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IN VITRO

FREE RADICAL SCAVENGING ACTIVITY

OF BAGO (Gnetum gnemon Linn.),

PAKO (Diplazium esculentum (Retz.) Sw.)

AND SALUYOT (Corchorus olitorius Linn.)

LEAF EXTRACTS1

1 Elden P. Jayag, +639486314925, eldenjayag92@gmail.com Andresito D. Acabal, 053-335-2619, aandresito@yahoo.com

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ABSTRACT

JAYAG, ELDEN P. April 2013. Visayas StateUniversity,Visca, Baybay City, Leyte.“IN VITRO FREE RADICALSCAVENGING ACTIVITY OF BAGO (Gnetum gnemon Linn.), PAKO(Diplazium esculentum (Retz.) Sw.) AND SALUYOT (Corchorus olitoriusLinn.) LEAF EXTRACTS”

Free radical scavenging activities (µmol Trolox

equivalent (TE)/ 100 g fresh weight) of Bago (Gnetum gnemon

Linn.), Pako (Diplazium esculentum (Retz.) Sw.) and Saluyot

(Corchorus olitorius Linn.) leaf extracts were measured by 1,1-

diphenyl-2-picrylhydrazil (DPPH) assay. Extraction was carried

out with and without heating using water, vinegar (5% acetic

acid) and ethanol as solvents.

Free radical scavenging activity was highest (380.29 ±

55.28 µmol TE/100 g) for C. olitorius Linn. and lowest (306.45 ±

43.35 µmol TE/100 g fresh weight) for D. esculentum (Retz.) Sw..

Ethanolic extract exhibited highest (357.39 ± 49.34 µmol

TE/100 g fresh weight) free radical scavenging activity and

water extract the least (249.32 ± 59.46 µmol TE/100 g fresh

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weight). Application of heat during extraction increased the

free radical scavenging activities.

Keywords: free radical, scavenging activity, indigenous

vegetables

CHAPTER I

INTRODUCTION

Nature and Importance of the Study

A free radical is reactive molecular species proficient

of independent existence having an unpaired electron in an

atomic orbital (Pravesh et al., 2011) and are unstable molecules.

Eventually, free radicals begin a chain reaction producing

more free radicals resulting in damage on the system (Abhishek

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et al., 2011). It can cause harm to other molecules by removing

electrons in such a way to accomplish stability (Hemalatha et

al., 2010).

Antioxidant defense system can be used to counteract free

radicals or reactive oxygen species (ROS) like superoxide

anion (O2.), hydroxyl radical (OH.) and peroxides (H2O2); its

defense system comprises of antioxidant enzymes and

antioxidant compounds (Pravesh et al., 2011). Antioxidants

prevent ROS toxicities by stopping the formation of ROS, by

scavenging reactive metabolites and transforming them to less

reactive molecules (Palaniswamy & Padma, 2011).

Antioxidant supplements or foods rich in medicinal plants

are utilized to assist the human body in reducing oxidative

harm by free radicals (Radha & Padma, 2011). The attack of the

free radicals can be stabilized by antioxidants; as a result,

it can lead to a defense of the system by any of the severe

disorder (Abhishek et al., 2011).

Candidate plants used in this study include Bago (Gnetum

gnemon Linn.), Pako (Diplazium esculentum (Retz.) Sw.) and Saluyot

(Corchorus olitorius Linn.). All of them contain nutrients that

could be possible sources of antioxidants needed by the body

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to fight against free radical formation which can cause damage

to the system. Bago is a multi-purpose tree species, its

edible young shoots are used in this study. It is native in

the Philippines and accepted as vegetable crop. Pako is an

edible plant that is widely distributed in the Philippines. It

contains several micronutrients and has antioxidant activity.

It contains several vitamins and minerals needed by the body

(McEvoy, 2010). C. olitorius Linn., commonly known as “Saluyot” is

one of the indigenous vegetables found in Philippines. It has

a lot of benefits like shielding the body against various

diseases. It contains high levels of the nutrients needed by

humans and its vital advantage is that it is high in

antioxidant which combines with free radicals in order to

stabilize the disorder in the system (Oboh et al., 2009;

Tindall, 1983).

