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PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Cocoa fresh beans aqueous extract as free radical quencher and ferric reducer

Pelita Perkebunan 38(2) 2022, 137

—

143

Cocoa Fresh Beans Aqueous Extract as Free Radical Quencher

and Ferric Reducer

Samuel Yap Kian Chee

1*)

, Arief Huzaimi Bin M.D. Yusof

, and Hui Han Chin

Cocoa Downstream Technology Division, Malaysian Cocoa Board, Lot 12621,

Kawasan Perindustrian Nilai, 71800 Nilai, Negeri Sembilan, Malaysia

Corresponding Author:

samuel@koko.gov.my

Received: 22 March 2022 / Accepted: 27 April 2022

Abstract

The presence of free radicals and oxidants in our body is either produced

by our body through normal cell metabolisms or from environment surrounding

us such as radiation, medication, pollutants or others. The free radicals and oxidants

can be harmful to our body when their accumulation posing oxidative stress in

the body. Cocoa beans are rich in polyphenols, which are functioning as antioxidant.

However, each antioxidant has difference response towards either free radicals

or other oxidants. The purpose of this research was to study the ability of cocoa

fresh beans aqueous extract functions as antioxidant between its ability to reduce

ferric to ferrous and number of free radicals removed from organic chemical compound

of 2,2-diphenyl-1-picrylhydrazyl (DPPH). A series concentration of cocoa fresh

beans aqueous were prepared. Its ability as ferric reducer was determined based

on ferric reducing antioxidant power (FRAP) assays and its ability as free radicals

scavenger was determined using DPPH assays. This study revealed that freeze

dried cocoa fresh beans aqueous extract of 50 ppm was able to reduce 3.2 x 10

of ferric molecules to ferrous. Concentration below 20 ppm generally was not

able to reduce Fe(III) to Fe(II). Whereas, at concentration of 45 ppm freeze dried

cocoa fresh beans aqueous extract was estimated able to remove 1.4 x 10

organic

nitrogen free radicals from DPPH assay. This study suggests that freeze dried

cocoa fresh beans aqueous extract has better performance in quenching free radicals

compared to reducing ferric into ferrous.

Keywords: cocoa, polyphenols, free radical quencher, ferric reduction

ISSN: 0215-0212 / e-ISSN: 2406-9574

INTRODUCTION

Free radicals and oxidants are produced

either from normal cell metabolisms in situ or

from external sources such as radiation,

medication and pollution. These free radicals

and oxidants are mostly destroyed by our

body system naturally. Nevertheless, when

too much of these free radicals and oxidants

present in our body and they cannot gradually

be destroyed, it can be harmful to the body

when their accumulation reaches the threshold

of oxidative stress level. This oxidative stress

can lead into the development of chronic and

degenerative illness such as cancer, autoimmune

disorders, aging, cataract, rheumatoid arthritis,

cardiovascular and neurodegenerative diseases

(Lien Ai et al., 2008). The purpose of taking anti-

oxidants is to boast our body mechanisms to

counteract this oxidative stress and consequently

to maintain healthy.

Generally, there are two types of antioxi-

dants, namely, non-enzymatic antioxidants and

enzymatic antioxidants (Gutpa, 2015). Plant

polyphenols, such as catechin and epicatechin,

DOI: 10.22302/iccri.jur.pelitaperkebunan.v38i2.502

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PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Samuel Yap et al.

are categorized as non-enzymatic antioxidants.

There are multiple sources of free radicals

and oxidants. This include, but not limited

to superoxide anion radical, singlet oxygen,

hydroxyl radical, nitric oxide, peroxynitrite,

hypochlorous acid, peroxyl radical, and lipid

peroxyl radical. Both oxidants and antioxi-

dants are exhibiting differently in terms of

their chemical and physical characteristics

(Brewer, 2011). Hence, each antioxidant, in

general, is responding in a different manner

towards different sources of radical or oxidant.

For instant, carotenoids are not good in quenching

peroxyl radicals relative to phenolics but are

exceptional good as singlet oxygen quencher

compared to most of the phenolics, which are

relatively ineffective (Satish & Dilipkumar,

2015).

