TOTAL PHENOLIC, FLAVONOID CONTENTS AND IN VITRO ANTIOXIDANT ACTIVITY OF SUAEDA MONOICA STEM FORSSK...

International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 2(2): March-April 2013

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES

Pharmaceutical Sciences Research Article……!!!

Received: 05-03-2013; Accepted: 13-03-2013

TOTAL PHENOLIC, FLAVONOID CONTENTS AND IN VITRO

ANTIOXIDANT ACTIVITY OF SUAEDA MONOICA STEM

FORSSK EX GMEL (CHENOPODIACEAE)

M. Packia Lincy, K. Paulpriya, V.R. Mohan*

Ethnopharmacology Unit, Research Department of Botany, V.O.Chidambaram College,

Tuticorin 628 008, Tamil Nadu, India.

KEYWORDS:

Mangrove, Suaeda

monoica, flavonoid,

Reducing Power.

For Correspondence:

V.R. Mohan *

Address:

Ethnopharmacology unit,

Research Department of

Botany, V.O.

Chidambaram College,

Tuticorin, Tamil Nadu.

628008.

Email:

[email protected]

ABSTRACT

Antioxidant activity of the stem of Suaeda monoica was studied for

its free radical scavenging property on different in vitro models eg

DPPH, hydroxyl, superoxide, ABTS radical cation scavenging and

reducing power by using different solvents. Total phenolic and

flavonoid contents were estimated. The methanol extract showed

promising free radical scavenging activity in dose dependent

manner. This antioxidant potency may be related to the presence of

antioxidant phenolic and flavonoid compounds present in the

extract. These results clearly indicate that Suaeda monoica stem is

effective against free radical mediated disease.

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International Standard Serial Number (ISSN): 2319-8141

1. INTRODUCTION :

Free radicals are chemical species which contains one or more unpaired electrons. They are

highly unstable and cause damage to other molecules by extracting electrons from them in

order to attain stability. They are formed inside the system, and are highly reactive and

potentially damaging transient chemical species. These radical are continuously produced in

the human body because they are essential for detoxification, chemical signaling, energy

supply and immune functions. Free radicals are regulated by endogenous antioxidant enzyme

system, but due to over production of free radicals by exposure to environmental oxidant

substances such as cigarette smoking, UV radiation etc or a failure in antioxidant defense

mechanism or damage to cell structures, the risk increases for many diseases such as

Alzheimer’s disease, mild congestive impairment, Parkinson’s disease, cardiovascular

disorders, liver diseases, ulcerative colitis, inflammation and cancer [1-2]. The body

possesses defense mechanism against free radical induced oxidative stress, which involve

preventive and repair mechanisms. Enzymatic antioxidants such as superoxide dismutase,

catalase, flutathione peroxidase etc and non-enzymatic antioxidants such as carotenoids,

ascorbic acid, phenolic compounds, flavonoids etc act by one or more mechanisms like

reducing activity, free radical scavenging and potential completing of pro-oxidant metals and

quenching of singlet oxygen. It is possible to reduce the risk of chronic diseases and prevent

the disease progression by either enhancing the body’s natural antioxidant defense or

supplementing with proven antioxidants. For this reason, discovery of natural antioxidants is

a major thrust area [3].

2. MATERIALS AND METHODS

The stem of S .monoica was collected from Tuticorin coast, Gulf of Mannar, Tamil Nadu.

The collected samples were cut into small fragments and shade dried until the fracture is

uniform and smooth. The dried plant material was granulated or powdered by using a

blender, and sieved to get uniform particles by using sieve No. 60. The final uniform powder

was used for the extraction of active constituents of the plant material.

2.1 Preparation of Plant extract

Freshly collected stem samples of S.monoica were dried in shade, and then coarsely

powdered separately in a willy mill. The coarse powder (100g) was extracted successively

with petroleum ether, benzene, ethyl acetate, methanol and ethanol, each 250 ml in a Soxhlet

apparatus for 24 hrs. All the extracts were filtered though Whatman No.41 filters paper. All

the extracts were concentrated in a rotary evaporator. The concentrated extracts were used for

in vitro antioxidant activity. The methanol extract was used for the estimation of total

phenolics and flavonoids.




