PHCOG J ORIGINAL ARTICLE

Comparative analysis of phenolic contents and

total antioxidant capacity of Moringa oleifera Lam.
Swati Vyas1, Sumita Kachhwaha1and S.L.Kothari1,2*
1
Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India–302004
2
Institute of Biotechnology, Amity University Rajasthan, Jaipur, Rajasthan, India-302019

ABSTRACT

Introduction: Accumulation of reactive species higher than permissible limits in biological systems may lead to various
degenerative disorders due to oxidative damage. Materials and Methods: Oxidation is a serious concern faced by the
food industry causing deterioration of shelved-food quality. Antioxidant compounds like polyphenolics scavenge such
free radicals and thus protect against oxidative stress. Consumption of polyphenol-rich plants as dietary component
confers protection against such cellular damage. Present study explores antioxidant capacity, total phenolic content
(TPC) and total flavonoid content (TFC)of different extracts prepared from various parts of Moringa oleifera Lam.
Results: Higher TPC, TFC and antioxidant activity was shown by methanolic extracts followed by aqueous, petroleum
benzene and chloroform extracts.The present study suggests that all the extracts might act as radical scavengers to
certain extent possibly due to presence of polyphenolic compounds. Conclusion: M. oleifera exhibits strong antioxidant
activity and could serve as prospective source of natural antioxidants to food and health industries.

Key words: Antioxidant activity, total phenolic content, total flavonoid content, sequential extract, oxidation.

INTRODUCTION with passage of time. Antioxidants (synthetic or natural;

endogenous or exogenous) are directly added to such
Oxidative stress results due to imbalanced homeostasis food products for combating lipid peroxidation which is
between the systematic manifestation of reactive oxygen major reason for deterioration of food. An antioxidant
species i.e. pro-oxidants and biological system’s ability to can be defined as "any substance that, when present at
readily detoxify the reactive intermediates i.e. anti-oxidants, low concentrations compared to those of an oxidizable
which leads to disruption of cellular redox signaling substrate, significantly delays or prevents oxidation of
pathways, nucleic acids, proteins, lipids, and membrane that substrate".4
integrity.1 Crucial etiological relation has been suggested
among oxidative stress and a number of degenerative Antioxidants significantly extend the shelf life of foods
disorders such as diabetes mellitus, cancer, atherosclerosis, containing lipids. Phenolic antioxidants form a radical
arthritis, neurodegenerative diseases and also in the ageing with low reactivity, due to delocalization of the unpaired
process.2 The brain is more vulnerable to oxidative damage electron over the aromatic ring, and exhibit no further
because of its elevated oxygen consumption resulting potential to react with lipids after hydrogen abstraction.5
into generation of large number of reactive intermediates, Consumption of food items rich in antioxidant is
despite of intricate network of defense mechanisms against recommended by several studies to augment array of free
oxidative stress.3 Shelved-food may undergo oxidation radical scavengers inside the body. To combat excess
of free radicals, nutraceutical antioxidants as dietary
*Corresponding author: supplements exerting positive pharmacological effects
Dr. Prof. S.L. Kothari,
on specific human diseases must be explored. However,
Department of Botany, University of Rajasthan, Jaipur, Rajasthan,
India–302004. Institute of Biotechnology Amity University Rajasthan, Jaipur, a concern about the safety of synthetic antioxidants
Rajasthan, India-302019. has increased interest in their replacement by natural
Phone: +91-9829179692
E-mail: [email protected]
antioxidants. The traditional medicinal system identifies
plants as great source of natural antioxidants. They are
DOI: 10.5530/pj.2015.7.5 likely to be promising, safe and effective with least side
effects and therefore, serve as leads for the development

44 Pharmacognosy Journal | Jan-Feb 2015 | Vol 7 | Issue 1

 Vyas et al., Antioxidant activity of Moringa oleifera Lam.