Objectives of the Study

This study was conducted with the following objectives:

1. To investigate which solvent (ethanol, vinegar and

water) is the best extractant in the determination of

free radical scavenging activities of the test plants;

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2. To determine the effect of heating during extraction on

the free radical scavenging activity of the plant

samples; and

3. To compare the free radical scavenging activities of the

leaf extracts.

Time and Place of the Study

This study was conducted at the Department of Pure and

Applied Chemistry, College of Arts and Sciences, Visayas State

University, Visca, Baybay City, Leyte from April 2012 to July

2012.

Scope and Limitation of the Study

This study was limited on the evaluation of the

antioxidant property of three (3) species of indigenous plants

namely: Bago (Gnetum gnemon Linn.), Pako (Diplazium esculentum

(Retz.) Sw.) and Saluyot (Corchorus olitorius Linn.) with varying

solvent extractant (water, vinegar and ethanol), and the

effect of heating on the antioxidant activity of the samples.

CHAPTER II

MATERIALS AND METHODS

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Preparation of Plant Extracts

Three species of indigenous plants namely: Bago (Gnetum

gnemon Linn.), Pako (Diplazium esculentum (Retz.) Sw.) and Saluyot

(Corchorus olitorius Linn.) were collected from different parts of

Leyte (Fig. 1). Leaves were separated from the plant and

washed with water to remove dirt.

In the preparation of the extracts, ten (10) grams each

of the three species of the leaves previously washed with

water were used. The ratio between the sample and the solvent

was 1:10 (w/v). Samples were homogenized using a blender with

100 mL of the solvent at room temperature. The solvents were

95% ethanol, vinegar (5% acetic acid) and water. The resulting

mixture was transferred into a small beaker, allowed to stand

for an hour at room temperature and filtered using Whatman 42

filter paper. To minimize degradation of pigments, the beaker

was wrapped with carbon paper and was stored overnight in the

refrigerator.

The same procedure was followed for the samples heated

10°C below the boiling point of the solvent. The extraction

was done in triplicate.

B CA

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Figure 1. The plant samples: (A) Bago, (B) Pako, and (C)

Saluyot

Determination of Free Radical Scavenging Activity

Using the DPPH radical scavenging assay, the antioxidant

activity of all extracts was determined. Stock solution of

DPPH (22.5 mg/L) was prepared using ethanol/water solvent and

the initial absorbance was measured at 517 nm by UV-Vis

spectrophotometer (Shimadzu Double-Beam Spectrophotometer, UV-

210A). To 3.9 mL of DPPH solution, 0.1 mL of sample extracts

was added in order to initiate the reaction. The resulting

mixture was then shaken for 5 minutes and was allowed to stand

at ambient temperature in the dark for one hour to complete

the reaction of the cellular antioxidants with DPPH.

Absorbance was read at 517 nm (Arbianti et al, 2007) using 95%

ethanol as blank and the antioxidant activity was calculated

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from the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic

acid (Trolox) standard curve.

The amount of the sample necessary to react with one-half

of the DPPH solution was expressed in terms of the micromole

equivalents of the standard Trolox per gram of the fresh

weight of sample or the Trolox units per gram or TE/100 g.

Analysis was done in triplicate for each plant.

TE/100 g = [(µTE) (Volume of solvent) / (Mass of sample)]

(100)

Experimental Design and Statistical Analysis

This study was conducted with the use of two factor-

factorial Complete Randomized Block Design (CRD) with the

following treatments. It was done in triplicate.

T1W = Bago + water

T1A = Bago + vinegar (5% Acetic acid)

T1E = Bago + ethanol

T2W = Pako + water

T2A = Pako + vinegar (5% Acetic acid)

T2E = Pako + ethanol

T3W= Saluyot + water

T3A= Saluyot + vinegar (5% Acetic acid)

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T3E= Saluyot + ethanol

Analysis of variance (ANOVA) in RCBD was used to determine the

significant difference among the treatments and Tukey’s

Honestly Significantly Different (HSD) test was used for

multiple comparisons of treatments.

