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Total phenolic, ﬂavonoid contents and in vitro antioxidant activity of leaf of

Suaeda monoica Forssk ex. Gmel (Chenopodiaceae)
Article · November 2012
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International Journal of Advanced Life Sciences (IJALS) ISSN
2277 – 758X
Nishanthini et al., IJALS, Volume (5) Issue (1) November - 2012. RESEARCH ARTICLE
Total phenolic, flavonoid contents and in vitro antioxidant activity of leaf of Suaeda
monoica Forssk ex. Gmel (Chenopodiaceae)
A. Nishanthini, A. Agnel Ruba, V.R. Mohan*
Ethnopharmacology Unit, Research Department of Botany, V.O.Chidambaram College,
Tuticorin - 628 008, Tamil Nadu, India.
Email : vrmohanvoc@gmail.com
Abstract
Antioxidant activity of petroleum ether, benzene, ethyl acetate,
methanol and ethanol extracts of the leaf of mangrove herb Suaeda monoica
have been tested using various antioxidant model systems viz, DPPH,
hydroxyl, superoxide, ABTS and reducing power. Methanol extract of
Suaeda monoica is found to possess higher DPPH and hydroxyl radical
Corresponding Author scavenging activity. Ethanol and methanol extracts of Suaeda monoica
V.R. Mohan
Ethnopharmacology Unit, Research exhibited highest superoxide and ABTS radical cation scavenging activity.
Department of Botany, Methanol extract of leaf of Suaeda monoica showed the highest reducing
V.O.Chidambaram College,
ability. This study indicates significant free radical scavenging potential of
Tuticorin - 628 008,
Tamil Nadu, India Suaeda monoica leaf which can be exploited for the treatment of various
Email : vrmohanvoc@gmail.com free radical mediated ailments.
Article History
Received on 26 September, 2012;
Revised in revised form 18 October,
Keywords: Mangrove, Suaeda monoica, Antioxidant activity, Methanol,
2012; Accepted 24 October, 2012 ABTS and DPPH.
Introduction
In addition to these ROS radicals in living organisms
Oxygen is the most vital element in our there are also other ROS monoradicals, such as the
environment for our survival. How can something so singlet oxygen (1O2), hydrogen peroxide (H2O2) and
vital be so toxic? The answer lies in the fact that the hypochlorous acid (HClO). It is accepted that ROS
problems may arise when the electron flow becomes play different roles in vivo. Some are positive and are
uncoupled (transfer of unpaired single electrons), related to their involvement in energy production,
generating free radicals. Free radicals are two types phagocytosis, regulation of cell growth, intercellular
known as “reactive oxygen species” (ROS) and signaling and synthesis of biologically important
“reactive nitrogen species” (RNS) (Bernatoniene et al., compound (Bernatoniene et al., 2011). However, ROS
2011 and Dolai et al., 2012). They contain superoxide may be very damaging, since they can attack lipids in
(O2-), Peroxyl (ROO), alkoxyl (RO), hydroxyl (HO) cell membranes, protein in tissues or enzymes, carbo-
and nitric oxide (NO). The hydroxyl and the alkoxyl hydrates and DNA, to induce oxidations, which may
free radicals are very reactive and rapidly attack the causes cancer, atherosclerosis, aging, immunesuppressant,
molecules in nearby cells (Powers and Jackson, 2008). inflammation, ischemic heart disease, diabetes, hair loss
Int. j. Adv. Lif. Sci., Available online on at www.

Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 34
2277 – 758X
and neurodegenerative disorders such as Alzheimer’s an attempt to evaluate the in vitro antioxidant activities
disease and Parkinson’s disease (Zadak et al., 2009 and from mangrove plants (Suaeda monoica) using different
Naskar et al., 2010). Antioxidants mediate their protective models viz, DPPH, Hydroxyl, Superoxide and ABTS.
effect by directly reaching with free radicals, quenching The present attempt has been made to find out the in
them and thereby prevent damage to cellular components, vitro antioxidant efficacy of various extract from leaf of
thus consequently hindering diseases (Rajkumar et al., Suaeda monoica.
2011 and Senthil kumar et al., 2012). Materials and Methods
Recently, interest has increased considerably in
The leaves of Suaeda monoica were collected
finding naturally occurring antioxidants to replace from Tuticorin coast, Gulf of Mannar (Lat 8º45’N;
synthetic antioxidants, which are being restricted due to Long 78º12’E), Tamil Nadu. The collected samples
their carcinogenicity (Abdel-Hameed, 2009). The anti- were cut into small fragments and shade dried until the
oxidant phytochemicals such as polyphenols, flavonoids fracture is uniform and smooth. The dried plant material
and related compounds found in medicinal plants have was granulated or powdered by using a blender, and
received increasing attention for their potential role in sieved to get uniform particles by using sieve No. 60.
prevention of human diseases (Upadhayay et al., 2010). The final uniform powder was used for the extraction
Some of the marine plants have been investigated and of active constituents of the plant material.
reported to have antioxidant and radical scavenging
Preparation of Plant extract
potential (Meenakshi et al., 2012; Vijayabaskar and
Freshly collected leaf samples of Suaeda monoica
Shiyamala, 2012).
were dried in shade, and then coarsely powdered separately
Suaeda monoica Forssk ex. Gmel belonging to
in a willy mill. The coarse powder (100g) was extracted
Chenopodiaceae family is a salt marsh mangrove herb
successively with petroleum ether, benzene, ethyl
similar to Suaeda maritima in appearance. It is an herb,
acetate, methanol and ethanol, each 250 ml in a Soxhlet
smaller in size. Leaves simple, succulent, linear, young
apparatus for 24 hrs. All the extracts were filtered
twigs are slender ribbed. The leaves have been used as
though Whatman No.41 filter paper. All the extracts
edible green leaves. Traditionally, the leaf from Suaeda
were concentrated in a rotary evaporator. The concentrated
monoica is known to use as a medicine for hepatitis and
extracts were used for in vitro antioxidant activity. The
scientifically it is reported to be used as ointment for
methanol extract was used for the estimation of total
wounds and possess antiviral activity, because of the
phenolics and flavonoids.
presence of triterpenoids and sterols (Ravikumar et al.,
2010 and Ravikumar et al., 2011). Mangrove and Estimation of Total phenolic content
mangrove associate plants are proved to have rich of Total phenolic content was estimated using the
high value secondary metabolites viz, saponins, alkaloids, Folin-Ciocalteu method (Lachman et al., 2000). Samples
polyphenols which show antibacterial, antifungal, (100µL) were mixed thoroughly with 2 mL of 2%
antiplasmodial and hepatoprotective activities Na2CO3. After 2 min. 100 µL of Folin-Ciocalteu
(Gnanadesigan et al., 2011). But, the studies revealed reagent was added to the mixture. The resulting mixture
with the in vitro antioxidant activities from mangrove was allowed to stand at room temperature for 30 min
plants are too limited. Hence, the present study was made and the absorbance was measured at 743 nm against a