of novel drugs to prevent and/or cure chronic human MATERIALS AND METHODS
diseases.6 Also, natural antioxidants may be utilized
as alternative to prevent deterioration of stored food Plant samples
products.
Different parts, namely, flowers (MoF), seeds
To determine the total antioxidant activity many (MoS), leaves (MoL), roots (MoR), bark (MoB) and gum
protocols either using commercially obtainable radicals (MoG) of M. oleifera (Voucher No. - RUBL 21189) were
(DPPH, ABTS, ORAC, TEAC, etc.) or metal ions (like collected from Jaipur, Rajasthan, India during full bloom
FRAP, LDL, etc.) are available.7 The antioxidant activity and were authenticated from Herbarium, Department of
measured depends substantially on various aspects such Botany, University of Rajasthan, Jaipur, Rajasthan, India.
as test system used, solvent system etc. Therefore, it is
Processing of plant samples
recommended to base any conclusions on at least two
different test systems.8 Thus, the present study employed All collected plant parts (except MoG) were rinsed thrice
two assays namely, DPPH and TEAC inhibition assays. using distilled water and excess water was dripped off.
Then plant samples were weighed and shade dried in a
Moringa oleifera Lam., popularly known as drum stick room with active ventilation and ambient temperature for
tree or horse radish tree or Saijna, is a member of a 10 days. Dried samples were weighed and powdered using
monogeneric family, the Moringaceae along with 12 a grinder. Finely grinded samples and MoG were stored at
other species. It is believed to be an aboriginal of Indian -20°C in dark air-tight containers till further use.
subcontinent that has become naturalized in the tropical
and subtropical areas around the world. It is a rapidly- Chemicals
growing perennial soft wood tree whose all parts are
edible and has long been consumed by humans.9 Almost 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
all the parts of this plant: root, bark, gum, leaf, fruit diammonium salt (ABTS), 2,2-Diphenyl-1-picrylhydrazyl
(pods), flowers, seed and seed oil are medicinally as (DPPH), aluminum chloride hydrate, Folin & Ciocalteu’s
phenol reagent (FCPR), gallic acid, potassium acetate,
well as functionally important food thereby giving the
potassium persulfate, quercetin, sodium carbonate,
name “Miracle Vegetable”.10 M. oleifera is an outstanding
petroleum benzene, chloroform, methanol were purchased
source of nutritional components. Leaves are highly rich
from Sigma-Aldrich, India.
in vitamin A, vitamin C, protein, calcium, magnesium,
potassium, iron, zinc, sodium, copper, manganese and Preparation of the extracts
phosphorus. This plant is known to have vast range of
medicinally important properties such as antimicrobial, The successive extracts from powdered samples (50g)
antiasthmatic, antioxidant, hepatoprotective, anti- packed in a Whatman® cellulose extraction thimbles
cancerous, spasmolytic, bradycardiac, hypotensive as well (Sigma-Aldrich) were prepared using different solvents
as cholesterol lowering and hypoglycemic effects.11 It also namely, petroleum benzene (PE), chloroform (C),
has numerous non-food product uses such as lumber, methanol (M) and water (AQ) via a Soxhlet apparatus
charcoal, textile industry, fencing, water clarification, fine- (Borosil). MoG (10g) was dissolved by constant stirring
machine lubricating oil, biogas, fertilizer, animal forage, using a magnetic stirrer (Digital Spinot, Tarsons) at 250 rpm
perfumery, hair-care products, etc.9 for 6 hours at 40 °C in the 20% M (100 mL) and filtered
using metal sieve. Then centrifugation (Eppendorf) was
To the best of our knowledge, there are only a few done at 1000 rpm for 10 minutes and the supernatant was
detailed data available on free radical scavenging and/ transferred to a flask. The residue was washed several times
or antioxidant properties of M. oleifera.10,12 The present with 20% M and the washings were added to the separated
supernatant. Finally, the solvents were evaporated with the
work is a comprehensive study that evaluates antioxidant
help of vacuum desiccator (Tarsons) to get a solid residue.
capacity, total phenolic and flavonoid content of
The solid residue was stored in dark colored glass bottles
successive extracts of various parts of M. oleifera at
at -20 °C till further use.
different concentrations using spectrophotometric assays.
Also, correlation between various study parameters was Assay for total phenolics (TPC)
determined to find out relationship between free radical
scavenging assays and phenolic compounds. The content of total phenolic compounds in plant extracts