Ability to reduce iron in ferric reducing

antioxidant power (FRAP) test has little re-

lationship to the radical quenching processes

(H transfer) as mediated by most antioxidants.

Nevertheless, reduced metals are active propa-

gators of radical chains through formation

of alkoxyl radical by hydroperoxide reduction

mechanism. Hence, it is interesting to evaluate

whether high FRAP values is correlating with

the tendency of polyphenols to become pro-

oxidants under some conditions.

2,2-diphenyl-1-picrylhydrazyl (DPPH)

assay is one of the few stable organic nitrogen

radicals, which is deep purple in color. This

assay is based on the measurement of the

reducing ability of antioxidants toward the

organic nitrogen radicals of DPPH. The de-

gree of antioxidant capability is based on

measurement of the loss of DPPH color at

520 nm after it is reacted with test compounds

monitored by an UV-Visible spectrometer.

Nevertheless, DPPH cannot be regarded to

the highly reactive and transient peroxyl radicals

which are involved in lipid peroxidation.

Studies showed that methods of extraction

and extraction mediums affecting greatly on

the free radicals quenching power of the

cocoa beans extract. A significant drop on

the free radical scavenging capability is observed

from the extract of fresh and dry beans compared

to roasted beans (Samuel Yap & Arief Huzaimi,

2020). Major phenolic compositions of the

fresh cocoa beans aqueous extract are catechin

and epicatechin (Samuel Yap, 2018). The

objective of this paper is to report the behaviors

of cocoa fresh beans aqueous extract towards

FRAP and DPPH antioxidant assays that are

commonly used in antioxidant study.

MATERIALS AND METHODS

The cocoa variety used in this study

was a commercial cocoa clone by MCB.

The supply of the cocoa materials were

from the cocoa research study plot at Bagan

Datuk, Perak of Malaysia.

Cocoa Phenolics Extraction

One gram of cocoa beans was added with

50 mL of distilled water, ground with food

processor in low speed for 3 seconds, incu-

bated at 80

C in an incubator shaker with

150 rpm orbital shaking mode for 15 minutes.

The extract was then filtered with filter paper

(Whatman no. 4). De-pulping processes were

carried out for fresh unfermented cocoa beans

and freeze dried prior to extraction.

FRAP Assay

A series concentration of cocoa fresh beans

aqueous extract from 1750 ppm to 500 ppm

with 250 ppm decreasing of concentrations

were prepared for FRAP analysis. Prior to

FRAP tests, a standard curve of Fe

from 0.2 µM to 2.0 µM versus absorbance

at 593 nm was plotted.

Two hundred milliliters of buffer (3.2 mL

of acetic acid mixed with 196.8 mL of distilled

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Cocoa fresh beans aqueous extract as free radical quencher and ferric reducer

water and 0.62 g of sodium acetate pH 3.6)

was added with 20 mL of TPTZ (2,4,6-tri(2-

pyridyl)-1,3,5-triazine) solution (0.063 g of

TPTZ in mixture of 78.8 µL of hydrochloric

acid) and with 20 mL of FeCl

(20 m mol

-1

). The mixture of buffer, TPTZ solution

and ferric chloride in the ratio of 10:1:1 re-

spectively, is known as FRAP assays. Samples/

standards (100 µL) were added with 3.0 mL

of FRAP assays and incubated for 30 minutes.

Absorbance was then measured by using

UV-Vis spectrophotometer at 593 nm against

the blank. Its reducing power from ferric

to ferrous was gauged in correlating to the

standard curve respectively.

Free Radicals Scavenging Capability

A series concentration of cocoa fresh

beans aqueous extract (500 ppm, 250 ppm,

125 ppm, 62.5 ppm, 31.25 ppm, 15.63 ppm,

and 7.81 ppm) were prepared from a stock

solution of 1,000 ppm.

Free radicals scavenging capability of

the extract were determined by drawing 0.5 mL

sample added with 5.0 mL 0.06 mM DPPH

solution and tris-buffer (pH 7.6), mixed well

and incubated in dark for 30 minutes prior

to measure with UV-Visible spectrophotometer

at 520 nm. Number of free radicals quenched,

rad

was calculated as the equation (1).