2.2 Estimation of Total phenolic content

Total phenolic content was estimated using the Folin-Ciocalteu method [4]. Samples (100µL)

were mixed thoroughly with 2 mL of 2% Na2CO3. After 2 min. 100 µL of Folin-Ciocalteu

reagent was added to the mixture. The resulting mixture was allowed to stand at room

temperature for 30 min and the absorbance was measured at 743 nm against a blank. Total

phenolic content was expressed as gram of gallic equivalents per 100 gram of dry weight (g

100g-1

DW) of the plant samples.

2.3 Estimation of Flavonoids

The flavonoids content was determined according to Eom et al [5]. An aliquot of 0.5ml of

sample (1mg/mL) was mixed with 0.1ml of 10% aluminium chloride and 0.1ml of potassium

acetate (1M). In this mixture, 4.3ml of 80% methanol was added to make 5mL volume. This

mixture was vortexed and the absorbance was measured spectrophotometrically at 415nm.

The value of optical density was used to calculate the total flavonoid content present in the

sample.

2.4 DPPH radical scavenging activity

The DPPH is a stable free radical and is widely used to assess the radical scavenging activity

of antioxidant component. This method is based on the reduction of DPPH in methanol

solution in the presence of a hydrogen donating antioxidant due to the formation of the non

radical form DPPH-H [6].

The free radical scavenging activity of all the extracts was evaluated by 1, 1-diphenyl-2-

picryl-hydrazyl (DPPH) according to the previously reported method [6]. Briefly, an 0.1mm

solution of DPPH in methanol was prepared, and 1mL of this solution was added to 3 ml of

the solution of all extracts in methanol at different concentration (50,100,200,400 &

800μg/mL).The mixtures were shaken vigorously and allowed to stand at room temperature

for 30 minutes. Then the absorbance was measured at 517 nm using a UV-VIS

spectrophotometer (Genesys 10S UV: Thermo electron corporation). Ascorbic acid was used

as the reference. Lower absorbance values of reaction mixture indicate higher free radical

scavenging activity. The capability to scavenging the DPPH radical was calculated by using

the following formula.

DPPH scavenging effect (% inhibition) = {(A0 –A1)/A0)*100}

Where, A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of

all of the extract samples and reference. All the tests were performed in triplicates and the

results were averaged.




2.5 Hydroxyl radical scavenging activity

The scavenging capacity for hydroxyl radical was measured according to the modified

method of Halliwell [7]. Stock solutions of EDTA (1mM), FeCl3 (10mM), Ascorbic Acid

(1mM), H2O2 (10mM) and Deoxyribose (10 mM), were prepared in distilled deionized water.

The assay was performed by adding 0.1mL EDTA , 0.01mL of FeCl3,0.1mL H2O2, 0.36mL

of deoxyribose, 1.0mL of the extract of different concentration (50,100,200,400

&800μg/mL)dissolved in distilled water,0.33mL of phosphate buffer (50mM , pH 7.9),

0.1mL of ascorbic acid in sequence . The mixture was then incubated at 370c for 1 hour.

1.0mL portion of the incubated mixture was mixed with 1.0mL of 10%TCA and 1.0mL of

0.5% TBA (in 0.025M NaOH containing 0.025% BHA) to develop the pink chromogen

measured at 532nm. The hydroxyl radical scavenging activity of the extract is reported as %

inhibition of deoxyribose degradation is calculated by using the following equation

Hydroxyl radical scavenging activity= {(A0 –A1)/A0)*100}




2.6 Superoxide radical scavenging activity

The superoxide anion scavenging activity was measured as described by Srinivasan et al[8].