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blank. Total phenolic content was expressed as gram of by using the following formula.
gallic equivalents per 100 gram of dry weight DPPH scavenging effect (% inhibition) = {(A0 –A1)
(g 100g-1DW) of the plant samples. /A0)*100}
Estimation of Flavonoids Where, A0 is the absorbance of the control reaction, and
The flavonoids content was determined A1 is the absorbance in presence of all of the extract
according to Eom et al. (2007). An aliquot of 0.5ml of samples and reference. All the tests were performed in
sample (1mg/mL) was mixed with 0.1ml of 10% triplicates and the results were averaged
aluminium chloride and 0.1ml of potassium acetate Hydroxyl radical scavenging activity
(1M). In this mixture, 4.3ml of 80% methanol was The scavenging capacity for hydroxyl radical
added to make 5mL volume. This mixture was vortexed was measured according to the modified method of
and the absorbance was measured spectrophoto metrically Halliwell et al. (1987). Stock solutions of EDTA (1mM),
at 415nm. The value of optical density was used to FeCl3 (10mM), Ascorbic Acid (1mM), H2O2 (10mM)
calculate the total flavonoid content present in the and Deoxyribose (10 mM), were prepared in distilled
sample. deionized water.
DPPH radical scavenging activity The assay was performed by adding 0.1mL
The DPPH is a stable free radical and is widely EDTA, 0.01mL of FeCl3,0.1mL H2O2, 0.36mL of deoxy-
used to assess the radical scavenging activity of ribose, 1.0mL of the extract of different concentration
antioxidant component. This method is based on the (50,100,200,400 and 800µg /mL) dissolved in distilled
reduction of DPPH in methanol solution in the presence water,0.33mL of phosphate buffer (50mM , pH 7.9),
of a hydrogen donating antioxidant due to the formation 0.1mL of ascorbic acid in sequence . The mixture was
of the non radical form DPPH-H (Shen et al., 2010). then incubated at 370c for 1 hour. 1.0mL portion of the
The free radical scavenging activity of all the incubated mixture was mixed with 1.0mL of 10%TCA
extracts was evaluated by 1, 1-diphenyl-2-picryl- and 1.0mL of 0.5% TBA (in 0.025M NaOH containing
hydrazyl (DPPH) according to the previously reported 0.025% BHA) to develop the pink chromogen
measured at 532nm. The hydroxyl radical scavenging
method (Shen et al., 2010). Briefly, an 0.1mm solution
activity of the extract is reported as % inhibition of
of DPPH in methanol was prepared, and 1mL of this
deoxyribose degradation is calculated by using the
solution was added to 3 ml of the solution of all extracts in
following equation
methanol at different concentration (50,100,200,400
and 800µg/mL). The mixtures were shaken vigorously Hydroxyl radical scavenging activity= {(A0 –A1)
and allowed to stand at room temperature for 30 /A0)*100}
minutes. Then the absorbance was measured at 517 nm Where, A0 is the absorbance of the control
using a UV-VIS spectro- photometer (Genesys 10S reaction, and A1 is the absorbance in presence of all of
UV: Thermo electron corporation). Ascorbic acid was the extract samples and reference. All the tests were
used as the reference. performed in triplicates and the results were averaged.
Lower absorbance values of reaction mixture Superoxide radical scavenging activity
indicate higher free radical scavenging activity. The The superoxide anion scavenging activity was
capability to scavenging the DPPH radical was calculated measured as described by Srinivasan et al. (2007). The

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Tris – HCL buffer (16 mM, pH 8.0), containing 0.5 mL of solution containing 50,100,200,400 and 800 µg/mL
of NBT (0.3mM), 0.5 ml NADH (0.936mM) solution, of extract was mixed with sodium phosphate buffer (5.0
1.0 mL extract of different concentration (125,250,500 mL, 0.2 M, pH 6.6) and potassium ferricyanide (5.0
&1000µg/ml), and 0.5 mL Tris – HCl buffer (16mM, mL, 1.0%): The mixture was incubated at 50oC for 20
pH 8.0). The reaction was started by adding 0.5 mL minutes. Then 5mL of 10% trichloroacetic acid was
PMS solution (0.12mM) to the mixture, incubated at added and centrifuged at 980 g (10 minutes at 5oC) in a
25oC for 5 min and the absorbance was measured at refrigerator centrifuge. The upper layer of the solution
560 nm against a blank sample, ascorbic acid. The (5.0 mL) was diluted with 5.0 mL of distilled water and
percentage inhibition was calculated by using the ferric chloride and absorbance read at 700 nm. The
following equation experiment was performed thrice and results were
Superoxide radical scavenging activity= {(A0 –A1) averaged.
/A0)*100} Statistical analysis
Where, A0 is the absorbance of the control Antioxidant activities like DPPH radical
reaction, and A1 is the absorbance in presence of all of scavenging activity, hydroxyl radical scavenging
the extract samples and reference. All the tests were activity, superoxide radical activity, ABTS radical
performed in triplicates and the results were averaged. cation scavenging activity and reducing powers were
Antioxidant Activity by Radical Cation (ABTS. +) estimated in triplicate determinations. Data were
ABTS assay was based on the slightly modified analyzed using the statistical analysis system SPSS
method of Huang et al., (2011). ABTS radical cation (SPSS software for windows release 17.5; SPSS Inc.,
(ABTS+) was produced by reacting 7mM ABTS solution Chicago IL, USA) Estimates of mean, standard error
for aforesaid parameters were calculated.
with 2.45 mM potassium persulphate and allowing the
mixture to stand in the dark at room temperature for 12- Results
16 h before use. The ABTS + Solution were diluted Total phenolic content and total flavonoid content
with ethanol to an absorbance of 0.70+0.02 at 734 nm. The total phenolic content and total flavonoid
After addition of 100µL of sample or trolox standard to content of the methanol extract of Suaeda monoica
3.9 mL of diluted ABTS+ solution, absorbance was leaf was found to be 0.92g100g-1 and 0.86g100g-1
measured at 734 nm by Genesys 10S UV-VIS (Thermo respectively.
scientific) exactly after 6 minutes. Results were expressed
DPPH radical scavenging activity
as trolox equivalent antioxidant capacity (TEAC).
DPPH radical scavenging activity of petroleum
ABTS radical cation activity = {(A0 –A1)/A0)*100}
ether, benzene, ethyl acetate, methanol and ethanol
Where, A0 is the absorbance of the control extracts of Suaeda monoica leaf was shown in Fig - 1.
reaction, and A1 is the absorbance in presence of all of The scavenging effect increases with the concentration
the extract samples and reference. All the tests were of standard and samples. Among the solvent tested,
performed in triplicates and the results were averaged. methanol extract exhibited highest DPPH radical
Reducing Power scavenging activity. At 800µg/mL concentration
The reducing power of the extract was determined methanol extract of Suaeda monoica possessed 114.32%
by the method of Kumar and Hemalatha (2011). 1.0 mL scavenging activity on DPPH.