............... (1)

where,

RSC (%) = percent of DPPH radicals quenched

= [(Abs(b) - Abs(S)/Abs(b)] x 100

N(DPPH) = available mole of DPPH in test solution x

Abs(b) = Absorbance for blank

Abs(S) = Absorbance for sample

NA = Avogadro constant = 6.022 x 10

mol

-1

RESULTS AND DISCUSSION

Antioxidant capability of the cocoa beans

extract is contributed by the presence of

phenolic contents in the cocoa beans. Previous

study reported that cocoa fresh beans aqueous

extract from the commercial clone by MCB

contains mainly flavanol monomer of epicatechin

and catechin (Samuel Yap, 2018). The anti-

oxidant capability of the cocoa fresh beans

aqueous extract was tested based on FRAP

and DPPH assays.

FRAP Assay

A ferrous standard curve from 0.2 µmol L

-1

to 2.0 µmol L

-1

versus absorbance value at

593 nm was constructed as in Figure 1.

Based on the methods outlined above,

the normalized concentration on each dilution

introduced in the test assays were calculated

based on equation (2) and tabulated in Table 1.

Normalized concentration, C

sample

x OC)/(V

sample

+ V

assays

) ............. (2)

Where:

sample

= amount of sample used in the test, mL

OC = original concentration of the sample from the

dilution series, ppm

assays

= total amount of test assays solution, mL

The reducing power of the cocoa beans

aqueous extract to reduce ferric to ferrous

were determined based on their absorbance

value in correlating to the value in the standard

curve above (Figure 1), and the results were

tabulated in Table 1.

Percentage of the ferric molecules re-

duced to ferrous by the cocoa fresh beans

aqueous extract (CFBAE) was determined

based on the equation (3) and results were

tabulated in Table I:

...................... (3)

Where,

FR(%) = percent of ferric reduced to ferrous

[Fe(II)] = concentration of Fe(II) as determined in correlation

with Figure 1

[Fe(III)] = available concentration of Fe(III) in the test

solution (3 mL FRAP assay)

= 1.67 mM

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Samuel Yap et al.

Number of ferric molecules reduced to

ferrous (Fe(III) Fe(II)) was calculated

based on the equation (4).

It could be noted from Table 1 that at the

normalized concentration below 20 ppm,

cocoa fresh beans aqueous extract basically

approaching zero reducing power.

Table 1. Reducing power from ferric to ferrous by cocoa fresh beans aqueous extract (CFBAE)

Series concentration of CFBAE (ppm)

Absorbance Fe(II) (µM) No. of ferric molecules

Original Normalized at 593 nm (Average of 3 readings) reduced to ferrous

1750 56.45 2.220 ± 0.002 1.84 3.32 x 10

1500 48.39 2.192 ± 0.007 1.80 3.25 x 10

1250 40.32 2.199 ± 0.002 1.81 3.26 x 10

1000 32.26 1.834 ± 0.008 1.29 2.33 x 10

875 28.23 1.722 ± 0.004 1.13 2.04 x 10

750 24.19 1.603 ± 0.007 0.96 1.73 x 10

625 20.16 1.274 ± 0.005 0.49 8.83 x 10

500 16.13 0.931 ± 0.006 0 0

Figure 1. Standard curve of ferrous concentrations vs absorbance at 593 nm

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2

.7H

O concentration (µM)

2.4

2.2

1.8

1.6

1.4

1.2

0.8

Abs

Y = 0.7018 X + 0.9295

= 0.9998

Fe(III) Fe(II) = .............................. (4)

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Cocoa fresh beans aqueous extract as free radical quencher and ferric reducer

Free Radicals Quenching

In this study, only 0.5 mL from each series

of dilutions were drawn and added into 5.0 mL

assays, hence, based on the equation (2),

normalized concentration in the reaction was

calculated as in Table 2 respectively. Average

absorbance value for blank was 2.057. The

power of the cocoa fresh beans aqueous extract

in quenching organic nitrogen radicals from

DPPH assay in term of percentage of free

radicals scavenging capability were calculated

based on equation (1) and tabulated in Table 2.