The superoxide anion radicals were generated in 3.0 ml of Tris – HCL buffer (16 mM, PH

8.0), containing 0.5 mL of NBT (0.3mM), 0.5 ml NADH (0.936mM) solution, 1.0 mL

extract of different concentration (50,100,200,400 & 800μg/mL), and 0.5 mL Tris – HCl

buffer (16mM, PH 8.0). The reaction was started by adding 0.5 mL PMS solution (0.12mM)

to the mixture, incubated at 25oC for 5 min and the absorbance was measured at 560 nm

against a blank sample, ascorbic acid. The percentage inhibition was calculated by using the

following equation

Superoxide radical scavenging activity= {(A0 –A1)/A0)*100}


all of the extract samples and reference. The entire test was performed in triplicates and the


2.7 Antioxidant Activity by Radical Cation (ABTS. +)

ABTS assay was based on the slightly modified method of Huang et al [9]. ABTS radical

cation (ABTS+) was produced by reacting 7mM ABTS solution with 2.45 mM potassium

persulphate and allowing the mixture to stand in the dark at room temperature for 12 - 16 h




before use. The ABTS + Solution were diluted with ethanol to an absorbance of 0.70+0.02 at

734 nm. After addition of 100μL of sample or trolox standard to 3.9 mL of diluted ABTS+

solution, absorbance was measured at 734 nm by Genesys 10S UV-VIS (Thermo scientific)

exactly after 6 minutes. Results were expressed as trolox equivalent antioxidant capacity

(TEAC).

ABTS radical cation activity = {(A0 –A1)/A0)*100}




2.8 Reducing Power

The reducing power of the extract was determined by the method of Kumar and Hemalatha

[10]. 1.0 mL of solution containing 50,100,200,400 &800μg/mL of extract was mixed with

sodium phosphate buffer (5.0 mL, 0.2 M, pH6.6) and potassium ferricyanide (5.0 mL, 1.0%):

The mixture was incubated at 50oC for 20 minutes. Then 5mL of 10% trichloroacetic acid

was added and centrifuged at 980 g (10 minutes at 5oC) in a refrigerator centrifuge. The

upper layer of the solution (5.0 mL) was diluted with 5.0 mL of distilled water and ferric

chloride and absorbance read at 700 nm. The experiment was performed thrice and results

were averaged.

2.9 Statistical analysis

Antioxidant activities like DPPH radical scavenging activity, hydroxyl radical scavenging

activity, superoxide radical activity, ABTS radical cation scavenging activity and reducing

powers were estimated in triplicate determinations. Data were analyzed using the statistical

analysis system SPSS (SPSS software for windows release 17.5; SPSS Inc., Chicago IL,

USA) Estimates of mean, standard error for aforesaid parameters were calculated.

3. RESULT:

3.1. Total phenolic content and total flavonoid content

The total phenolic content and total flavonoid content of the methanol extract of S. monoica

stem was found to be 0.35 g 100g-1

and 0.60 g 100g-1

respectively.

3.2 DPPH radical scavenging activity:

The effect of petroleum ether, benzene, ethyl acetate, methanol and ethanol extracts of

S.monoica stem and standard ascorbic acid on DPPH radical scavenging activity were

compared and shown in Figure 1. The scavenging effect increases with the concentration of

standard and samples. At 800µg/mL concentration of methanol, ethanol and ethyl acetate

extracts of S. monoica stem possessed 116.22%, 103.62% and 92.68% scavenging activity on




DPPH respectively. All the concentration of S. monoica stem extracts showed higher activity

except petroleum ether and benzene extracts than the standard ascorbic acid. The scavenging

ability decreased in the order of methanol >ethanol >ethyl acetate >petroleum ether>benzene.