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Fig – 2. Hydroxyl radical scavenging activity of

different extracts of Suaeda monoica
Fig - 1. DPPH radical scavenging activity of
different extracts of Suaeda monoica
Hydroxyl radical scavenging activity

Hydroxyl radical scavenging activity of
petroleum ether, benzene, ethyl acetate, methanol and
ethanol extracts of Suaeda monoica leaf was shown in
Fig 2. Methanol extract showed very potent activity. At
800µg/mL concentration, methanol extract of Suaeda
monoica possessed 66.81% scavenging activity on
hydroxyl radical.
Fig. - 3. Superoxide radical scavenging activity of
Superoxide radical scavenging activity different extracts of Suaeda monoica
The Suaeda monoica leaf extracts were
subjected to the superoxide scavenging assay and the
results were shown in Fig 3. It indicates that ethanol
extract of Suaeda monoica leaf (800µg/mL) exhibited
the maximum superoxide scavenging activity of
94.84% which is higher than the standard ascorbic acid
whose scavenging effect is 76.11%.
ABTS radical cation scavenging activity
The Suaeda monoica leaf extracts were
subjected to the ABTS radical cation scavenging
activity and the results were presented in Fig. - 4. The
methanol extract exhibited potent ABTS radical cation Fig. - 4. ABTS radical cation scavenging activity
of different extracts of Suaeda monoica
scavenging activity in concentration dependent manner.

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At 800µg/mL concentration, Suaeda monoica leaf