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 Vyas et al., Antioxidant activity of Moringa oleifera Lam.

was determined by Folin Ciocalteu’s method13 with slight of corresponding solvent with each set of experiment.
modifications. One mL of plant extract (10 g/L) was Inhibition activity was calculated in following way:
mixed with 3 mL of 10% FCPR (w/v) and 2 mL of 20%
sodium carbonate (w/v). This mixture was incubated for % Inhibition = [(AB - AS) / AB] x 100
30 minutes on water bath (40 °C) then cooled to room
temperature and absorption was read at 765 nm. Gallic where, AB - absorption of blank sample; AS - absorption
acid was taken as standard and calibration curve was of tested extract solution.
prepared using its various aliquots. TPC was calculated by
Trolox equivalent antioxidant capacity (TEAC) assay
the following formula:
ABTS radical cation decolorization test16 is another
P = c * V/e
spectrophotometric method widely used for the assessment
where; P—total content of phenolic compounds, mg/g of antioxidant activity.
plant extract, in gallic acid equivalents (GAE); c—the
Four mL of 2 mM ABTS cation solution were mixed with
concentration of gallic acid established from the calibration
100 µL test extract and the decrease of absorption was
curve, mg/mL; V— the volume of extract, mL; e—the
measured spectrophotometrically at 734 nm during 6 min.
weight of pure plant extract, g.
The degree of decolorization as percentage inhibition
Assay for total flavonoids (TFC)
of the ABTS radical cation is determined as a function
For determining the total flavonoid content pharmacopeia of concentration and time and calculated relative to the
method14 was followed with minor modifications. One reactivity of trolox as a standard, under the same conditions.
mL of plant extract (10 g/L) was mixed with 1 mL Therefore, this assay is often referred to as the Trolox
of 10% aluminium trichloride (w/v) and 1mL of 1M equivalent antioxidant capacity (TEAC) assay.
potassium acetate, then volume was raised up to 10 mL
Statistical analysis
with the corresponding solvent. This mixture was then
shaken vigorously and incubated for 30 minutes at room Analyses were run in triplicates and the results were
temperature. The absorption was read at 415 nm. Quercetin expressed as mean values with standard error mean.
was used as positive control. The absorption of quercetin Correlation coefficients (R) to determine the relationship
solutions was measured under the same conditions to plot among different antioxidant assays, TPC and TFC were
calibration curve. calculated using MS Excel software (CORREL statistical
function).
F = a * V/e

where; F — total content of flavonoid compounds, mg/g RESULTS AND DISCUSSION

plant extract, in Quercetin equivalent (QE); a —the
concentration of gallic acid established from the calibration Extraction method and solvent selection
curve, mg/mL; V— the volume of extract, mL; e—the
weight of pure plant extract, g. In this study, Soxhlet apparatus was used to achieve
sequential extraction of the plant material to assure recovery
DPPH radical scavenging assay of even compounds with limited solubility in a solvent. Four
different solvents (PE, C, M and AQ) in order of increasing
The radical scavenging activity15 of the plant extracts so polarity were employed for this purpose. This proved to
prepared was measured spectrophotometrically using be advantageous method due to two reasons; firstly, this
stable DPPH. One mL of different concentrations of the provided a comparative data to analyze effectiveness of
extracts was added to a 3 mL of a 0.004% DPPH solution extraction solvent having active principles; secondly, this
(w/v). After shaking vigorously, the mixture was allowed abridges identification of the active compounds in the
to stand undisturbed for 30 minutes in complete dark. The crude extracts so obtained via their further fractionation.
absorbance of the resulting solution was measured at 517 nm
with a UV/visible light spectrophotometer (Shimadzu UV Content of phenolic and flavonoid compounds
1700, Kyoto, Japan). Blank sample was prepared following
same steps but test sample was replaced with same amount Diversity of secondary metabolites is produced by plants

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 Vyas et al., Antioxidant activity of Moringa oleifera Lam.