3.3 Hydroxyl radical scavenging activity

The effect of petroleum ether, benzene, ethyl acetate, methanol and ethanol extract of S.

monoica stem and standard ascorbic acid on hydroxyl radical scavenging activity were


standard and samples. At 800µg/mL concentration of benzene, methanol and ethyl acetate

extracts of S. monoica stem showed 78.41%, 73.16 % and 70.12 % scavenging activity on

hydroxyl radical respectively. All the concentration of S. monoica stem extracts showed

higher activity except petroleum ether and ethanol extracts than the standard ascorbic acid.

Hydroxyl radical scavenging activity of extracts were in following order benzene>

methanol> ethyl acetate> petroleum ether>ethanol.

3.4 Superoxide radical scavenging activity

The S. monoica stem extracts were subjected to be superoxide scavenging assay and the

results were shown in Figure 3. It indicates that methanol, benzene and ethanol extract of S.

monoica stem (800µg/mL) exhibited the maximum superoxide radical scavenging activity of

116.92%, 112.16 % and 102.86 % respectively, which is higher than the standard ascorbic

acid whose scavenging effect is 76.11 %. Superoxide radical scavenging activity of extracts

were in following order methanol>benzene> ethanol> petroleum ether >ethyl acetate.

3.5 ABTS radical cation scavenging activity:

The effect of S. monoica stem extracts and standard trolox on ABTS radical cation were


standard and samples. At 800µg/mL concentration of methanol, ethanol, ethyl acetate and

petroleum ether extracts of S. monoica stem possessed 95.22 %, 85.92 %, 78.21 % and 73.16

% scavenging activity on ABTS. All the concentration of S. monoica stem extracts showed

higher activity except benzene extract than the standard trolox. ABTS radical cation

scavenging activity were in following order methanol>ethanol> ethyl acetate >petroleum

ether>benzene.

3.6 Reducing power:

The reducing power of S. monoica stem extracts was compared with the standard ascorbic

acid. The reducing power increases with the increasing concentration.




The reducing power of the petroleum ether, benzene, ethyl acetate, and methanol and ethanol

extracts of S. monoica stem was shown in Figure 5. At 800µg/mL concentration of ethanol

and methanol extracts of S.monoica stem showed higher reducing power than the ascorbic

acid.

3.7 IC50 values:

IC50 values of petroleum ether extract of S.monoica stem and standard ascorbic acid for

DPPH, hydroxyl, superoxide radical scavenging and trolox for ABTS radical cation

scavenging were found to be 16.36µg/mL and 19.24µg/mL; 16.08µg/mL and 20.63µg/mL;

25.88µg/mL and 24.04µg/mL and 25.88µg/mL and 24.04µg/mL respectively. IC50 values of

benzene extract of S. monoica stem and standard ascorbic acid for DPPH, hydroxyl,

superoxide radical scavenging and trolox for ABTS radical cation scavenging were found to

be 16.05µg/mL and 19.24µg/mL; 19.62µg/mL and 20.63µg/mL; 28.04µg/mL and

24.04µg/mL and 18.19µg/mL and 20.84µg/mL respectively. IC50 values of ethyl acetate

extract of S. monoica stem and standard ascorbic acid for DPPH, hydroxyl, superoxide

radical scavenging and trolox for ABTS radical cation scavenging were found to be

17.22µg/mL and 19.24 µg/mL; 15.93µg/mL and 20.63µg/mL; 22.84µg/mL and 24.04µg/mL

and 21.93µg/mL and 20.84µg/mL respectively. IC50 values of methanol extract of S. monoica

stem and standard ascorbic acid for DPPH, hydroxyl, superoxide radical scavenging and

trolox for ABTS radical cation scavenging were found to be 29.91µg/mL and 19.24µg/mL;

16.84µg/mL and 20.63µg/mL; 29.81µg/mL and 24.04µg/mL and 27.16µg/mL and

20.84µg/mL respectively. IC50 values of ethanol extract of S. monoica stem and standard

ascorbic acid for DPPH, hydroxyl, superoxide radical scavenging and trolox for ABTS

radical cation scavenging were found to be 27.84µg/mL and 19.24µg/mL; 15.41µg/mL and

20.63µg/mL; 28.17µg/mL and 24.04 µg/mL and 23.56µg/mL and 20.84µg/mL respectively

(Table 1).