Results showed a useful normalized

concentration of cocoa beans aqueous extract

was 22.5 ppm and above where more than

50% of the available free radicals were

quenched. At normalized concentrations of

lower than 2 ppm, cocoa beans aqueous

extract might lose its quenching ability. A

similar profiles were reported by Azizah et al.

(2007) but with higher cocoa beans extract

concentration, perhaps due to the direct uses

of cocoa beans extract without freeze dried as

mentioned in their methodology of the study.

Azizah et al. (2007) reported that no significant

free radicals scavenging activity from DPPH

assays was detected at the cocoa beans extract

concentration below 0.62 mg mL

-1

and rapidly

increased from 0.62 to 2.5 mg mL

-1

DPPH versus FRAP

Figure 2 shows that cocoa fresh beans

aqueous extract has better performance in

organic nitrogen free radicals scavenging

capability compared to ferric reduction to

ferrous. Ferric reducing antioxidant power

(FRAP) assay is conducted at acidic environ-

ment of pH 3.6 to maintain its iron solubility.

Reaction at low pH decreases the ionization

potential and consequently, initiates electron

transfer and increases the redox potential,

causing a shift in the dominant reaction

mechanism (Spiegel et al., 2020; Zhong &

Shahidi, 2015). However, at lower pH condi-

tion, the antioxidant activity of the phenols

decreases due to the decreasing of its electron-

donating ability upon deprotonation (Arzu

et al., 2016; Pekal & Pyrzynska, 2015).

The other reasons maybe due to the higher

steric inaccessibility by the phenolic compounds,

mainly catechin and epicatechin, found in

cocoa fresh beans aqueous extract (Samuel

Yap, 2018). In TPTZ solution, ferric ion is

trapped in between the tripyridylniazine ligands

of the ferric triphyridylniazine complex, causing

the antioxidant compounds of catechin and

epicatechin having less room to interact with

the trapped ferric ion. Whereas compared

to DPPH, it has less steric effect to access

Table 2. Free radicals scavenging capability from the series of concentrations for cocoa fresh beans aqueous extract (CFBAE)

Series concentration of CFBAE (ppm)

Absorbance at 520 nm

% Scavenging No. of free radicals quenched

Original Normalized

(Average of 3 readings)

1000 90.9 0.388 ± 0.002 81.1 1.47 x 10

500 45 0.476 ± 0.004 76.9 1.39 x 10

250 22.5 0.987 ± 0.004 52.0 9.39 x 10

125 11.25 1.393 ± 0.002 32.3 5.84 x 10

62.5 5.63 1.752 ± 0.004 14.8 2.67 x 10

31.25 2.81 1.896 ± 0.006 7.8 1.41 x 10

15.63 1.41 2.074 ± 0.007 -0.8 -

7.81 0.70 2.067 ± 0.005 -0.5 -

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Samuel Yap et al.

its organic nitrogen free radicals by the phenolic

compounds contain in the cocoa fresh beans

aqueous extract.

CONCLUSIONS

Cocoa fresh beans aqueous extract (CFBAE)

performed better in quenching the nitrogen

organic radicals from DPPH assay if compared

to its ability to reduce ferric to ferrous from

FRAP assay. A normalized CFBAE concen-

tration of 22.5 ppm and above was needed to

quench more than 50% of the available free

radicals in a 0.0003 mM DPPH solution and

lost its quenching power at concentration lower

than 2 ppm. However, to function as ferric reducer,

higher concentration was needed as at 20 ppm

and lower, cocoa fresh beans aqueous extract

basically was unable to reduce ferric into ferrous

from a FRAP assay.

ACKNOWLEDGMENT

The authors would like to thanks the

Government of Malaysia to fund this project

via RMK11 Development Project. A special

thanks to Dr. Tee Yei-Kheng in providing

cocoa fresh beans relentlessly for this project.

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Figure 2. Relationship between effective concentrations of CFBAE and number of DPPH free

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FRAP assays

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