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CHAPTER III

RESULTS AND DISCUSSION

Free Radical Scavenging Activity of G. gnemon L., D. esculentum(Retz.) Sw. and C. olitorius L. Leaf Extracts

The free radical scavenging activity was determined using

1,1-diphenyl-2-picrylhydrazil (DPPH) assay as in vitro models for

Bago (Gnetom gnemon Linn.), Pako (Diplazium esculentum (Retz.) Sw.)

and Saluyot (Corchorus olitorius Linn.) vegetable samples. During

the reaction, DPPH molecule accepts an electron or hydrogen

radical in order to form a stable diamagnetic molecule (Gupta

et al., 2009). Basically, DPPH is a long-lived organic nitrogen

radical having a deep purple color. The purple chromogen

radical is reduced by antioxidant compounds to the

corresponding yellow hydrazine (Karadag et al., 2009). The

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antioxidants found in plant samples react with the stable free

radical DPPH that is converted to 1,1-diphenyl-2-

picrylhydrazine indicated by the change in color (Deore et al.,

2009) as shown in Figure 2, 3 and 4.

Figure 2a. Color of DPPH solution with various G.gnemon Linn. leaf extracts obtained without heating

Figure 2b. Color of DPPH solution withvarious G. gnemon Linn. leaf extracts

obtained with heating

Figure 3a. Color of DPPH solution with various D.esculentum (Retz.) Sw.

leaf extracts obtained without heating

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Figure 3b. Color of DPPH solution with various D. esculentum(Retz.) Sw.

leaf extracts obtained with heating

Figure 4a. Color of DPPH solution with various C.olitorius Linn. leaf extracts obtainedwithout heating

Figure 4b. Color of DPPH solution with various C.olitorius Linn. leaf extracts obtained withheating

In this particular assay, the free radical scavenging

activities of G. gnemon Linn., D. esculentum (Retz.) Sw. and C.

olitorius Linn. leaf extracts were determined using Trolox as

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standard and expressed in terms of Trolox equivalent per 100

grams sample or TE/100 g. Using the established λmax at 517 nm,

the bleaching of DPPH color signifies a decrease in

absorbance. A lower value of absorbance observed at 517 nm

indicates higher free radical scavenging activity of the plant

samples (Sanchez-Moreno, 2002; Mohammedi et al., 2011; Julkunen-

Tiito et al., 1985; Marinova et al., 1997)

Leaves of G. gnemon Linn., D. esculentum (Retz.) Sw. and C.

olitorius Linn. are known to contain compounds that act as

antioxidants which can scavenge free radical in order to form

a more stable molecule. Potential antioxidant components of

plants include Vitamin C and E, carotenoids, lycopenes (Gupta

et al., 2009) and other phytochemicals, namely: alkaloids,

flavonoids, tannins and phenolic compounds (Afolayan et al.,

2012). In addition, almost all minerals are potential

antioxidants which include selenium, iron, copper, zinc and

manganese (Mcdowell et al., 2007; Sharma et al., 2006). As reported

by Mcdowell et al., (2007), principal antioxidants vitamins are

vitamin E, C and β-carotene.

Table 1 shows the average free radical scavenging

activity of the three indigenous plant samples. A significant

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difference in free radical scavenging capability of the plant

samples was observed. Using ethanol as a solvent and without

heating, C. olitorius Linn. (380.29 ± 55.28 µmol TE/100 g) gives

the highest free radical scavenging activity followed by G.

gnemon Linn. (306.45 ± 43.35 µmol TE/100 g) and D. esculentum

(Retz.) Sw. (258.73 ± 90.02 µmol TE/100 g). The same trend

was observed in heated samples. C. olitorius Linn. is known to

have high levels of carotenoids, vitamins and minerals as

reported by Ibrahim et al. (2011). This crop is an excellent

source of vitamins A and C (Tindall, 1983; Adebayo, 2010;

Adeniyi, 2012), fiber and minerals, including calcium and

iron, which contribute to its high free radical scavenging

activity. On the other hand, G. gnemon Linn. and D. esculentum

(Retz.) Sw. contains low levels of vitamin C and minerals (Hoe

et al., 1999) which partly explains its low free radical

scavenging activity.

