage to
various biomolecules including proteins, lipids, lipoproteins
and DNA [3]. This oxidative damage is a crucial etiological
factor implicated in several chronic human diseases such as
diabetes mellitus, cancer, atherosclerosis, arthritis, neurode-
generative diseases and also in the ageing process [4, 5].
Based on growing interest in free radical biology and the
lack of effective therapies for most chronic diseases, the
usefulness of antioxidants in protection against these
diseases is warranted. Epidemiological studies have found
that the intake of antioxidants such as vitamin C reduces the
risk of coronary heart disease and cancer [6]. The
antioxidants may mediate their effect by directly reacting
with ROS, quenching them and/or chelating the catalytic
metal ions [7]. Several synthetic antioxidants, e.g., BHA
(butylated hydroxy anisole) and BHT (butylated hydroxy
toluene) are commercially available but are quite unsafe
and their toxicity is a problem of concern [8]. Natural
antioxidants, especially phenolics and flavonoids, are safe
and also bioactive.
304 Plant Foods Hum Nutr (2009) 64:303–311

The use of traditional medicine is widespread, and plants Environment & Forests, Botanical Survey of India, Coim-
still present a large source of natural antioxidants that might batore, Tamilnadu, Government of India. (BSI/SC/5/25/05-
serve as leads for the development of novel drugs [9]. 06/Tech-908). The leaves were procured (both mature and
Moringa oleifera commonly known as (family: Moringa- tender) fresh for each estimation (Fig. 1).
ceae) horse radish tree or drumstick tree is both nutritional
and medicinal with some useful minerals, vitamins, amino Chemicals
acids, etc. [10]. A native of the sub-Himalayan regions of
North West India Moringa oleifera is indigenous to many 2, 2-Diphenyl-2-picryl hydrazyl (DPPH) was obtained from
countries in Africa, Arabia, South East Asia, the Pacific, Sigma–Aldrich Co., St. Louis, USA. Naphthyl ethylene
Caribbean Islands and South America. Although there are diamine dihydrochloride (NEDD) was from Roch-Light
12 varieties of Moringa species Moringa oleifera is the best Ltd., Suffolk, UK, ascorbic acid, nitro blue tetrazolium
known of all species of the genus Moringaceae [11]. Almost (NBT) and butylated hydroxyl anisole (BHA) were from SD
all the parts of this plant: root, bark, gum, leaf, fruit (pods), Fine Chemicals Ltd., Mumbai, India. Sodium nitroprusside
flowers, seed and seed oil have been used for various and Silymarin were from Ranbaxy Laboratories Ltd.,
ailments in the indigenous medicine of South Asia, including Mohali, India. Sulphanilic acid used was from E-Merck
the treatment of inflammation and infectious diseases along (India) Ltd., Mumbai, India. All chemicals used were of
with cardiovascular, gastrointestinal, hematological and analytical grade.
hepatorenal disorders [12]. The flowers and roots are used
in folk remedies, for tumors, the seeds for abdominal tumors, Preparation of the Extract
leaves applied as poultice to sores, rubbed on temples for
headaches and are said to have purgative properties [13]. The leaves were chopped to small pieces and dried in
Moringa oleifera is called “Miracle Vegetable” because it is shade. The dried leaves were powdered and passed through
both a medicinal and a functional food [14]. Administration
of Moringa oleifera leaf extract inhibited the growth of
pathogenic gram positive and gram negative bacteria [15]
and exerted chemo-modulatory effect against skin papillo-
magnesis in mice [16]. Moringa oleifera has the highest
proportion of essential amino acids and significant quantities
of minerals [17] when analyzed. Moringa oleifera is rich in
compounds like glucosinolates and isothiocyanates [18] and
the stem bark has been reported to contain alkaloids namely
Moringinine and Moringine [19]. Flowers contain pigments
such as alkaloids, kaempferol, rhamnetin, isoquercitrin and
kaempferritin [20]. Although much has been learned about Mature Leaves
the nutritional value of Moringa oleifera additional knowl-
edge remains to be secured. Therefore in recent years;
considerable attention has been directed towards identifica-
tion of plants with antioxidant ability that may be used for
human consumption. In view of the several ethno botanical
uses of Moringa oleifera described above, it was proposed to
screen its successive extracts for the in vitro antioxidant
activity using standard procedures at two different stages of
maturity.