possessed 92.84 % scavenging activity on ABTS which
is higher than the standard trolox whose scavenging
activity is 69.04%.
Reducing power
Figure. 5 showed the reducing ability of different
solvent extracts of Suaeda monoica leaf compared to
ascorbic acid. Absorbance of the solution was increased
when the concentration increased. A higher absorbance
indicates a higher reducing power. Among the solvent
tested, ethanol extract exhibited higher reducing
activity. Fig. - 5. Reducing power ability of different extracts of
IC50 value Suaeda monoica
IC50 values of petroleum ether extract of
Suaeda monoica leaf and standard ascorbic acid for DPPH, µg/mL; 24.16µg/mL and 24.04µg/mL and 22.62µg/mL
hydroxyl, superoxide radical scavenging and trolox for and 20.84µg/mL respectively. IC50 values of ethanol
ABTS radical cation scavenging were found to be extract of Suaeda monoica leaf and standard ascorbic
20.14µg/mL and 19.24µg/mL; 18.69µg/mL and 20.63 acid for DPPH, hydroxyl, superoxide radical scavenging
µg/mL; 20.86 µg/mL and 24.04µg/mL and 16.84µg/mL and trolox for ABTS radical cation scavenging were
and 20.84µg/mL respectively. IC50 values of benzene found to be 28.83µg/mL and 19.24µg/mL; 15.88µg/mL
extract of Suaeda monoica leaf and standard ascorbic and 20.63µg/mL; 26.88µg/mL and 24.04 µg/mL and
acid for DPPH, hydroxyl, superoxide radical scavenging 18.36µg/mL and 20.84µg/mL respectively (Table - 1).
and trolox for ABTS radical cation scavenging were Table – 1. IC50 values of different solvent extracts of
found to be 19.63µg/mL and 19.24µg/mL; 16.69 Suaeda monoica leaf
µg/mL and 20.63µg/mL; 19.13µg/mL and 24.04µg/mL
IC50 (µg/mL)
and 14.93µg/mL and 20.84µg/mL respectively. IC50 Solvent
DPPH Hydroxyl Superoxide ABTS
values of ethyl acetate extract of Suaeda monoica leaf radical
and standard ascorbic acid for DPPH, hydroxyl, superoxide Petroleum 20.14 18.69 20.86 16.84
radical scavenging and trolox for ABTS radical cation ether
Benzene 19.63 16.69 19.13 14.93
scavenging were found to be 18.12µg/mL and 19.24
Ethyl 18.12 18.51 22.84 17.13
µg/mL; 18.51µg/mL and 20.63µg/mL; 22.84µg/mL acetate
and 24.04 µg/mL and 17.13µg/mL and 20.84µg/mL Methanol 29.16 19.13 24.16 22.62
respectively. IC50 values of methanol extract of Suaeda Ethanol 28.83 15.88 26.88 18.36
monoica leaf and standard ascorbic acid for DPPH, Ascorbic 19.24 20.63 24.04 -
hydroxyl, superoxide radical scavenging and trolox for acid
ABTS radical cation scavenging were found to be Trolox - - - 20.84
29.16µg/mL and 19.24 µg/mL; 19.13µg/mL and 20.63 * *All values are mean of triplicate determinations

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Discussion phenolic compounds etc serve as sources of antioxidants

and do scavenging activity (Ghasemi et al., 2009). In
Phenolics are secondary metabolites that play a
this study, it is evident that the extract of the study
role in the maintenance of the human body. The presence
species, Suaeda monoica leaf possess effective
of phytoconstituents, such as phenols, flavonoids and
antioxidant activity. In vivo antioxidant activity of the
tannin in plants, indicates the possibility of antioxidant
petroleum ether, benzene, ethyl acetate, methanol and
activity and this activity will helps in preventing a
ethanol extracts of Suaeda monoica leaf was
number of diseases through free radical scavenging
investigated in the present study by DPPH, hydroxyl,
activity. Phenolic compounds are commonly found in
superoxide and ABTS radical cation scavenging activities
plants and have been reported to have several biological
and their reducing ability. These methods have proven
activities including antioxidant properties. Many
the effectiveness of the extracts in comparison to that of
studies focused on the biological activities of phenolic
the reference standard antioxidant, ascorbic acid and
compounds, which have potential antioxidants and free
trolox.
radical scavengers (Meenakshi et al., 2012).
The DPPH is a stable free radical, which has
Phenolic compounds are well known as been widely accepted as a tool for estimating free
antioxidant and scavenging agents free radicals radical scavenging activities of antioxidants. DPPH is a
associated with oxidative damage. Phenolic compounds stable free radical and accepts an electron or hydrogen
have attracted much interest recently because in vitro radical to become a stable diamagnetic molecule
studies suggest that they have a variety of beneficial (Kalaivani and Mathew, 2010). The reduction capability
biological properties like anti-inflammatory, antitumor of DPPH radical is determined by the decrease in
and antimicrobial activities. Studies have attributed that absorbance at 517nm induced by antioxidants. The
antioxidant properties are due to the presence of experimental data of the extract revealed that the
phenols and flavonoids (Turkoglu et al., 2007; Jose and extract is likely to have the effects of scavenging free
Radhamary, 2012). radicals. From the result, in the present study a dose
Antioxidant activity of phenolic compounds is dependent relationship in the DPPH radical scavenging
based on their ability to donate hydrogen atoms to free activity.
radicals. In addition, they possess ideal structural properties Among the oxygen radicals, hydroxyl radical is
for free radical scavenging properties. Flavonoids are the most reactive and induces severe damage to
important secondary metabolites of plant modulating adjacent biomolecules (Awah et al., 2010). In the
lipid peroxidation involved in atherogenesis, thrombosis present study, the hydroxyl radical scavenging activity
and carcinogenesis. It has been confirmed that pharma- of different extracts of Suaeda monoica leaf was
cological effects of flavonoids is correlating with their assessed by the deoxyribose degradation method. In
antioxidant activities (Mbaebie et al., 2012). deoxyribose method, the sugar is degraded on exposure
Free radicals and other reactive species are to hydroxyl radical generated by Fenton reaction. The
thought to play an important role in many human diseases. resulting complex mixture of products is heated under
Radical scavenging activities are very important due to acid condition; malondialdehyde (MDA) is formed and
the deleterious role of free radicals in biological systems. detected by its ability to react with thiobarbituric acid
Many secondary metabolites which include flavonoids, to form a pink chromogen. In the deoxyribose method,