4. DISCUSSION:

Phenolic compounds are considered to be the most important antioxidants of plant materials.

They constitute one of the major groups of compounds activity as primary antioxidants or

free radical terminators. Antioxidant activity of phenolic compounds is based on their ability

to donate hydrogen atoms to free radicals. In addition they possess ideal structural properties

[11]. Phenolic compounds have attracted much interest recently because in vitro and in vivo

studies suggested that they have a variety of beneficial biological properties like anti-

inflammatory, antitumor and antimicrobial activities [12]. Studies have attributed that

antioxidant properties are due to the presence of phenols and flavonoids [13].




The antioxidant activity of the phenolic, tannins, flavonoid compounds which are very

common both in edible and inedible plants are attributed to its redox properties. With their

properties they can act as reducing agents, hydrogen donators and singlet oxygen quenchers.

They also have metal chelating properties. Polyphenolic scavenging action is mainly due to

their hydroxyl groups. They are very important plant constituents which can product body

from different type of oxidative stress [14-17]. Flavones glycosides have also been reported

as story antioxidants and potent hypoglycemic agents. Flavonoids are reported to be involved

in anti-inflammatory activity of plants. Flavanoids, the major group of phenolic compounds

are reported for their antimicrobial, antiviral and spasmolytic activity. Flavonoids are able to

scavenge hydroxyl radicals, superoxide anion radicals and lipid peroxy radicals, which

highlights many of the flavonoids health- promotic functions in organism. They are important

for prevention of diseases associated with oxidative damage of membrane, proteins and

DNA. Flavonoids, in human diet may reduce the risk of various cancers, as well as

preventing menopausal symptoms. Flavonoids, on the other hand, are potent water soluble

antioxidants and free radical scavengers, which prevent oxidative cell damage and have

strong anticancer activities [18].

Free radicals are chemically unstable atoms that cause damage to lipid cells, proteins and

DNA as a result of imbalance between the generation of relative oxygen species (ROS) and

the antioxidant enzymes [19]. They are known to be the underlying cause of oxidative stress

which is grossly implicated in the pathogenesis of various diseases such as cancer, diabetes,

cardiovascular diseases, aging and metabolic syndrome [20]. Examples of their radicals

include superoxide anions, hydroxyl, and nitric oxide and hydrogen peroxide radicals. There

radicals can be scavenged by the protective role of natural and synthetic antioxidant agents.

The use of natural antioxidant has gained much attention from consumers because they are

considered safer than synthetic antioxidants. Recently, there has been a worldwide trend

towards the use and injection of natural antioxidants present in different parts of plants due to

their phytochemical [21]. For this reason, discovery of natural antioxidants is major thrust

area. The antioxidant activities of the petroleum ether, benzene, ethyl acetate, and methanol

and ethanol extracts of S. monoica stem were investigated against various in vitro models.

Since, free radicals are of different chemical entities, it is essential to test the extract against

many free radicals to produce its antioxidant activity. Hence, a large number of in vitro

methods were used for the scavenging IC50 values obtained were compared were compared

with the standard used.




The reaction of plant extract with purple coloured DPPH radical converted the radical to α, a

diphenyl –β picryl hydrazine due to the extract antioxidant property. The degree of

discoloration indicates the potential of the plant extract to scavenge free radical due its ability

to donate hydrogen proton. The concentration- dependent curve of DPPH radical scavenging

activity of S .monoica stem compared well with ascorbic acid.

The hydroxyl radical is one of representative reactive oxygen species generated in the body.

These radicals are produced through various biological reactions; one of the common

reactions is the Iron (II) based Fenton reaction. In the present study, among the tested

extracts, benzene exhibited the strongest hydroxyl radical scavenger activity (78.41% at

800µg/ml) highest ethanol extract showed the lowest activity (62.17% at 800µg/ml). The

radical scavenging capacity may be attributed to phenolic compounds in seed extracts with

the ability to accept electrons, which can combine with free radical competitively to decrease

hydroxyl radical [22].