Materials and Methods

Plant Material

The Moringa oleifera leaves were purchased from the

Horticulture Research Institute, Periyakulam Tamilnadu, Tender Leaves

and Agricultural University, India. The plant specimen was Fig. 1 Moringa oleifera leaves used in the study at two different
authenticated by the office of the Joint Director, Ministry of stages of growth
Plant Foods Hum Nutr (2009) 64:303–311
sieve no. 20 and extracted (100 g) successively with 600 ml
of water in a soxhlet extractor for 18–20 h. The extracts
were concentrated to dryness under reduced pressure and
controlled temperature (40–50 °C). The yield (w/w) of the
extract from fresh leaves was about 10%. The extracts were
prepared in duplicate and all analysis was carried out in
triplicates.
Determination of Enzymatic Antioxidants
Advances in nutrition research during the past few decades
have changed scientists’ understanding of the contribution
of vegetarian diets to human health and disease. Phyto-
chemicals, the bioactive non-nutrient plant compounds in
fruits, vegetables, grains and other plant foods have been
linked to reductions in the risk of major chronic diseases.
Fruits and vegetables contain a wide variety of antioxidant
phytochemicals such as phenolics and carotenoids that may
help to protect the cellular systems from oxidative damage
and lower the risk of chronic diseases. Many medicinal
plants that were discovered by early people are still in use
today and medicines are still being discovered in plants
[21].
Catalase activity was measured according to the method
where [22] one unit of catalase was defined as the amount
of enzyme required to decompose 1 μM of H2O2 in 1 min.
The reaction was initiated by the addition of 1.0 ml of
freshly prepared 20 mM H2O2. The rate of decomposition
of H2O2 was measured spectrophotometrically at 240 nm
for 1 min. The enzyme activity was expressed as units/g.
One unit is the amount of enzyme activity required to
decrease the absorbance at 240 nm by 0.05 units. The
activity of SOD was measured according to the principle
where [23] superoxide dismutase (SOD) was assayed based
on the inhibition of production of nitro blue tetrazolium
formazone. SOD activity was then measured at 560 nm.
The enzyme activity was expressed as units/g. Where one
unit is the amount of enzyme that gives 50% inhibition of
the extent of NBT reduction in 1 min.
GPX activity was measured according to procedure
where the [24] reaction mixture consisted of sodium
phosphate buffer, pH 7.0, 1 mM sodium azide, 1 U/ml of
reduced glutathione, extract, 0.25 mM H2O2 in a total
volume of 1 ml. The tubes were incubated at 37 °C for
3 min. The reaction was terminated by TCA, and to the
residual glutathione content, disodium hydrogen phosphate
and DTNB solution were added. Yellow colour developed
at 412 nm was recorded at 25 °C. One unit of GPx activity
was expressed as μg of glutathione consumed/min.
GST activity was assayed accordingly [25] with some
modifications. The reaction was carried out in 0.1 M
potassium phosphate buffer (pH 6.5), 1 mM GSH, and
1 mM 1-chloro-2, 4-dinitrobenzene in a 50 μl sample. An
305
increase in absorbance was monitored with a wavelength of
340 nm at 25 °C for a 4 min time period, and the activity of
enzyme was expressed as μmol/min/g.
Determination of Non-enzymatic Antioxidants
The method described by Zakaria et al. [26] was followed
for the estimation of total carotenoids. The total carotenoids
in the sample can be extracted in petroleum ether and
estimated in UV/visible spectrophotometer at 450 nm.
Ascorbic acid, a scavenger of oxy radicals was assayed
by the method where [27] ascorbate is converted to
dehydroascorbate by the treatment with activated charcoal.
Dehydro ascorbic acid then reacts with 2,4-dinitrophenyl
hydrazine to form osazones, which dissolves in sulphuric
acid to give an orange colored solution, whose absorbance
can be measured spectrophotometrically at 540 nm.
The method described by Rosenberg [28] was followed
for the estimation of tocopherol. Emmerie-Engel reaction is
based on the reduction of ferric to ferrous ions by
tocopherols, which then forms a red color with 2, 2′-
dipyridyl. Tocopherols and carotenes are first extracted with
xylene and the extinction read at 460 nm to measure
carotenes. A correction is made for this after adding ferric
chloride and read at 520 nm.