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Suaeda monoica leaf extracts shows potent hydroxyl Afolayan, 2011; Sajeesh et al., 2011 and Poongothai
radical scavenging activity which can be comparable to et al., 2011). Reductones are also reported to react with
the standard ascorbic acid. The scavenging activity may certain precursors of peroxide, thus preventing peroxide
be due to the presence of various phytochemicals including formation. The data obtained in the present study
polyphenols and flavonoids in Suaeda monoica leaf suggest that it is likely to contribute significantly
extracts. towards the observed antioxidant effects. Like the
Superoxide radical is known to be a very harmful antioxidant activity, the reducing power of the extract
species to cellular components as a precursor of more increase with increasing concentration.
reactive species (Halliwal and Gutteridge, 2007). The The results from various free radicals scavenging
superoxide radical is known to be produced in vivo and systems reveal that all the extracts of Suaeda monoica
can result in the formation of hydrogen peroxide via leaf have significant antioxidant activity. The extracts
dismutation reaction. The ethanol extract is found to be are found to have different levels of antioxidant activity
an efficient scavenger of superoxide radical generated in all the methods tested. IC50 values obtained were
in alkaline DMSO system. The results clearly indicate comparable to that of the standards used i.e, ascorbic
that the Suaeda monoica leaf extracts has a noticeable acid and trolox. According to this study, a significant
effect as scavenging superoxide radical. antioxidant activity was found. Total phenol and
ABTS radical scavenging activity is relatively flavonoid contents determination indicate the high
recent one, which involves a more drastic radical, content of phenols and flavonoids and these compounds
could be major contributors to antioxidant activity.
chemically produced and is often used for screening
Therefore, further investigations need to be carried out
complex antioxidant mixtures such as plant extracts,
to isolate and identify the antioxidant compounds
beverages and biological fluids. The ability in a wide
present in the plant extract. Furthermore, the in vivo
pH range raised the interest in the use of ABTS for the
antioxidant activity of this extract needs to be assessed
estimation of antioxidant activity (Huang et al., 2011).
prior to clinical use.
The methanol extract showed potent antioxidant
activity in ABTS method, which is higher than that of Acknowledgement
standard trolox. Here, the Suaeda monoica leaf extracts The Authors wishes to thank Dr. R. Sampatharaj,
radical scavenging activity showed a direct role of its Honorary Advisor, Samsun Clinical Research Laboratory,
phenolic compounds in free radical scavenging. Tirupur, for their assistance in animal studies.
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Corresponding Author : V.R. Mohan, Ethnopharmacology Unit, Research Department of Botany, V.O.Chidambaram
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Total phenolic, ﬂavonoid contents and in vitro antioxidant activity of leaf of

Article · November 2012

Nishanthini A. Veerabahu Ramasamy Mohan

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Fig – 2. Hydroxyl radical scavenging activity of

Hydroxyl radical scavenging activity

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At 800µg/mL concentration, Suaeda monoica leaf

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Discussion phenolic compounds etc serve as sources of antioxidants

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