Table 1. Free radical scavenging activity of G. gnemon Linn., D.esculentum (Retz.)

Sw. and C. olitorius Linn. leaf extracts obtained with andwithout heating

during extraction

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Values are average of sample measurements and represented as mean and ± standard deviation.Values with different letters differ significantly at α = 0.05, Tukey’s HSD

G. gnemon Linn., D. esculentum (Retz.) Sw. and C. olitorius Linn.

may possess free radical scavenging activity. However in

comparison with other leafy vegetables, these samples have

less free radical scavenging activity (Fig. 5).

Figure 5. Free radical scavenging activities of somevegetables

The free radical scavenging activities of Moringa oleifera Linn.,

3495 µmol TE/100 g (Dańas, 2012), Ipomea batatas Linn., 3239 µmol

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TE/100 g (Lazona, 2012), Basella spp., 2759 µmol TE/100 g

(Dahan, 2012) and taro were 2620 µmol TE/100 g (Cuabo, 2012)

respectively using water as solvent. Moreover, the free

radical scavenging activity of Chili pepper leaves is

approximately 215 µmol TE/100 g (Cardańo, 2013).

Effect of Extracting Solvent

Free radical scavenging activities depend on the nature

of the extracting solvents due to the availability of

different extractable antioxidant components that have

different chemical characteristics and polarities (Mohammedi et

al., 2011).

Table 2 shows the average free radical scavenging

activity as influenced by the solvent system. Results revealed

that ethanol (357.39 ± 49.34 µmol TE/100 g) has the highest

ability to extract plant antioxidants followed by vinegar (5%

acetic acid) (338.77 ± 89.42 µmol TE/100 g) and water (249.32

± 59.46 µmol TE/100 g) in samples not subjected to heating.

The same trend was observed in heated samples. Result implies

that ethanol is the most effective extractant in the

determination of free radical scavenging activity. The present

finding is in agreement with the result of Cuabo (2012), Pole

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(2012), Salas (2012) and Perez (2012). On the other hand,

acetic acid extracts possessed higher free radical scavenging

activity than that of water extracts. Significant difference

was observed in all of the unheated samples using different

solvents. In heated samples, no significant difference between

ethanol and acetic acid as solvent but in water extracts,

there is a significant difference.

Table 2. Free radical scavenging activity of samples using

different solvents

Values are average of sample measurements and represented as mean and ± standard deviation. Values with different letters differ significantly at α = 0.05, Tukey’s HSD

Effect of Extraction Temperature

To test whether heating affects the free radical

scavenging activity of the G. gnemon Linn., D. esculentum (Retz.)

Sw. and C. olitorius Linn., extraction was carried out at

temperatures 10oC less than the boiling point of the solvents.

Results revealed that the free radical scavenging activity of

extracts increased with increasing temperature which is in

agreement with findings of Navarro et al., (2010). As reported by

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Yoshioka et al., (1990), bioactive compounds that are associated

with free radical scavenging activity such as polyphenols,

flavonoids and other components are usually extracted at

higher temperature which partly explains the results. Thus,

heating enhances the extraction process, extracting the

components which cannot be extracted at room temperature. In

extraction without heating, extracts showed that C. olitorius Linn.

has the highest free radical scavenging activity. However, the

ethanolic extract possessed the highest ability to extract

components upon heating. These results may infer that the test

plants are best prepared through heating to optimize the

availability of their antioxidant compounds.

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CHAPTER IV

CONCLUSIONS

The following conclusions were attained in the conduct of this

study:

1. Ethanol was the most effective extracting solvent in the

determination of free radical scavenging activity of G.

gnemon Linn., D. esculentum (Retz.) Sw. and C. olitorius Linn.;

2. Heating caused a significant increase in the free radical

scavenging activity of all the plant samples; and,

3. C. olitorius Linn. exhibited the highest free radical

scavenging activity.

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