DPPH (2, 2-Diphenyl-1-Picrylhydrazyl)—Scavenging
Activity
The free radical scavenging activity of the extract was
measured in terms of hydrogen donating or radical
scavenging ability using the stable free radical DPPH
[29]. One milliliter solution of the extract in methanol was
added to 0.5 ml of 0.15 mM DPPH solution in methanol.
The contents were mixed vigorously and allowed to stand
at 20 °C for 30 min. The absorbance was read at 517 nm.
IC50 value (the concentration required to scavenge 50%
DPPH free radicals) was calculated. The capability to
scavenge the DPPH radical was calculated using the
following equation:
DPPH scavenging effect ð%Þ ¼ ½ðA0 A1 =A0 Þ 100
;
where A0 was the absorbance of the control reaction and
A1 the absorbance in the presence of the sample.
Scavenging of Superoxide Radical
To the reaction mixture containing 0.1 ml of NBT (1 mg/mL
solution in DMSO) and 0.3 ml of the extracts, the compound
and standard in dimethyl sulphoxide (DMSO), 1 ml of
alkaline DMSO (1 ml DMSO containing, 5 mM NaOH in
0.1 ml water) was added to give a final volume of 1.4 ml and
the absorbance was measured at 560 nm [30].
306
Scavenging of Nitric Oxide Radical
Scavenging of nitric oxide (NO) radical was determined by
incubating sodium nitroprusside (SNP) (5 mM, in PBS)
with different concentrations of Moringa oleifera leaf
extract at 25 °C. After 120 min, 0.5 ml of the incubation
solution was withdrawn and mixed with 0.5 ml of griess
reagent [31]. The absorbance was measured at 550 nm.
Percent inhibition of the nitric oxide generated was
measured by comparing the absorbance values of control
and test preparations. Cur cumin was used as a reference
standard.
Lipid Peroxidation (LPO)
LPO was induced and assayed in goat liver homogenates
according to the method where [32] the reaction mixture, in
a total volume of 1.0 ml, contained 0.58 ml phosphate
buffer (0.1 M, pH 7.4), 0.2 ml of liver homogenate (10%,
w/v), 0.2 ml ascorbic acid (100 mM) and 0.02 ml ferric
chloride (100 mM) was incubated at 37 °C in a shaking
water bath for 1 h. The reaction was stopped by the addition
of 1.0 ml TCA (10%, w/v). Then, 1.0 ml of TBA (0.67%,
w/v) was added and all the tubes were placed in a boiling
water bath for 20 min. At the end, the tubes were shifted to
an ice-bath and centrifuged at 2,500 × g for 10 min.
The amount of malondialdehyde formed in each of the
samples was assessed by measuring the optical density of
the supernatant at 535 nm against a reagent blank. The
molar extinction coefficient for MDA was taken to be
1.56×105 M−1 cm−1.
DNA Damage Using λ DNA
The extent of DNA damage induced in λ DNA was
followed by the difference in migration pattern on agarose
gel [33]. The reaction was conducted in a total volume of
30 μl containing 5 μl of tris buffer, 5 μl of λ phage DNA
and 5 μl of plant extract prepared in tris buffer. Then, 10 μl
of H2O2 and 5 μl of FeCl3 were added and incubated at
37 °C for 30 min. The reaction mixture was then mixed
with 6 μl of gel loading dye, loaded into 1% agarose gel
and run at 100 V for 15 min in a submarine gel
electrophoretic apparatus. The DNA was visualized and
photographed using an Alpha Digidoc digital gel docu-
mentation system.
Total Phenolics and Flavonoids
Antioxidant compounds generally contain phenolic group(s)
and hence, the amounts of phenolic compounds in the
extracts of the leaves were estimated by using Folin–
Ciocalteau reagent [34]. In a series of test tubes, 0.4 ml of
Plant Foods Hum Nutr (2009) 64:303–311
the extract in methanol was taken, mixed with 2 ml of
Folin–Ciocalteau reagent and 1.6 ml of sodium carbonate.