Figure 1: Total phenolic content of different test extracts. Each value is expressed as mean ± standard error. {Petroleum ether (PE),
chloroform (C), methanol (M), water (AQ) extracts; Moringa oleifera flowers (MoF), seeds (MoS), leaves (MoL), roots (MoR), bark
(MoB) and gum (MoG)); Gallic acid equivalent (GAE), Total phenolic content (TPC)}.

and phenolic compounds are one of its most important radicals via formation of resonance-stabilized phenoxyl
groups. Phenolics possessing at least one aromatic ring radicals. Flavonoids are probably the most important class
(C6) bearing one or more hydroxyl groups posses’ ideal of natural phenolics and have ability to donate electrons
structural chemistry to scavenge free radicals. In general, or hydrogen atoms readily, so they can directly scavenge
phenolic compounds act as potential metal chelators reactive oxygen species.17 Therefore, TPC and TFC were
as well as inhibit lipid per-oxidation by quenching free investigated (Figure 1 and 2).

Figure 2: Total flavonoid content of different test extracts. Each value is expressed as mean ± standard error. {Petroleum ether
(PE), chloroform (C), methanol (M), water (AQ) extracts; Moringa oleifera flowers (MoF), seeds (MoS), leaves (MoL), roots (MoR),
bark (MoB) and gum (MoG); Quercetin equivalent (QE), Total flavonoid content (TFC)}.

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 Vyas et al., Antioxidant activity of Moringa oleifera Lam.

The TPC (mg/g), determined from regression equation 3 and 4). Both of them are spectrophotometric electron
of calibration curve (y = 0.0018x - 0.0469, R² = 0.99) and transfer-based assays which were employed to measure the
expressed in GAE, varied between 7.44 and 144.77. TPC capacity of an antioxidant in the reduction of an oxidant,
was in order M> AQ> PE> C which was well correlated which changes color when reduced. The degree of color
with previous studies about higher recovery of phenolic change (either an increase or decrease of absorbance
compounds in M followed by AQ, PE and C.18 M extract of the probe at a given wavelength) is correlated to the
of MoL showed the highest value (144.77 mg/g in GAE) concentration of antioxidants in the sample.
whereas C extract of MoS showed the lowest value (7.44
mg/g in GAE). 20 % M extract of MoG possessed high The present study reveals that all extracts exhibit
TPC (108.11mg/g in GAE). It was observed that M and radical scavenging activity to certain extent and their
AQ extracts were richer in TPC which suggests presence of distinct activities are probably derived from diversity
polar phenolic compounds. Also, TPC was relatively higher of phytochemicals reacting uniquely with various free
in PE extracts than C extracts which indicates presence of radicals.19 Also, different solvents with different polarities
some non-polar phenolic compounds. were used in our work that likely extracts different classes
of compounds. The highest radical scavenging activity
The TFC (mg/g), determined from regression equation was exhibited by M extracts followed by AQ, PE and
of calibration curve (y = 0.6942x - 0.0042, R² = 0.99) C in all samples. Previous studies have suggested that
and expressed in QE, varied between 2.51 and 57.64.
extracts obtained from polar solvents were likely to show
Significantly higher results were found in M and AQ extracts
higher antioxidant activity.20 It can be noted that flower
followed by C and PE extracts. Highest concentration of
extract displayed highest antioxidant activity followed by
flavonoids was observed in 20% M extract of MoG whereas
leaf, root, gum, bark, and seed. A possible explanation
C extract of MoS revealed lowest TFC. C extracts of MoL
might be that flowers contain variety of compounds (such
exhibited high TFC (56.42 mg/g in QE) followed by MoR
as anthocyanins, reductones, etc.) which exert higher
(38.812 mg/g in QE) which suggests presence of less polar
antioxidant activities. On the other hand, leaf having
compounds in M. oleifera.
longer life span in comparison with other plant parts and
Antioxidant activity being site of energy production faces high magnitude of
oxidative damage therefore, requires greater production of
Results of TEAC and DPPH assays were revealed (Figure antioxidants and their capacity to act as shield.21