Endogenously, superoxide could be produced in large compounds by various biological

processes. It is known to the very harmful to cellular components as a precursor of the most

reactive oxygen species (ROS), contributing to tissue damage and various disease[23]. In the

present study, methanol extract of S.monica showed 116.92% superoxide inhibition at the

concentration of 800µg/ml followed by the benzene extract (112.16%). All the tested

extracts, exhibited higher ability in scavenging superoxide anion radical, when compared to

the standard ascorbic acid (76.11% at 800µg/ml).The results revealed that the S.monica stem

extracts have superoxide radical scavenging activity which can be of significant interest in

health point of view in reducing the level of superoxide radical which is elevated during

oxidative stress in the body.

The decolorization of ABTS radical reflects the capacity of an antioxidant species to donate

electrons or hydrogen atoms to inactivate this radical species. The ABTS radical cation is

generated from the reaction of ABTS with potassium persulfate overnight in water [24]. In

the present study, methanol extract is highly potent in neutralizing ABTS cation radicals

(95.22% at 800µg/ml) phenolics of this plant extracts are probably involved in the ABTS

radical scavenging activity. This study indicated that the extracts have the hydrogen donating

ability and could serve as free radical scavengers by activity as primary antioxidants.

The transformation of Fe3+

into Fe2+

in the presence of various fractions was measured to

determine the reducing power activity.




The reducing ability if a compound generally depends on the presence of reductores, which

exert the antioxidant activity by breaking the free radical chain by donating a hydrogen atom

[25]. In the present study, the reducing power of ethanol extract of stem of S.monica was very

potent and the reducing power of the extract was increased with increasing concentration.

From the results obtained in the present study, it is concluded that all the presently studied

extracts of S.monica stem exhibits high antioxidant and free radical scavenging activities

presence of adequate amount of phenolic and flavonoid compounds may account for this fact.

So these findings of present study suggest that this plant have a potential source of natural

antioxidant. Further studies are warranted for the isolation and characterization of antioxidant

compounds, and also in vivo studies are needed for understanding their mechanism of action

as antioxidants.

ACKNOWLEDGEMENT

The authors are thankful to Dr.R. Sampathraj, Honorary Director, Dr. Samsun Clinical

Research Laboratory, Thiruppur for providing necessary facilities to carry out this work. The

second author, V.R.M. gratefully acknowledges and expresses his sincere thanks to

University Grants Commission, New Delhi for providing financial assistance to this Major

Research Project (F39-429/2010 (HRP).

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Fig 1: DPPH radical scavenging activity of different extracts of Suaeda monoica




Fig 2: Hydroxyl radical activity of different extracts of Suaeda monoica.

Fig 3: Superoxide radical scavenging activity of different extracts of Suaeda monoica

Fig 4: ABTS radical cation scavenging activity of different extracts of Suaeda monoica.




Fig 5: Reducing power ability of different extract of Suaeda monoica.

Table No1. IC50 values of different solvent extracts of Suaeda monoica.*

*All the Values are Mean by Triplicates determines.


Solvent

IC50 (µg/mL)

DPPH Hydroxyl Superoxide ABTS

Petroleum

ether 16.36 16.08 25.88

20.17

Benzene 16.05 19.62 28.04 18.19

Ethyl acetate 17.22 15.93 22.84 21.93

Methanol 29.91 16.84 29.81 27.16

Ethanol 27.84 15.41 28.17 23.56

Ascorbic acid 19.24 20.63 24.04 -

Trolox - - - 20.84


TOTAL PHENOLIC, FLAVONOID CONTENTS AND IN VITRO ANTIOXIDANT ACTIVITY OF SUAEDA MONOICA STEM FORSSK...

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