After shaking, it was kept for 2 h and the absorbance was
measured at 750 nm using a Shimadzu-UV-160 spectropho-
tometer. Using gallic acid monohydrate, a standard curve
was prepared. The linearity obtained was in the range of
1–10 μg/ml. Using the standard curve, the total phenolic
compounds content was calculated and expressed as gallic
acid equivalent in mg/g of extracts.
Flavonoids were extracted and estimated by the method
where [35] an aliquot of the extract was pipetted out and
evaporated to dryness. 4.0 ml of vanillin reagent was added
and heated for 15 min in a boiling water bath. The standard
was also treated in the same manner. The optical density
was read at 340 nm. The values are expressed as mg
flavonoids/g leaf.
TLC of Alkaloids, Phenolics and Flavonoids
The extracted fractions of Moringa oleifera leaves of both
mature and tender are subjected to TLC on silica gel G60
F254 plates (Merck) as described [36]. The alkaloid fraction
was developed with CH2Cl2: ethanol: 28% NH4OH
(85:14:1) and sprayed with Dragendroff’s reagent. Phe-
nolics were separated with acetic acid: chloroform (45:55)
and flavonoids with n-butanol:acetic acid:water (4:1:5) and
both were detected with vanillin-H2SO4 (10% vanillin in
ethanol: concentrated sulphuric acid in 2:1 ratio) spray
reagent. The Rf values of the spots were calculated as the
ratio of the distance traveled by the solute to that by the
solvent front.
Statistical Analysis
The data were subjected to statistical analysis to verify and
evaluate the difference between the antioxidant activities of
the two leaf extracts. The data were expressed as mean ±S.
D (n=6) where ‘n’ represents the no. of samples. Results
were analyzed statistically by one-way ANOVA, followed
by post hoc analysis using Fischer’s LSD, Sigma Stat
statistical package (Version 3.1). The difference was
considered significant if P<0.05.
Results
Enzymatic Antioxidants and Non-enzymatic Antioxidants
The leaves of Moringa oleifera, which is commonly
consumed in the Indian diet, were analyzed for the levels/
activities of non-enzymatic and enzymatic antioxidants. The
leaves were analyzed at two different stages of growth,
namely tender and mature, in order to study whether a
Plant Foods Hum Nutr (2009) 64:303–311
difference in the levels existed in the different stages of
growth. The results showed (Tables 1 and 2) that the mature
leaves possessed higher activities of enzymatic antioxidants
and a higher level of the non-enzymatic antioxidants studied.
The results indicate that the mature leaf extract significantly
exhibited best values of enzymatic and non-enzymatic
antioxidants.
DPPH Free Radical Scavenging Activity
DPPH scavenging activity has become routine in establish-
ing the antioxidant activity of herbal extracts and phyto-
chemicals. The amount of sample needed to decrease the
initial DPPH concentration by 50% is a parameter widely
used to measure antioxidant activity. The scavenging effects
of mature and tender leaf extracts on the DPPH radical are
illustrated in Table 3. Moringa oleifera leaf extract
significantly reduced DPPH radicals. In comparison, the
positive control, Trolox® had an IC50 of 2.14±0.12 µg/ml.
The DPPH scavenging ability of the extract may be
attributed to its hydrogen donating ability and mature leaf
extract showed significant scavenging activity.
Superoxide Anion and Nitric Oxide Radical Scavenging
Activity
Moringa oleifera leaf extract scavenged O2− significantly
as shown in Table 3. The mature leaf extract (12 µg) was
found to be better scavenger than the tender leaf extract.