Figure 3: The effects of different test extracts on the inhibition of the ABTS cation. Each value is expressed as mean ± standard
error. {Petroleum ether (PE), chloroform (C), methanol (M), water (AQ) extracts; Moringa oleifera Leaves (MoL), seeds (MoS), flowers
(MoF) and gum MoG); Trolox equivalent antioxidant capacity (TEAC) assay}.

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 Vyas et al., Antioxidant activity of Moringa oleifera Lam.

Figure 4: The effects of different test extracts of Moringa oleifera on the inhibition of the DPPH. Each value is expressed as mean ±
standard error. {Petroleum ether (PE), chloroform (C), methanol (M), water (AQ) extracts; 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
assay}.

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 Vyas et al., Antioxidant activity of Moringa oleifera Lam.

Figure 5: Correlation matrix representing relationship between study variables{Petroleum ether (PE), chloroform (C), methanol
(M), water (AQ) extracts; Total phenolic content (TPC); Total flavonoid content (TFC); 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay;
Trolox equivalent antioxidant capacity (TEAC) assay}.

On comparing various parameters of the study, good due to their hydroxyl groups, were the major contributors
correlation was observed (Figure 5). Data so obtained to free radical scavenging ability of the extracts.The overall
revealed good correlation between polyphenolic content result of the present study certainly provides promising
with their antioxidant activity, implying plausible baseline information to ascertain the potency of the crude
contribution of phenolics and flavonoids to the radical extracts of M. oleifera as a potential source of natural
scavenging activity of these plants extracts.15, 20, 22, 23 Strong antioxidants. This suggests that these extracts possess
correlation was observed on comparing results of two reasonable prospect as a source of natural antioxidants
radical scavenging tests. This can be explained due to to be used by food industry. They have great potential
fact that both the assays are based on similar underlying in health-related area via preventing or treating diseases
mechanism i.e. electron donating ability.7 caused by the oxidative stress and might be extensively
used for the treatment of degenerative diseases. However,
further investigation is suggested to identify and isolate
CONCLUSION
individual components forming antioxidative system and
utilize such agents with high efficacy and activity to develop
Numerous studies are going on globally to identify their application for pharmaceutical and food industries.
pharmacologically potent antioxidant compounds with In addition, in vivo pharmacological studies should also be
low profile of side effects for food and health industry. conducted.
Antioxidants may be derived from various sources like
plants, animals and synthetic chemical preparations.
However, synthetic antioxidants cause safety issues ACKNOWLEDGEMENTS
whereas animal-derived antioxidants face ethical issues.
Ethnobotany along with traditional medical systems The authors wish to express their gratitude to the
provides lots of prospects to find active and therapeutically Department of Science and Technology, India through
useful compounds from plants. M. oleifera is a medicinally the “Innovation in Science Pursuit for Inspired Research
important and commonly consumed plant. Therefore, (INSPIRE)-Fellowship” to Ms. Swati Vyas for financial
antioxidant capacities of its various parts were compared support. Special thanks to Dr. Rohit Jain and Dr. Pooja
in the present study. In vitro assessment of polyphenolic Sharma for their suggestions and improvisations. Ms. Swati
compounds and various antioxidant activities showed Vyas heartily thank her family and friends for their constant
positive correlation, indicating that polyphenols may be support and encouragement.

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 Vyas et al., Antioxidant activity of Moringa oleifera Lam.

CONFLICT OF INTEREST Res. 2012; 6: 4368-74.

12. Siddhuraju P, Becker K. Antioxidant properties of various
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