IC50 value of the standard ascorbic acid was achieved at
9 µg concentration. Moringa oleifera leaf extract also
quenched NO released by a NO donor, SNP (sodium nitro
prusside). The extract effectively decreased the release of
NO. In comparison the IC50 value of the standard curcumin
was achieved at 51 µg concentration. It is evident from this
Table 1 Activities of enzymatic antioxidants in Moringa oleifera
leaves
Parameter Matured leaves Tender leaves
SOD (Ua/g) 14.64±0.01d 13.64±0.02
CAT (Ub/g) 106.84±0.07d 71.06±0.02
GPx (Uc/g) 163.68±2.36d 142.22±0.37
GST (U#/g) 0.30±0.008d 0.2±0.008d
Values are mean ± SD of triplicates
a
1 Unit = Amount of enzyme that gives 50% inhibition of the extent
of NBT reduction in 1 min
b
1 Unit = Amount of enzyme required to decrease the absorbance at
240 nm by 0.05 units
c
1 Unit = Change of absorbance/minute at 430 nm
d
Statistically significant (P<0.05) compared to tender leaves
#
1 Unit = µmol of CDNB conjugated/min
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Table 2 Levels of non-enzymatic antioxidants in Moringa oleifera
leaves
Parameter Matured leaves Tender leaves
Ascorbic acid (mg/g) 6.60±0.01a 5.81±0.01
Tocopherol (μg/g) 6.53±0.01a 5.63±0.008
Total carotenoids (mg/g) 92.38±0.11a 85.20±0.14
Values are mean ± SD of triplicates
a
Statistically significant (P<0.05) compared to tender leaves
observation that Moringa oleifera leaves possess active
components that seem to contribute to radical scavenging
activity. However the activity remains to be below the
standards as observed.
Lipid Peroxidation
The lipids in membrane are continuously subjected to oxidant
challenges. Oxidant induced abstraction of a hydrogen atom
from an unsaturated fatty acyl chain of membrane lipids
initiates the process of LPO, which propagates as a chain
reaction. In the process, cyclic peroxides, lipid peroxides and
cyclic end peroxides are generated, which ultimately are
fragmented into aldehydes like MDA. MDA forms a pink
chromogen with TBA that absorbs at 535 nm. Moringa
oleifera leaf extract inhibited the amount of MDA generated
(and thus lipid per oxidation) in liver homogenate which is
presented in Table 3. Thus, the decrease in the MDA level in
the leaf extracts indicates the role of the extracts as an
antioxidant where mature leaf extract showed higher
inhibition than the tender leaf extract.
Extent of DNA Damage
The gel pattern of λ DNA exposed to H2O2 in vitro in the
presence and the absence of leaf extracts of Moringa
oleifera is presented in Fig. 2. From the migration pattern, it
can be deduced that H2O2 damaged λ DNA leading to
shredding causing the absence of specific band in H2O2
treated DNA. Mature and tender leaf extracts of Moringa
oleifera significantly reduced the DNA damage induced by
H2O2 to λ DNA. Moringa oleifera leaf extracts, by
themselves, did not cause any DNA damage as evident
from the intact DNA band. The striking observation that
could be made was that the extent of DNA damage induced
by H2O2 was completely reverted by the leaf extracts both
mature and tender.
Amount of Total Phenolic Compounds
In the present study the total phenolic compounds of the
successive extracts were expressed as gallic acid equivalent
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Table 3 Antioxidant profiles of Moringa oleifera (Concentration in micrograms (μg /ml) needed for 50% inhibition)
Antioxidant and radical scavenging activities of Moringa oleifera extract
λDNA damage assay
DPPH scavenging activity
Superoxide scavenging activity
Nitric oxide scavenging activity
LPO inhibition
Values are mean ± SD of triplicates
a
Statistically significant (P<0.05) compared to tender leaves
in mg/g and the flavonoids were expressed as quercetin
equivalent in mg/g of plant material. Mature leaf extract
had the highest phenolic content followed by tender extract.
An analysis of the data given in Table 4 reveals that the
observed in vitro antioxidant activity of the successive
extracts of Moringa oleifera correlates with its phenolic
content. Since polyphenols are responsible for the antiox-
idant activity, the obtained amount of total polyphenols in
the extract indicated the extract that it possess a high
antioxidant activity.
TLC Analysis
Phytochemical screening was conducted in order to identify
the chemical nature of active principles possibly rendering
antioxidant protection. Qualitative analysis of the extracts
revealed the presence of phenolics, flavonoids and trace
amounts of alkaloids, in both mature and tender leaves.
Agarose gel electrophoretic pattern showing protection of Moringa oleifera leaf extracts of H2O2
induced.
Strand breaks in λ DNA as a function of concentration. Lane 1- λDNA control. Lane 2 - H2O2
exposed.
Lane 3&4 – exposed to H2O2 in the presence of tender and mature leaf extracts.
Lane 5&6 –exposed to tender and mature leaf extracts.
Fig. 2 Migration pattern of λ DNA treated with H2O2 with and
without the leaf extracts. Agarose gel electrophoretic pattern showing
protection of Moringa oleifera leaf extracts of H2O2 induced strand
breaks in λ DNA as a function of concentration. Lane 1—λ DNA
control. Lane 2—H2O2 exposed. Lane 3&4—exposed to H2O2 in the
presence of tender and mature leaf extracts. Lane 5&6—exposed to
tender and mature leaf extracts
Plant Foods Hum Nutr (2009) 64:303–311
IC 50 (µg/ml) extract/standard
Mature leaf extract Tender leaf extract
72.45±0.23a 82.63±0.51
18.15±0.92a 19.12±0.75
12.71±0.15a 15.51±0.25
56.77±0.45a 65.88±0.65
25.32±0.54a 30.15±0.23
Since phenolics and flavonoids were found to be maximum,
the phenolics and flavonoid fractions were subjected to
TLC in different solvent mixtures, acetic acid : chloroform
(1:9) for phenolics, and n-butanol : acetic acid : water
(4:1:5) for flavonoids and developed with the respective
spraying reagent as described in methodology. Mature
leaves showed two spots (Rf values 0.68 and 0.85) for
phenolics and two spots (Rf values of 0.75, 0.80) for
flavonoids and a single spot for alkaloids (Rf value of 0.72).
Tender leaves also showed two spots (Rf values of 0.68,
0.84) for phenolics and two spots (Rf values of 0.76 and
0.85) for flavonoids and a faint spot for alkaloids. The
phenolic compounds may contribute directly to the anti-
oxidative action [37]. Thus, the antioxidant properties of
Moringa oleifera may possibly be attributed to the phenolic
compounds present.
Discussion
Plant extracts and plant-derived antioxidants can elicit a
number of in vivo effects such as promotion of increased
synthesis of endogenous antioxidant defenses or them-
selves acting directly as antioxidants [38]. It is also
reported that the composition of antioxidants varies
widely with several factors like the stage of maturity,
variety, climatic conditions, part of the plant analyzed,
post-harvest handling, processing, and storage [39]. The
results in the present study also show that the
antioxidants vary with the stage of maturity in Moringa
oleifera leaves. Zimmermann and Zentgraf [40] have
reported that the components of both the enzymatic and
the non-enzymatic antioxidant system correlate well with
oxidative stress during senescence and plant development.
SOD and CAT activities declined in Triticum aestivum
leaves upon senescence [41] and with maturity of
blackberry [42]. The effects of senescence and aging on
the expression of antioxidant gene products have been
studied in many plants which report the variation in levels
Plant Foods Hum Nutr (2009) 64:303–311
Table 4 Total phenolic & flavonoid contents in Moringa oleifera leaf extract
Extract/standard contents (mg/g)
Mature leaf extract
Tender leaf extract
Values are means of triplicates ± standard deviation. Phenolics expressed as mg gallic acid equivalents (GAE)/g plant material. Flavonoids
expressed as mg equivalents of quercetin/g plant material
of antioxidants [43, 44]. In the present study, the activities
of all the enzymatic antioxidants studied showed higher
values at the mature stage than the tender stage. Thus, in
light of the reports, it is clear that the response of
enzymatic antioxidant components to maturity process is
not uniform in all the plants.
Similarly, higher levels of total phenolics, total flavo-
noids and antioxidant capacity were observed in spinach
leaves at the mid-maturity stage, compared to the immature
stage [45]. The levels of chlorophyll and carotenoids in the
leaves of Zea mays have been shown to be dependent on
age [46]. Total carotenoids increased with maturity in
pineapple [47] and in guava [48]. Chlorophyll content
increased with maturity in Cistus chesli plants [49]. In a
sweet pepper variety; it was shown that red ripe fruits had
the highest content of vitamin C and provitamin A
compared to the immature green peppers [50]. β-carotene
and α-tocopherol increased with maturity in the leaves of
Pistacia lentiscus, while ascorbate levels showed no
difference [51]. Our results show that the non-enzymatic
antioxidants increase with maturity in Moringa oleifera
leaves. From our results, it can be deduced that the best
stage of Moringa oleifera suited for consumption is the
mature stage, when the maximum benefit of the antioxidant
content can be derived.
The antioxidants react with DPPH, a purple colored
stable free radical and convert it into a colorless α-α-
diphenyl-β-picryl hydrazine. Antioxidants, on interaction
with DPPH, either transfer an electron or hydrogen atom to
DPPH, thus neutralizing its free radical character [52].
Moringa oleifera leaf extract significantly reduced DPPH
radicals. The degree of discoloration indicates the scaveng-
ing potential of the antioxidant extract, which is due to the
radical scavenging ability. Superoxide anion radical (O2−) is
a precursor to active free radicals that have the potential of
reacting with biological macromolecules and there by
inducing tissue damage [53]. NO, is a key signaling
molecule in physiological and pathological conditions and
when commonly consumed vegetables were screened for
their inhibitory effect on NO production, the aqueous
extracts of several of them exhibited over 80% inhibition
[54]. Therefore, Moringa oleifera leaf extracts can signif-
icantly scavenge free radicals and hence inhibit cellular
damage.
309
Total phenolic contents Total flavonoid
45.81±0.02 27±0.03
36.02±0.01 15±0.02
LPO has been used as a reliable marker of oxidative
stress, both in vitro and in vivo. Several plant extracts have
been shown to inhibit LPO as measured by the levels of
TBARS. Polyphenols other than vitamin E have been
known to exert powerful antioxidant effect in vitro. They
inhibit lipid per oxidation by acting as chain-breaking
peroxyl-radical scavengers, and can protect LDL from
oxidation [55]. It has been found in the present study that
the Moringa oleifera leaf extract contains polyphenols,
therefore the antioxidant effects of the leaf extract may
depend on its phenolic components.
DNA contains reactive group in its bases that are highly
susceptible to free radical attack [56]. H2O2 plays an
important role in the generation of free radical induced
DNA damage, and mutations [57]. Many studies have
reported the protection against oxidative DNA damage by
herbal extracts and formulations. Oxidative damage medi-
ated as single strand breaks in super coiled PTZ18U
plasmid DNA has been reported to be suppressed by 6-
gingerol (a phenolic compound in ginger) [58]. Methanolic
extracts of Nelumbo nucifera inhibited H2O2-induced
damage to pUC18 DNA [59]. Oxidative DNA damage
has been shown to be reverted upon treatment with many
antioxidants. A reduction in the basal levels of oxidative
DNA damage upon treatment with 4-coumaric and proto-
catechuic acids were reported [60]. The results of the
present study are also in agreement with the above reports.
These findings support the use of the leaves of Moringa
oleifera to protect against oxidative DNA damage.
Natural antioxidants that are present in herbs are
responsible for inhibiting or preventing the deleterious
consequences of oxidative stress. Herbs contain free radical
scavengers like polyphenols, flavonoids and phenolic
compounds. A number of scientific reports indicate certain
terpenoids, steroids and phenolic compounds such as
tannins, coumarins and flavonoids have protective effects
due to its antioxidant properties [61]. Phenolics are the
most wide spread secondary metabolite in plant kingdom.
These diverse groups of compounds have received much
attention as potential natural antioxidant in terms of their
ability to act as both efficient radical scavengers and metal
chelator. It has been reported that the antioxidant activity of
phenol is mainly due to their redox properties, hydrogen
donors and singlet oxygen quenchers [62]. Several studies
310
have shown that the higher antioxidant activity associated
with medicinal plants is attributed to the total phenolic
compounds [63]. Thus, the outcome of the present study
highlights the antioxidant effects rendered by mature and
tender leaf extracts of Moringa oleifera leaves under
oxidative stress conditions.
Conclusion
Overall, it could be concluded that Moringa oleifera leaves
bear a potent antioxidant activity. The analysis revealed
only minor differences in the antioxidant activity in the two
maturity stages, mature and tender leaves. Their constituents
scavenge free radicals and exert a protective effect against
oxidative damage induced to cellular macromolecules. The
antioxidant potential may be attributed to the presence of
polyphenolic compounds and consumption of both mature
and tender leaves, and might be equally beneficial to human
antioxidant protection system against oxidative damage.
These results are encouraging enough to pursue character-
ization of these fractions.
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